A Novel Salicylaldehyde Dehydrogenase from Alpine Soil Metagenomes Reveals a Unique Catalytic Mechanism

A Novel Salicylaldehyde Dehydrogenase from Alpine Soil Metagenomes Reveals a Unique Catalytic Mechanism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Novel Salicylaldehyde Dehydrogenase from Alpine Soil Metagenomes Reveals a Unique Catalytic Mechanism Shamsudeen Umar Dandare, Aliyu Ibrahim Dabai, Deepak Kumaresan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6604919/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Nov, 2025 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted 5 You are reading this latest preprint version Abstract Metagenomic approaches have revolutionised the discovery of novel enzymes with biotechnological potential from unexplored environments. Here, we report the identification and comprehensive characterisation of a novel salicylaldehyde dehydrogenase (SALD AP ) from an alpine soil metagenome. Phylogenetic analysis revealed that SALD AP is the first experimentally characterised alphaproteobacterial SALD, forming a distinct evolutionary clade among known bacterial SALDs. The recombinant enzyme showed strict specificity for NAD⁺ and exceptional catalytic efficiency toward aromatic aldehydes, with benzaldehyde as the preferred substrate. SALD AP was most active under mildly alkaline conditions (optimum pH 8.0) and tolerated a range of chemical environments, though high concentrations of certain metal ions and solvents were inhibitory. Kinetic analysis demonstrated that SALD AP binds and oxidises aromatic substrates much more efficiently than aliphatic aldehydes, with catalytic efficiencies exceeding 10⁶ M⁻¹ s⁻¹ for aromatics. The enzyme was stabilised by the simultaneous presence of substrate and cofactor, as shown by differential scanning fluorimetry. Molecular docking with the crystal structures of SALD AP and Pseudomonas NahF revealed that SALD AP utilises a unique arrangement of active site residues (ASN-137, ARG-145, GLU-238, and CYS-272) to mediate catalysis. Based on structural and docking data, we propose a distinct catalytic mechanism for SALD AP , in which ASN-137 plays a central role in substrate binding and stabilisation. This study expands the functional diversity of the ALDH superfamily. It establishes a new paradigm for aromatic aldehyde oxidation, providing valuable insights for the engineering of ALDHs for environmental bioremediation and synthetic biology applications. Metagenomics Alpine soil microbiome Aldehyde dehydrogenase Aromatic aldehydes Catalytic mechanism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Microbial enzymes are key players in the degradation and detoxification of environmental pollutants, including persistent aromatic hydrocarbons such as polycyclic aromatic hydrocarbons (PAHs). Among these, aldehyde dehydrogenases (ALDHs) represent a structurally conserved but functionally diverse superfamily of NAD(P) + -dependent enzymes responsible for oxidising aldehydes to their corresponding carboxylic acids, a key step in microbial aromatic compound catabolism [ 1 , 2 ]. In the aerobic breakdown of PAHs by bacteria, salicylaldehyde (SAL) emerges as an important metabolic intermediate that links the upper and lower catabolic pathways [ 3 ]. Salicylaldehyde dehydrogenase (SALD; EC 1.2.1.65), a member of the ALDH superfamily, catalyses the NAD(P) + -dependent oxidation of SAL to salicylate, facilitating its entry into central carbon metabolism via the catechol or gentisate pathways [ 4 ]. While ALDHs share a conserved domain and catalytic core, they exhibit remarkable diversity in substrate range, oligomeric state, and cofactor preference [ 5 , 6 ]. Despite the central importance of SALDs in aromatic hydrocarbon metabolism, only a handful have been structurally or biochemically characterised to date, mainly from Pseudomonas and related genera. The in vivo activity of SALD has been documented in several PAH-degrading microorganisms [ 7 – 11 ], but detailed in vitro biochemical characterisation remains limited. Zhao and colleagues [ 12 ] purified and partially characterised two SALDs (NahV and NahF) from P. putida ND6. Additional studies have examined SALDs from Pseudomonas sp. strain C6 [ 13 ] and Alteromonas naphthalenivorans [ 14 ]. Notably, Coitinho and colleagues reported the first crystal structure and kinetic analysis of a broad-substrate specificity SALD (NahF) from P. putida G7 [ 1 ]. More recently, we determined the crystal structure of the first metagenome-derived SALD [ 15 ], which shares only 43% amino acid identity with the well-characterised NahF from P. putida G7, highlighting the sequence diversity within this enzyme family. The rise of metagenomic sequencing has enabled access to novel enzyme diversity from extreme or underexplored environments. Alpine soils, shaped by glaciation and harsh climatic conditions, represent unique reservoirs of new microbial enzymes with potentially distinct biochemical properties. While recent metagenomic studies have begun to reveal the functional diversity of such environments [ 16 , 17 ], no SALD from a metagenomic source has been fully characterised until now. In this study, we report the comprehensive characterisation of a novel salicylaldehyde dehydrogenase (SALD AP ) identified from an alpine soil metagenome. Through integrated phylogenetic, structural, kinetic, ligand-binding, and molecular docking analyses, we show that SALD AP is the first experimentally characterised alphaproteobacterial SALD and exhibits a unique catalytic mechanism involving a strictly conserved asparagine residue. SALD AP displays strict NAD + dependence, high catalytic efficiency for aromatic aldehydes, and is stabilised by substrate and cofactor. These findings expand the functional and mechanistic landscape of the ALDH superfamily and provide a foundation for engineering novel biocatalysts for environmental and synthetic biology applications. Materials and Methods Soil Sample Collection and Metagenomic Library Construction This procedure has been described previously [ 17 ]. In brief, soil samples were aseptically collected at mid-depth from different horizons (Ah, Bw, Cox, Cu) in glacial moraines from the Guil and Po River valleys of the French and Italian Alps, respectively. Samples were transported on ice and stored at -20 o C. Total DNA was extracted using the PowerSoil DNA extraction kit (Mo Bio) according to the manufacturer’s instructions, with minor modifications. DNA concentrations were determined using a Quantus Fluorometer (Promega). DNA libraries were prepared and sequenced in paired-end mode on an Illumina MiSeq at the University of Cambridge DNA sequencing facility. Raw metagenome data are available in the NCBI Sequence Read Archive (Bioproject number: PRJNA490486). Gene Mining, Assembly, and Phylogenetic Analysis To identify aldehyde dehydrogenases (ALDHs), a vanillin dehydrogenase from Pseudomonas putida KT2440 (NP_745497.1) served as a reference for BLAST searches against merged Illumina datasets. Sequence data from different soil horizons and nearby sample sites were combined to maximise coverage. Discontinuous MagaBLAST (dc-megablast) was used with an E-value threshold of 1.0e-5 and a minimum 70% identity. Hit sequences were extracted and used as seeds for gene-targeted assembly using the PRICE de novo assembler [ 18 ], which builds longer contigs from paired-end reads. PRICE was run for ten assembly cycles, with only contigs matching initial BLAST hits retained. Contigs longer than the expected gene length were analysed for open reading frames (ORFs) with NCBI’s ORF Finder (bacterial code). The quality and novelty of each ORF were confirmed by BLAST. The assembled SALD AP sequence is available in NCBI with the GenBank accession PV600639. Protein sequences were aligned with Clustal Omega [ 19 ], and poorly aligned regions were trimmed with trimAl [ 20 ]. A phylogenetic tree was constructed using FastTree [ 21 ], and visualised in iTOL [ 22 ]. Cloning, Expression, and Purification of SALD Primers designed based on the assembled SALD AP sequence were used to amplify the gene from alpine soil metagenomic DNA. The PCR product was cloned into the pLATE51 vector and expressed as previously described [ 15 ]. The recombinant plasmid (pLATE51-SALD AP ) was transformed into chemically competent E. coli BL21 (DE3) cells. Cultures were grown to OD₆₀₀ ≈ 0.6 at 30°C, induced with 1 mM IPTG, and incubated for 6 hours. Cells were harvested, resuspended in lysis buffer, and lysed by sonication. The soluble fraction was purified by Co²⁺-affinity chromatography and dialysed. The recombinant protein was expressed with an N-terminal 6xHis tag, and all experiments were performed with the 6xHis-SALD AP . Enzyme activity assay SALD AP activity was measured as described by Coitinho et al. (2016). Reactions were performed at 25°C, monitoring NAD⁺ reduction to NADH at 340 nm. The assay mixture contained 200 µM salicylaldehyde (SAL), 200 µM NAD⁺, and 1–2 µM purified enzyme in 1 mL of sodium phosphate buffer (pH 8.0). NADH formation was confirmed by colorimetric detection of salicylate. Initial reaction rates were calculated from the linear region of the absorbance curve using linear regression. The NADH extinction coefficient (6,220 M⁻¹ cm⁻¹) was used for concentration conversion [ 23 ]. Substrate Specificity and Steady-State Kinetics Optimal NAD⁺ and enzyme concentrations were established by varying NAD⁺ concentrations (0–1000 µM) with 1 µM enzyme and 200 µM salicylaldehyde, then varying enzyme (0–1 µM) at the optimal NAD + concentration. Steady-state kinetics were performed at 25°C with 50 µM NAD⁺ and varying substrate (2–1500 µM) in 100 mM Tricine buffer (pH 8.0, 1 mL volume). For phenolic and long-chain aldehydes, 1.7% DMSO was included as a solvent carrier, a concentration shown to have minimal effect on ALDH activity [ 24 ].​ Initial rates were calculated as described above. kinetic parameters (Kₘ, K i , Vₘₐₓ) were obtained by fitting rate data to the Michaelis-Menten and substrate inhibition models (equations 1 and 2) using GraphPad Prism 9.0. Turnover number (k cat ) was calculated using Eq. 3 below, where [E o ] is the total enzyme concentration [ 25 , 26 ] $$\:v=\frac{{V}_{max}\left[S\right]}{{K}_{m}+\left[S\right]}\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(1\right)$$ $$\:v=\frac{{V}_{max}\left[S\right]}{{K}_{m}+\left[S\right]+\frac{{\left[S\right]}^{2}}{{K}_{i}}}\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(2\right)$$ $$\:{k}_{cat}=\frac{{V}_{max}}{{\left[E\right]}_{o}}\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(3\right)$$ Where V max is the maximum velocity, k m is the Michaelis-Menten constant, K i is the substrate inhibition constant, and [ S ] is the substrate concentration. Differential Scanning Fluorimetry (DSF) DSF was performed with SYPRO Orange dye to assess thermal stability and ligand effects. Enzyme (5–7 µM) in 50 mM HEPES buffer (pH 7.4) was mixed with substrates, NAD⁺, and Ca²⁺ as required. Substrates were prepared in DMSO, kept below 1%. SYPRO Orange was added immediately before measurement. Triplicate reactions were run in 0.2 mL PCR tubes using a Rotor-Gene Q cycler (Qiagen), with temperature ramped from 25°C to 95°C at 1°C/5 s. Fluorescence (excitation 460 nm, emission 510 nm) was used to monitor protein unfolding. The melting temperature (Tₘ) was determined from the first derivative of the fluorescence (ΔF/ΔT). Molecular Docking Docking was performed with AutoDock Vina 1.1.2 with UCSF Chimera 1.12 as the interface. Crystal structures of SALD AP (PDB ID: 6QHN) and P. putida G7 NahF (PDB ID: 4JZ6) were prepared by removing non-standard residues and ligands, adding hydrogens, and assigning Gasteiger charges.​ Ligands were built in Chimera from PubChem SMILES, energy-minimised, and converted to .pdbqt format. Docking used grid boxes centred on residues within 6 Å of the co-crystallised ligands. Default parameters were used for docking. Binding interactions and hydrogen bonds were visualised in Chimera and PyMOL 2.6. Dissociation constants (K d ) were calculated from binding affinities (ΔG) using: $$\:{K}_{d}={e}^{\left(\frac{\varDelta\:G}{RT}\right)}\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(4\right)$$ where R is the gas constant (1.987 cal mol − 1 K − 1 ) and T is the temperature (298.15 K) [ 27 ].​ Results and Discussion Sequence and Phylogenetic Analysis of Assembled Salicylaldehyde Dehydrogenase (SALD AP ) After ten assembly cycles, 215 contigs were recovered, with only five exceeding 1,000 nucleotides. Analysis revealed two major ORFs (Table S1 ), and only the second contig encoded a full-length gene with both start and stop codons. BLAST analysis showed this gene shares just 82% nucleotide identity with known sequences in NCBI and contains an aldehyde dehydrogenase superfamily (ALDH-SF) and a salicylaldehyde dehydrogenase DoxF-like (ALDH SaliADH) domains (Figure S1 ), confirming it as a bona fide SALD enzyme. Phylogenetic analysis placed SALD AP within a distinct alphaproteobacteria cluster (including Croceicoccus, Erythrobacter, Kordiimonas and Novosphingobium ), separate from clusters of beta- ( Ralstonia, Paraburkholderia and Polaromonas ) and Gammaproteobacteria ( Pseudomonas and Alteromonas ) (Fig. 1 ). This distinct phylogenetic position highlights SALD AP as a novel representative of the SALD family. Previous survey[ 14 ] showed most bacterial SALDs are distributed among Betaproteobacteria (31.33%), Gammaproteobacteria (21.58%), and Alphaproteobacteria (16.80%); however, to our knowledge, no alphaproteobacterial SALD has yet been characterised, motivating our study. Multiple sequence alignment revealed that SALD AP , NahF, and NahV share 40% amino acid identity, with key conservation at catalytic and cofactor-binding domains. Notably, the cofactor binding domain, which conforms to the Rossmann fold (residues 128–221) contains a glycine-rich motif (GSTXVG, residues 216–221), analogous to the G 1 XXXXG 2 motif in other NAD + -dependent dehydrogenases (Fig. 2 ) [ 5 , 28 ]. The invariant catalytic cysteine (Cys272), as well as substrate-binding residues Asn137, Arg145, Glu238 and Leu239, are conserved. Aromatic residues which may be implicated in substrate recognition (Trp83, Phe87 and Phe262) [ 1 ] are also preserved. Isolation of SALD AP from Soil Metagenome and Recombinant Production Specific primers designed from the SALD AP sequence enabled its targeted amplification from alpine metagenomic DNA (Figure S2a), where it was present in all samples tested. The gene was cloned and optimally expressed, yielding soluble protein purified to homogeneity with Co 2+ -affinity resin (Figure S2b). From 1 L of culture, approximately 2 mg/mL (40 µM) purified protein was obtained. SDS-PAGE analysis indicated a monomeric molecular weight of 49.6 kDa, determined by measuring and comparing the distances travelled by the protein band and the size markers (Figure S2c). SALD AP is a NAD + -dependent Aldehyde Dehydrogenase The purified enzyme’s activity was assayed using SAL as substrate, with NAD + and NADP + as possible cofactors. SALD AP showed strong activity with NAD + but none with NADP + . This selectivity reflects a common pattern among ALDHs, where NAD + is typically used in oxidative degradation, while NADP + is reserved for reductive biosynthesis [ 29 ]. Sequence analysis revealed that, unlike the well-characterised Pseudomonas putida NahF enzyme, which accepts both cofactors [ 1 ] SALD AP contains a glycine in place of an arginine in the nucleotide binding site (Fig. 2 ). While this substitution may contribute to cofactor selectivity, broader studies suggest that cofactor discrimination depends on the overall architecture of the binding pocket, rather than individual residues [ 29 ]. SALD AP has an Alkaline pH Optimum and Is Stable Over a Wide Temperature Range The optimal pH for recombinant 6xHis-SALD AP was pH 8.0, with a bell-shaped activity profile across pH 5.3–11.1. Activity dropped by over 30% with a one-unit shift from the optimum, indicating high sensitivity to pH changes. At least 40% and 35% activity loss was observed at pH 7.0 and 9.0, respectively (Fig. 3 a). This behaviour is consistent with classical SALDs from Pseudomonas species [ 1 , 13 ] and other reported ALDHs [ 30 – 32 ]. Thermostability studies showed that 6xHis-SALD AP retained more than 60% activity after 1 h at 40 o C, but was quickly inactivated at higher temperatures, where it showed marginal activity at 50 o C after 1 h, and complete inactivation after 30 mins at 60 o C (Fig. 3 b). Compared to NahF from P. putida , which remains active longer at elevated temperatures [ 1 ], SALD AP is less thermostable but remains robust under moderate conditions. Effect of Metal Ions, Organic Solvents, Detergents, and Inhibitors on SALD AP Activity The activity of SALD AP was affected by several metal ions, solvents, detergents, and inhibitors. At 1 mM, Fe 2+ and Ca 2+ increased activity by 31% and 17%, respectively, while K + and La3 + had negligible effects. In contrast, Zn 2+ , Ni + , Mn 2+ , Rb + , Co 2+ , Cu 2+ , Na + , and Mg 2+ inhibited activity to varying extents, with Zn 2+ causing more than 67% inhibition. At 50 mM, most metal ions except Na + caused substantial inhibition. Notably, the inhibitory effect depended on the salt type; for example, CuCl 2 caused total inactivation, while CuSO 4 retained 15% activity. These results are presented in Table 1 . These findings differ somewhat from previous reports on Pseudomonas SALDs [ 1 , 12 ], which showed different metal ion sensitivities. Table 1 Effect of metal ions on the activity of recombinant 6xHis-SALD AP Metal Ions Metal Salts Relative activity (%) 1 mM 50 mM None None 100 100 Ca 2+ Calcium chloride 117 40 K + Potassium chloride 101 84 Mg 2+ Magnesium chloride 99 61 Na + Sodium chloride 95 101 Cu 2+ Copper (II) sulphate 51 15 Cu 2+ Copper (II) chloride 50 0 Fe 2+ Ferrous sulfate 131 0 La 3+ Lanthanum (III) chloride 101 0 Co 2+ Cobalt chloride 96 0 Rb + Rubidium chloride 92 0 Mn 2+ Manganese (II) chloride 88 0 Ni + Nickel (II) chloride 87 0 Zn 2+ Zinc sulphate 33 0 The activity of SALD AP was assayed using 100 mM Tricine buffer (pH 8.0), 50 µM NAD + , 70 µM salicylaldehyde and 1 µM enzyme. Values reported are relative to the activity of the control (set as 100%). SALD AP activity was also affected by organic solvents (Table 2 ). At low concentrations, ethanol and DMSO enhanced activity by ∼35% and 18%, respectively, while methanol and isopropanol had no effect. At moderate concentrations (10% v/v), activity decreased for all solvents, but higher concentrations (50% v/v) of solvents inhibited the enzyme, except DMSO, which retained more than 30% activity. These results are consistent with evidence that low concentrations of DMSO can stabilise protein structure and function [ 33 ]. Table 2 Effect of organic solvents, detergents and potentially inhibitory compounds on SALD AP Compound Final Conc. Relative activity (%) Compound Final Conc. Relative activity (%) Organic solvents (% v/v) Methanol 1 99.4 ± 0.7 Ethanol 1 134.6 ± 0.8 10 68.0 ± 0.6 10 86.5 ± 1.0 50 0.0 ± 0.0 50 0.0 ± 0.0 Isopropanol 1 99.5 ± 0.8 DMSO 1 118.1 ± 0.4 10 82.1 ± 0.8 10 97.2 ± 0.8 50 0.0 ± 0.0 50 33.4 ± 0.2 Detergents (% w/v) Triton X-100 1 107.6 ± 5.0 Tween-20 1 102.8 ± 7.5 10 68.9 ± 6.1 10 71.4 ± 4.8 Enzyme inhibitors EDTA (µM) 10 115.8 ± 0.9 DTT (mM) 0.5 102.4 ± 0.6 20 114.3 ± 0.7 1 101.4 ± 0.6 50 111.4 ± 1.0 5 96.8 ± 0.7 80 106.4 ± 1.1 10 95.3 ± 0.6 100 104.2 ± 0.8 20 93.4 ± 0.5 500 90.0 ± 0.6 50 77.2 ± 0.4 The activity of SALD AP was assayed using 100 mM Tricine buffer (pH 8.0), 50 µM NAD + , 70 µM salicylaldehyde and 1 µM enzyme. Values reported are relative to the activity of the control (set as 100%) and are from triplicate measurements presented as mean ± SD. Detergents (Tween-20, Triton X-100) ànd reducing/chelating agents had minimal effects except at their highest concentrations, where the detergents showed 30% reduction in activity while EDTA and DTT showed 10 and 23% reduction, respectively. The inability of EDTA to inhibit SALD AP indicates the absence of metal ions that are reversibly coordinated within the active site of the enzyme. However, very high concentrations (50 mM) of chelators (EDTA and EGTA) have been shown to reduce the activity of SALDH [ 13 ]. Collectively, these findings demonstrate that SALD AP is generally tolerant to a broad range of chemical environments but is inhibited by high levels of certain metals or solvents. This pattern likely reflects the chaotropic or kosmotropic effects of these compounds on protein structure and stability [ 34 ]. The SALD AP has Broad Substrate Specificity and High Catalytic Efficiency for Aromatic Aldehydes SALD AP displayed activity towards both aliphatic and aromatic aldehydes, with notably higher activity against aromatic substrates (Table 3 ; Figure S3). Catalytic efficiency, assessed by specificity constant ( k cat / k m ), was greatest for aromatic aldehydes, with benzaldehyde being the preferred substrate. The k m values for aromatics were in the low micromolar range (0.76–13.14 µM), indicating high affinity, whereas k m values for aliphatic aldehydes were substantially higher (230–5697 µM). SALD AP thus binds and processes aromatic aldehydes much more efficiently than aliphatic ones. The substrate preference (benzaldehyde > 3-hydroxybenzaldehyde > salicylaldehyde) matches previous reports on Pseudomonas SALDs [ 1 , 13 , 14 ]. The activity of an enzyme towards an aromatic substrate depends on the size and the position of the substituent(s) attached to the aromatic ring, which in turn affects several factors that play crucial roles in activity. These factors include hydrogen bonding, hydrophobicity, electronic and steric effects [ 35 , 36 ]. Turnover number (k cat ) was highest for benzaldehyde (120.5 s − 1 ), while short-chain aliphatic aldehydes, propionaldehyde and crotonaldehyde exhibited much lower rates of 5.9 and 5.6 s − 1 , respectively. This represents ∼20-fold lower k cat ap than benzaldehyde. Long-chain aliphatic substrates showed intermediate turnover. For comparison, although Pseudomonas NahF and SALDH have higher affinity for benzaldehyde, their highest k cat ap values were observed with salicylaldehyde [ 1 , 13 ]. Table 3 Substrate specificities and steady state kinetic parameters of 6xHis-SALD AP for aromatic and aliphatic aldehydes. Substrate K m (µM) V max (µM s − 1 ) K cat ap (s − 1 ) K cat ap / K m 10 3 (M − 1 s − 1 ) 2-Hydroxy-benzaldehyde (Salicylaldehyde) 13.14 ± 1.66 0.72 ± 0.03 48.0 ± 2.0 3700 ± 430 3-Hydroxy-benzaldehyde 0.99 ± 0.29 1.41 ± 0.08 94.3 ± 5.3 95200 ± 27300 Benzaldehyde 0.76 ± 0.29 1.81 ± 0.12 120.5 ± 8.0 158500 ± 63700 Cyclohexane carboxaldehyde 62.15 ± 7.65 4.24 ± 0.23 42.4 ± 2.3 700 ± 80 Propionaldehyde 5685 ± 937.4 3.28 ± 0.28 5.9 ± 0.5 1.03 ± 0.17 Butyraldehyde 4473 ± 411.5 7.53 ± 0.33 26.9 ± 1.2 6.01 ± 0.54 Isobutyraldehyde 5697 ± 367.0 6.63 ± 0.22 23.7 ± 0.8 4.16 ± 0.26 Crotonaldehyde 3319 ± 524.0 1.58 ± 0.11 5.6 ± 0.4 1.70 ± 0.26 Valeraldehyde 1450 ± 118.2 7.39 ± 0.24 26.4 ± 0.9 18.2 ± 1.40 Hexaldehyde 230.4 ± 57.6 5.97 ± 0.37 21.3 ± 1.3 92.5 ± 22.2 Heptaldehyde 504.6 ± 62.05 7.51 ± 0.44 26.8 ± 1.6 53.2 ± 6.4 Octaldehyde 469.1 ± 51.35 5.18 ± 0.26 51.8 ± 2.6 110 ± 12.0 Decanaldehyde 1224 ± 298.2 5.33 ± 0.50 53.3 ± 5.0 43.5 ± 10.6 The activity of SALD AP was assayed using varied enzyme and substrate concentrations in 100 mM Tricine buffer (pH 8.0) and 50 µM NAD + at 25 o C. Values of Kinetic parameters reported are those returned from non-linear fitting using Eq. 1 and are shown as ± standard errors derived from this process. Refer to Table S2 and Figure S3 for additional kinetic data and individual plots, respectively. Overall, SALD AP catalysed aromatic substrate oxidation with catalytic efficiencies ( k cat ap /k m ) exceeding 10 6 M − 1 s − 1 , markedly higher than those for aliphatic substrates. The enzyme’s high catalytic efficiency for aromatics and broad specificity suggest adaptation for aromatic compound metabolism in soil environments. To better visualise the catalytic trends, the K cat ap , and K cat ap /K m for SALD AP listed in Table 3 are presented graphically in Fig. 4 . Substrate inhibition was observed for some substrates (Table S2), especially benzaldehyde and 3-hydroxybenzaldehyde, a phenomenon also reported for other ALDHs [ 1 , 13 , 35 , 37 ]. This may serve regulatory functions in metabolic pathways [ 38 ], though its mechanism remains unclear. Ligands Stabilise SALD AP Against Heat Denaturation Differential scanning fluorimetry (DSF) was used to assess the thermal stability of SALD AP and the effect of ligands (substrates and cofactors) on its melting temperature (T m ). The untreated enzyme exhibited a T m of ∼68 o C, with higher stability observed in the presence of both substrate and cofactor (Table 4 ). Aromatic aldehydes and NAD + , especially when combined, significantly increased the T m (up to 71 o C), indicating that ligand binding stabilises SALD AP . The enzymes T m were higher than those reported for yeast ALDHs [ 39 , 40 ], suggesting greater intrinsic stability. Neither NAD⁺ nor salicylaldehyde alone significantly stabilised the enzyme, highlighting that both are required for maximal thermal stability. Enhanced stability was also more pronounced with aromatic aldehydes and longer-chain aliphatic substrates; short-chain aliphatic aldehydes provided little or no stabilisation, paralleling trends in catalytic efficiency (Table 3 ; Figure S3). Substrates that did not stabilise SALD AP (propionaldehyde, crotonaldehyde) also showed low activity and weak binding. Calcium, previously shown to enhance activity, did not affect the enzyme’s thermal stability, either alone or in combination with NAD⁺ and/or substrate. Low concentrations of DMSO (1%) significantly (p < 0.05) decreased T m by about 1.2°C; this destabilisation was partially reversed by calcium and completely masked by NAD⁺ or substrate. This is consistent with reports that DMSO can destabilise certain protein–ligand complexes [ 41 ]. DSF melting curves revealed a biphasic pattern with two peaks, corresponding to ligand-free and ligand-bound states (Figure S4). The rightward peak (higher T m ) was favoured when both substrate and cofactor were present, while DMSO favoured the lower T m . Biphasic melting behaviour, similar to that observed for coenzyme A binding to WcbI from Burkholderia pseudomallei [ 42 ] may reflect multiple oligomeric or binding states of SALD AP . In contrast, yeast ALDHs exhibit monophasic melting under comparable conditions [ 39 , 40 ], possibly due to differences in subunit organisation. Table 4 Thermal stability of 6xHis-SALD AP showing its melting temperatures (T m o C) upon interaction with different ligands. Aromatic Substrate/Cofactor T m o C Aliphatic Substrate/Cofactor T m o C Ligand/Cofactor T m o C Untreated 67.9 ± 0.17 Propionaldehyde 68.8 ± 0.29 Salicylaldehyde only 67.7 ± 0.29 NAD + 68.7 ± 0.25 Crotonaldehyde 68.8 ± 0.25 Calcium only 67.8 ± 0.17 Benzaldehyde 69.7 ± 0.40* Butyraldehyde 68.9 ± 0.12* NAD + + Ca 2+ 68.5 ± 0.50 Salicylaldehyde 70.3 ± 0.35* Isobutyraldehyde 69.2 ± 0.25* NAD + + Sal + Ca 2+ 69.6 ± 0.36* 3-hydroxybenzaldehyde 70.6 ± 0.36* Valeraldehyde 69.3 ± 0.25* DMSO only 66.7 ± 0.25* 4-hydroxybenzaldehyde 70.0 ± 0.30* Hexaldehyde 70.9 ± 0.40* DMSO + Calcium 67.3 ± 0.29 3,4-dihydroxybenzaldehyde 71.0 ± 0.00* Heptaldehyde 70.3 ± 0.25* NAD + + DMSO 68.6 ± 0.12 Cyclohexane carboxaldehyde 71.0 ± 0.50* Octaldehyde 69.7 ± 0.29* NAD + + DMSO + Calcium 68.8 ± 0.06 Vanillin 69.6 ± 0.35* Decanaldehyde 69.6 ± 1.22* NAD + + DMSO + Ca 2+ + Sal 69.9 ± 0.17* The T m o C of SALD AP bound to aldehyde substrates and cofactor were measured in the presence of 2 mM substrates and 1.5 mM NAD + . The T m o C of the enzyme bound to cofactor only was measured in the presence of 1.5 mM NAD + . All experiments were carried out with 6 µM enzyme. The values indicate means of triplicate measurements ± standard deviation. All results were compared with the T m o C of the untreated enzyme (control) for statistical significance using one-way ANOVA and Dunnett’s multiple comparison post-test. (*) indicates a statistically significant difference (p < 0.05) between the test and the control. Overall, the SALD AP melting pattern shows that when its cofactor and/or substrates bind, the protein favours the peak to the right, signifying denaturation at the ligand-bound melting temperature (Figures S4a, b, c, and d), conferring significant thermal stability to SALD AP . Conversely, in the presence of inhibitors such as DMSO, the ligand-free melting temperature to the left is favoured (Figure S4e). Molecular Docking Elucidates Substrate Specificity and Catalytic Mechanism in SALD AP and its Structural Analogue NahF The crystal structure of SALD AP , previously resolved by our group, revealed high structural similarity to Pseudomonas putida NahF, despite low sequence identity [ 15 ]. Being the only two SALD crystal structures in the Protein Data Bank (PDB), we performed molecular docking with 17 aldehyde substrates (aromatic and aliphatic) to elucidate substrate interactions and specificity. Docking results (Table 5 ) showed that aromatic substrates bind more tightly to both SALD AP and NahF, as reflected by lower binding energies (more negative ΔG) and dissociation constants (K d ), compared to short-chain aliphatic substrates. Binding affinity for aliphatic substrates improved with increasing chain length. NahF exhibited generally higher affinity for aromatic substrates than SALD AP , while affinities for aliphatic substrates were comparable between the two enzymes. Interestingly, for most substrates, SALD AP formed a key hydrogen bond between the aldehyde group and ASN137, whereas NahF typically interacted via CYS284. The different positioning of substrate relative to active-site residues suggests distinct mechanistic strategies, despite structural similarity (see Figure S5). Table 5 Summary of in silico docking results with the crystal structures of SALD AP and NahF. SALD AP (PDB: 6QHN) NahF (PDB: (PDB: 4JZ6) Substrate ΔG (Kcal/mol) K D (µM) Interacting residue H-bond (Å) ΔG (Kcal/mol) K D (µM) Interacting residue H-bond (Å) Benzaldehyde -5.2 154.2 ASN137 2.2 -5.3 130.2 CYS284 2.0 Salicylaldehyde -5.4 110.0 ASN137 2.2 -5.4 110.0 CYS284 2.0 3-hydroxybenzaldehyde -5.2 154.2 ASN137 1.9 -6.1 33.8 CYS284 1.9 4-hydroxybenzaldehyde -5.2 154.2 ASN137 2.0 -5.7 66.3 CYS284 2.0 3,4-dihydroxybenzaldehyde -5.1 182.5 ASN137 1.9 -5.7 66.3 CYS284 2.2 Vanillin -5.3 130.2 ASN137 2.0 -6.0 40.0 CYS284 2.1 2-naphthaldehyde -6.6 14.5 ASN137 1.8 -7.1 6.2 ARG157 2.4 Cyclohexane carboxaldehyde -5.0 216.1 ASN137 2.2 -5.2 154.2 SER208 2.4 Propionaldehyde -2.9 7482.9 ASN137 2.2 -2.9 7482.9 CYS284 2.0 Butyraldehyde -3.3 3809.2 ASN137 2.2 -3.4 3217.6 ARG157 2.4 Isobutyraldehyde -3.4 3217.6 ASN137 2.3 -3.4 3217.6 - - Crotonaldehyde -3.3 3809.2 ASN137 2.2 -3.6 2295.7 CYS284 2.0 Valeraldehyde -3.7 1939.1 ASN137 2.2 -3.8 1637.9 CYS284 2.0 Hexaldehyde -4.1 987.1 ASN137 2.2 -4.1 987.1 CYS284 2.1 Heptaldehyde -4.4 594.9 ASN137 2.2 -4.3 704.3 CYS284 2.1 Octanaldehyde -4.7 358.5 ASN137 2.2 -4.7 358.5 ARG157 2.4 Decanaldehyde -5.1 182.5 ASN137 2.1 -5.0 216.1 CYS284 2.7 ΔG is the binding energy. K D is the substrate dissociation constant, and H-bond is the hydrogen bond distance between the substrate substituent and the interacting residue. Structural features underlying substrate specificity include the presence of an “aromatic box” [ 43 ]. In SALD AP , Ala138 and Phe433 fulfil this role, which is more consistent with a catalytic than a binding function. Additionally, the presence of a less bulky residue (Ile141 in SALD AP , Val153 in NahF) at the position analogous to Met177 in S. cerevisiae Ald6p likely enables accommodation and turnover of aromatic and cyclic substrates, as supported by mutagenesis studies in yeast ALDHs [ 40 ]. Overall, the docking analysis demonstrates that both SALD AP and NahF preferentially bind and catalyse aromatic substrates. However, they use different active site residues to engage substrates, potentially reflecting mechanistic divergence within this enzyme family. Proposed Catalytic Mechanism of SALD AP The catalytic mechanism of ALDHs typically involves hydride transfer from the substrate aldehyde to NAD + , via a tetrahedral intermediate stabilised by an invariant catalytic cysteine. Upon cofactor binding, this cysteine forms a thiohemiacetal intermediate with the substrate. Hydride transfer yields a thioacyl-enzyme and NADH, after which a conserved glutamate activates a water molecule to hydrolyse the thioester, regenerating the cysteine and releasing the carboxylic acid product [ 1 , 5 , 44 ]. Numerous mutagenesis studies support the central role of the invariant cysteine as the catalytic thiol [ 45 – 48 ]. In NahF, the proposed mechanism follows this canonical pathway, with CYS-284 acting as the catalytic thiol and ARG-157 aiding in the positioning of the nucleophilic water molecule [ 1 ]. Our docking studies support this model for NahF. However, for SALD AP , docking and structural analysis reveal that substrate binding is primarily mediated by ASN-137, a strictly conserved residue which lies close to the catalytic cysteine and NAD + [ 6 , 49 ]. Based on our data, we propose a catalytic mechanism for SALD AP involving ASN-137 , ARG-145, GLU-238 and CYS-272 (Fig. 5 a, 5 b); the bolded residues are strictly conserved across ALDHs. Electron density maps and docking place the substrate carbonyl oxygen near ASN-137, supporting its central role in catalysis (Fig. 5 c). Upon cofactor binding, the catalytic cysteine becomes activated. Furthermore, aldehyde substrate binding reduces active site solvation, thereby increasing catalytic thiol nucleophilicity [ 48 ]. The thiol group attacks the substrate carbonyl ( I ), forming a transient tetrahedral intermediate. Stabilised by H-bonding between the ASN-137 side chain amide and the substrate’s carbonyl oxygen. A hydride transfer to NAD + occurs ( II ), forming NADH and a thioester intermediate ( III ). The thioester is positioned and ready for base catalysis . GLU-238 activates a water molecule for nucleophilic attack on the thioester, generating a second, transient tetrahedral intermediate ( IV ). The nucleophilic water molecule is well-positioned by ARG-145, as clearly seen in the electron density map (Fig. 5 c). The weak intermediate collapses to yield the product and regenerate the thiolate ( V ). Once the product dissociates, the regenerated enzyme becomes ready for another turnover. While broadly consistent with the established ALDH paradigm, this mechanism uniquely highlights ASN-137’s key role in substrate binding and stabilisation and identifies ARG-145 as critical for water activation and positioning—roles not emphasised in previous models [ 49 ]. The spatial arrangement and proximity of these four active-site residues (Fig. 5 b) provide a strong mechanistic framework for SALD AP catalysis, though further studies will be required for experimental validation. Conclusion In this study, we have identified and comprehensively characterised a novel salicylaldehyde dehydrogenase (SALD AP ) from an alpine soil metagenome. This enzyme expands the known diversity of the ALDH superfamily by representing the first experimentally characterised alphaproteobacterial SALD, distinguished by high affinity and catalytic efficiency for aromatic aldehydes. Detailed structural, biochemical, and phylogenetic analyses reveal that SALD AP exhibits a unique substrate-binding mode, and a distinct catalytic mechanism centred on a strictly conserved asparagine residue, ASN-137, in concert with ARG-145, GLU-238, and CYS-272. Unlike previously described SALDs, SALD AP displays pronounced specificity for NAD⁺ and broad tolerance to various chemical environments but is uniquely stabilised by the combined presence of substrate and cofactor. Molecular docking and DSF studies confirm the central role of key active-site residues in substrate recognition and enzyme stability. The catalytic mechanism proposed for SALD AP differs from classical paradigms by emphasising the dual roles of ASN-137 and ARG-145 in catalysis and water activation. Overall, this work not only enriches our understanding of ALDH family evolution and function but also introduces a new mechanistic paradigm for aldehyde oxidation. These findings may inform future engineering of ALDHs for biocatalysis, environmental detoxification, and synthetic biology applications. Declarations Authors Contributions SUD: Conceptualisation; Methodology; formal analysis and investigation; writing-original draft preparation; writing - review and editing; Funding acquisition. AID: Methodology; writing – review and editing. DK: Supervision; writing – review and editing. CCRA: Conceptualisation; resources; supervision; funding acquisition; writing – review and editing. Funding The Commonwealth Scholarships Commission UK supported this work with a PhD studentship (NGCA-2014-78) awarded to S.U.D. Competing Interests The authors have no competing interests to declare that are relevant to the content of this article. Data Availability All the experimental data and other results that support the findings of this study are presented in the manuscript and supplementary files. Metagenomes and assembled sequences are available in the NCBI database under the Bioproject code and accession number specified in the manuscript. Ethical Approval Not applicable Consent to Participate Not applicable Consent to Publish Not applicable References Coitinho, J. B., Pereira, M. S., Costa, D. M. A., Guimarães, S. L., Araújo, S. S., Hengge, A. C., … Nagem, R. A. P. (2016). Structural and kinetic properties of the aldehyde dehydrogenase NahF, a broad substrate specificity enzyme for aldehyde oxidation. Biochemistry , 55 (38), 5453–5463. https://doi.org/10.1021/ACS.BIOCHEM.6B00614/ASSET/IMAGES/LARGE/BI-2016-00614J_0007.JPEG Wang, W., Wang, L., & Shao, Z. (2018). Polycyclic aromatic hydrocarbon (PAH) degradation pathways of the obligate marine PAH degrader Cycloclasticus sp. strain P1. Applied and Environmental Microbiology , 84 (21). https://doi.org/10.1128/AEM.01261-18/SUPPL_FILE/ZAM021188824S1.PDF Ghosal, D., Ghosh, S., Dutta, T. K., & Ahn, Y. (2016). Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons (PAHs): A review. Frontiers in Microbiology , 7 (AUG), 208111. https://doi.org/10.3389/FMICB.2016.01369/PDF Mallick, S., Chakraborty, J., & Dutta, T. K. (2011). Role of oxygenases in guiding diverse metabolic pathways in the bacterial degradation of low-molecular-weight polycyclic aromatic hydrocarbons: A review. Critical Reviews in Microbiology , 37 (1), 64–90. https://doi.org/10.3109/1040841X.2010.512268 Liu, Z. J., Sun, Y. J., Rose, J., Chung, Y. J., Hsiao, C. D., Chang, W. R., … Wang, B. C. (1997). The first structure of an aldehyde dehydrogenase reveals novel interactions between NAD and the Rossmann fold. Nature structural biology , 4 (4), 317–326. https://doi.org/10.1038/NSB0497-317 Perozich, J., Nicholas, H., Wang, B., Lindahl, R., & Hempel, J. (1999). Relationships within the aldehyde dehydrogenase extended family. Protein Science : A Publication of the Protein Society , 8 (1), 137. https://doi.org/10.1110/PS.8.1.137 Davies, J. I., & Evans, W. C. (1964). Oxidative metabolism of naphthalene by soil pseudomonads. The ring-fission mechanism. Biochemical Journal , 91 (2), 251. https://doi.org/10.1042/BJ0910251 Shamsuzzaman, K. M., & Barnsley, E. A. (1974). The regulation of naphthalene metabolism in pseudomonads. Biochemical and Biophysical Research Communications , 60 (2), 582–589. https://doi.org/10.1016/0006-291X(74)90280-0 Schell, M. A. (1983). Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7. Journal of bacteriology , 153 (2), 822–829. https://doi.org/10.1128/JB.153.2.822-829.1983 Grund, E., Denecke, B., & Eichenlaub, R. (1992). Naphthalene degradation via salicylate and gentisate by Rhodococcus sp. strain B4. Applied and environmental microbiology , 58 (6), 1874–1877. https://doi.org/10.1128/AEM.58.6.1874-1877.1992 Rossello-Mora, R. A., Lalucat, J., & Garcia-Valdes, E. (1994). Comparative biochemical and genetic analysis of naphthalene degradation among Pseudomonas stutzeri strains. Applied and Environmental Microbiology , 60 (3), 966. https://doi.org/10.1128/AEM.60.3.966-972.1994 Zhao, H. B., Li, Y. J., Chen, W., & Cai, B. L. (2007). A novel salicylaldehyde dehydrogenase-NahV involved in catabolism of naphthalene from Pseudomonas putida ND6. Chinese Science Bulletin , 52 (14), 1942–1948. https://doi.org/10.1007/S11434-007-0296-8/METRICS Singh, R., Trivedi, V. D., & Phale, P. S. (2014). Purification and characterization of NAD+-dependent salicylaldehyde dehydrogenase from carbaryl-degrading pseudomonas sp. Strain C6. Applied Biochemistry and Biotechnology , 172 (2), 806–819. https://doi.org/10.1007/S12010-013-0581-8/TABLES/3 Jia, B., Jia, X., Hyun Kim, K., Ji Pu, Z., Kang, M. S., & Ok Jeon, C. (2017). Evolutionary, computational, and biochemical studies of the salicylaldehyde dehydrogenases in the naphthalene degradation pathway. Scientific Reports 2017 7:1 , 7 (1), 1–13. https://doi.org/10.1038/srep43489 Dandare, S. U., Ha kansson, M., Anders Svensson, L., Timson, D. J., & Allen, C. C. R. (2022). Expression, purification and crystallization of a novel metagenome-derived salicylaldehyde dehydrogenase from Alpine soil. Acta crystallographica. Section F, Structural biology communications , 78 (Pt 4), 161–169. https://doi.org/10.1107/S2053230X22002345 Young, J. M., Skvortsov, T., Kelleher, B. P., Mahaney, W. C., Somelar, P., & Allen, C. C. R. (2019). Effect of soil horizon stratigraphy on the microbial ecology of alpine paleosols. Science of The Total Environment , 657 , 1183–1193. https://doi.org/10.1016/J.SCITOTENV.2018.11.442 Dandare, S. U., Young, J. M., Kelleher, B. P., & Allen, C. C. R. (2019). The distribution of novel bacterial laccases in alpine paleosols is directly related to soil stratigraphy. The Science of the total environment , 671 , 19–27. https://doi.org/10.1016/J.SCITOTENV.2019.03.250 Ruby, J. G., Bellare, P., & DeRisi, J. L. (2013). PRICE: Software for the targeted assembly of components of (Meta) genomic sequence data. G3: Genes, Genomes, Genetics , 3 (5), 865–880. https://doi.org/10.1534/G3.113.005967/-/DC1 Sievers, F., & Higgins, D. G. (2018). Clustal Omega for making accurate alignments of many protein sequences. Protein Science , 27 (1), 135–145. https://doi.org/10.1002/PRO.3290 Capella-Gutiérrez, S., Silla-Martínez, J. M., & Gabaldón, T. (2009). trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics , 25 (15), 1972–1973. https://doi.org/10.1093/BIOINFORMATICS/BTP348 Price, M. N., Dehal, P. S., & Arkin, A. P. (2010). FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments. PLOS ONE , 5 (3), e9490. https://doi.org/10.1371/JOURNAL.PONE.0009490 Letunic, I., & Bork, P. (2021). Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Research , 49 (W1), W293–W296. https://doi.org/10.1093/NAR/GKAB301 Horecker, B. L., & Kornberg, A. (1948). THE EXTINCTION COEFFICIENTS OF THE REDUCED BAND OF PYRIDINE NUCLEOTIDES. Journal of Biological Chemistry , 175 (1), 385–390. https://doi.org/10.1016/S0021-9258(18)57268-9 Parajuli, B., Kimble-Hill, A. C., Khanna, M., Ivanova, Y., Meroueh, S., & Hurley, T. D. (2011). Discovery of Novel Regulators of Aldehyde Dehydrogenase Isoenzymes. Chemico-biological interactions , 191 (1–3), 153. https://doi.org/10.1016/J.CBI.2011.02.018 Copeland, R. a, & Wiley, a J. (2000). ENZYMES A Practical Introduction. The Communications of the TeX Users Group , 7 (2), 103–145. Retrieved from http://pubs.acs.org/doi/abs/10.1021/jm020467f Johnson, K. A., & Goody, R. S. (2011). The Original Michaelis Constant: Translation of the 1913 Michaelis-Menten Paper. Biochemistry , 50 (39), 8264. https://doi.org/10.1021/BI201284U Atkins, P. W. (1989). Physical chemistry (p. 263). Oxford: Oxford University Press. Hempel, J., Nicholas, H., & Lindahl, R. (1993). Aldehyde dehydrogenases: widespread structural and functional diversity within a shared framework. Protein science : a publication of the Protein Society , 2 (11), 1890–1900. https://doi.org/10.1002/PRO.5560021111 Carugo, O., & Argos, P. (1997). NADP-dependent enzymes. I: Conserved stereochemistry of cofactor binding - PubMed. Proteins , 28 (1), 10–28. Raj, S. M., Rathnasingh, C., Jung, W. C., Selvakumar, E., & Park, S. (2010). A novel NAD+-dependent aldehyde dehydrogenase encoded by the puuC gene of klebsiella pneumoniae DSM 2026that utilizes 3- hydroxypropionaldehyde as a substrate. Biotechnology and Bioprocess Engineering , 15 (1), 131–138. https://doi.org/10.1007/S12257-010-0030-2/METRICS Li, X., Li, Y., Wei, D., Li, P., Wang, L., & Feng, L. (2010). Characterization of a broad-range aldehyde dehydrogenase involved in alkane degradation in Geobacillus thermodenitrificans NG80-2. Microbiological research , 165 (8), 706–712. https://doi.org/10.1016/J.MICRES.2010.01.006 Alam, M. F., Laskar, A. A., Choudhary, H. H., & Younus, H. (2016). Human Salivary Aldehyde Dehydrogenase: Purification, Kinetic Characterization and Effect of Ethanol, Hydrogen Peroxide and Sodium Dodecyl Sulfate on the Activity of the Enzyme. Cell biochemistry and biophysics , 74 (3), 307–315. https://doi.org/10.1007/S12013-016-0742-9 Landreh, M., Alvelius, G., Johansson, J., & Jörnvall, H. (2014). Protective effects of dimethyl sulfoxide on labile protein interactions during electrospray ionization. Analytical Chemistry , 86 (9), 4135–4139. https://doi.org/10.1021/AC500879C/SUPPL_FILE/AC500879C_SI_001.PDF Cray, J. A., Russell, J. T., Timson, D. J., Singhal, R. S., & Hallsworth, J. E. (2013). A universal measure of chaotropicity and kosmotropicity. Environmental Microbiology , 15 (1), 287–296. https://doi.org/10.1111/1462-2920.12018 MacKintosh, R. W., & Fewson, C. A. (1988). Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Substrate specificities and inhibition studies. Biochemical Journal , 255 (2), 653. Retrieved from https://pmc.ncbi.nlm.nih.gov/articles/PMC1135276/ Rietveld, E. C., de Zwart, M., Cox, P. G. F., & Seutter-Berlage, F. (1987). Substituent effects during the rat liver aldehyde dehydrogenase catalyzed oxidation of aromatic aldehydes. Biochimica et biophysica acta , 914 (2), 162–169. https://doi.org/10.1016/0167-4838(87)90059-8 Shrivastava, R., Basu, A., & Phale, P. S. (2011). Purification and characterization of benzyl alcohol- and benzaldehyde- dehydrogenase from Pseudomonas putida CSV86. Archives of microbiology , 193 (8), 553–563. https://doi.org/10.1007/S00203-011-0697-6 Reed, M. C., Lieb, A., & Nijhout, H. F. (2010). The biological significance of substrate inhibition: A mechanism with diverse functions. BioEssays , 32 (5), 422–429. https://doi.org/10.1002/BIES.200900167 Datta, S., Annapure, U. S., & Timson, D. J. (2016). Characterization of Cd36_03230p, a putative vanillin dehydrogenase from Candida dubliniensis. RSC Advances , 6 (102), 99774–99780. https://doi.org/10.1039/C6RA22209A Datta, S., Annapure, U. S., & Timson, D. J. (2017). Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii. Bioscience reports , 37 (2). https://doi.org/10.1042/BSR20160529 Cubrilovic, D., & Zenobi, R. (2013). Influence of dimehylsulfoxide on protein-ligand binding affinities. Analytical Chemistry , 85 (5), 2724–2730. https://doi.org/10.1021/AC303197P/SUPPL_FILE/AC303197P_SI_001.PDF Vivoli, M., Novak, H. R., Littlechild, J. A., & Harmer, N. J. (2014). Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry. Journal of Visualized Experiments : JoVE , (91), 51809. https://doi.org/10.3791/51809 Riveros-Rosas, H., González-Segura, L., Julián-Sánchez, A., Díaz-Sánchez, Á. G., & Muñoz-Clares, R. A. (2013). Structural determinants of substrate specificity in aldehyde dehydrogenases. Chemico-biological interactions , 202 (1–3), 51–61. https://doi.org/10.1016/J.CBI.2012.11.015 Wang, M. F., Han, C. L., & Yin, S. J. (2009). Substrate specificity of human and yeast aldehyde dehydrogenases. Chemico-biological interactions , 178 (1–3), 36–39. https://doi.org/10.1016/J.CBI.2008.10.002 Wenzl, M. V., Beretta, M., Griesberger, M., Russwurm, M., Koesling, D., Schmidt, K., … Gorren, A. C. F. (2011). Site-Directed Mutagenesis of Aldehyde Dehydrogenase-2 Suggests Three Distinct Pathways of Nitroglycerin Biotransformation. Molecular Pharmacology , 80 (2), 258–266. https://doi.org/10.1124/MOL.111.071704 González-Segura, L., Mújica-Jiménez, C., & Muñoz-Clares, R. A. (2009). Reaction of the catalytic cysteine of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa with arsenite-BAL and phenylarsine oxide. Chemico-Biological Interactions , 178 (1–3), 64–69. https://doi.org/10.1016/J.CBI.2008.10.049 Farres, J., Wang, T. T. Y., Cunningham, S. J., & Weiner, H. (1995). Investigation of the Active Site Cysteine Residue of Rat Liver Mitochondrial Aldehyde Dehydrogenase by Site-Directed Mutagenesis’. Biochemistry , 34 , 2592–2598. Retrieved from https://pubs.acs.org/sharingguidelines Kitson, T. M., Hill, J. P., & Midwinter, G. G. (1991). Identification of a catalytically essential nucleophilic residue in sheep liver cytoplasmic aldehyde dehydrogenase. The Biochemical journal , 275 ( Pt 1) (Pt 1), 207–210. https://doi.org/10.1042/BJ2750207 Hempel, J., Perozich, J., Chapman, T., Rose, J., Boesch, J. S., Liu, Z. J., … Wang, B. C. (1999). Aldehyde Dehydrogenase Catalytic Mechanism. Advances in Experimental Medicine and Biology , 463 , 53–59. https://doi.org/10.1007/978-1-4615-4735-8_7 Supplementary Files SALDAPSupplementary.docx Cite Share Download PDF Status: Published Journal Publication published 03 Nov, 2025 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted Editorial decision: Accept with revisions 27 Jul, 2025 Reviewers agreed at journal 14 May, 2025 Reviewers invited by journal 14 May, 2025 Editor invited by journal 12 May, 2025 First submitted to journal 09 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6604919","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":456787395,"identity":"de8049f6-124e-40c9-9727-3e77a0f9fbe7","order_by":0,"name":"Shamsudeen Umar Dandare","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYHACAwYGNiDF3sDADBFgI1YLzwGStUgkEKlFvoF54+eCMrt8fsnH26QLGOzkGSTSEvBbcYCtWHrGuWTLmbPTyqRnMCQbNkikHSDgKh4Dad42ZgOD2zlm0jwMzAkMEukNBBzGY/ybt63ewP7mGZCWesJaGA7wmAFtOWxgIMED0nIYqIWQww6zlVnznDtuIHEmrdh6hsFxwzaeZwn4HdbevPk2T1m1AX/74Y23Cyqq5fnZ0wzwO4wZyUZ4HBENCBg+CkbBKBgFIxYAAB54NpHnPvuwAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9517-2504","institution":"Queen's University Belfast","correspondingAuthor":true,"prefix":"","firstName":"Shamsudeen","middleName":"Umar","lastName":"Dandare","suffix":""},{"id":456787396,"identity":"63927667-637f-4b06-8018-75c1629b4644","order_by":1,"name":"Aliyu Ibrahim Dabai","email":"","orcid":"","institution":"Agri-Food and Biosciences Institute","correspondingAuthor":false,"prefix":"","firstName":"Aliyu","middleName":"Ibrahim","lastName":"Dabai","suffix":""},{"id":456787397,"identity":"f98ed431-46ee-4340-b7e1-a439491da723","order_by":2,"name":"Deepak Kumaresan","email":"","orcid":"","institution":"University of Birmingham Edgbaston campus: University of Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Deepak","middleName":"","lastName":"Kumaresan","suffix":""},{"id":456787398,"identity":"8c7cdc4a-08fa-4180-b288-5344a8d89d3a","order_by":3,"name":"Christopher C.R. Allen","email":"","orcid":"","institution":"Queen's University Belfast","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"C.R.","lastName":"Allen","suffix":""}],"badges":[],"createdAt":"2025-05-06 16:07:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6604919/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6604919/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12010-025-05445-4","type":"published","date":"2025-11-03T15:56:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83031783,"identity":"5e6ab473-9239-4a32-8aa8-542b92127733","added_by":"auto","created_at":"2025-05-19 09:16:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":228575,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationship of metagenome-derived SALD (bold letters) and other bacterial salicylaldehyde dehydrogenases (465 – 483 amino acids). The tree was computed using the maximum likelihood method (1000 bootstrap replicates) and the Jones-Taylor-Thornton (JTT) matrix-based model for computing evolutionary distances. The tree was drawn to scale with branch lengths measured in the number of substitutions per site. Entries with red stars have been shown to have activity.\u003c/p\u003e","description":"","filename":"Fig1Phylogenetictree.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6604919/v1/7d80183ec2b7e85ba56cae9c.jpg"},{"id":83031780,"identity":"eac1605b-bc36-4b2d-9d12-b95f2213491e","added_by":"auto","created_at":"2025-05-19 09:16:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":232190,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment (MSA) of novel SALD\u003csub\u003eAP\u003c/sub\u003e with class 2 ALDHs.\u0026nbsp; NahF: Salicylaldehyde dehydrogenase from \u003cem\u003ePseudomonas putida\u003c/em\u003e G7 and NahV: Salicylaldehyde dehydrogenase from \u003cem\u003ePseudomonas putida \u003c/em\u003eND6. Identical residues are boxed in red, while similar residues are in yellow. Green rectangles represent critical conserved residues in the catalytic domain. The GXXXXG motif is shown in green.\u003c/p\u003e","description":"","filename":"Fig2MSA.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6604919/v1/1bdc2cb62cad00bcdf9241f8.jpg"},{"id":83033239,"identity":"43cb8567-e6d3-4bda-8993-12bcfebaac53","added_by":"auto","created_at":"2025-05-19 09:24:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63072,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eEffect of pH on the activity of 6xHis-SALD\u003csub\u003eAP\u003c/sub\u003e for the oxidation of salicylaldehyde at 25\u003csup\u003eo\u003c/sup\u003eC, in the presence of 200 µM NAD\u003csup\u003e+\u003c/sup\u003e. Activities are reported as values relative to the activity of 1 µM protein (set as 100%) at pH 8.0. Values represent the means of triplicate measurements (mean ± standard deviation). \u003cstrong\u003e(b) \u003c/strong\u003eRelative activity of SALD\u003csub\u003eAP\u003c/sub\u003e under standard assay conditions after preincubation of the enzyme at different times. Data points are the average of triplicate measurements; error bars represent ± SD.\u003c/p\u003e","description":"","filename":"Fig3abpHthermostability.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6604919/v1/e172a97d3d5a0dfe9662105d.jpg"},{"id":83031782,"identity":"d05d1d6a-2e04-45f4-b42a-509bf467f0a5","added_by":"auto","created_at":"2025-05-19 09:16:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":138040,"visible":true,"origin":"","legend":"\u003cp\u003eLogarithmic plots of (a) K\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cem\u003eap\u003c/em\u003e\u003c/sup\u003e and (b) K\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cem\u003eap\u003c/em\u003e\u003c/sup\u003e/k\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e for the oxidation of aromatic and aliphatic aldehydes by 6xHis-SALD\u003csub\u003eAP\u003c/sub\u003e in the presence of 50 µM NAD+ at 25 oC.\u003c/p\u003e","description":"","filename":"Fig4abCatalyticefficiencies.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6604919/v1/15b657f1683c5599a7e0f2b3.jpg"},{"id":83031784,"identity":"8f2df012-fb72-4962-bb04-72bf210be725","added_by":"auto","created_at":"2025-05-19 09:16:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":94491,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eProposed reaction mechanism of novel SALD\u003csub\u003eAP\u003c/sub\u003e based on its tertiary structure, molecular docking of various aldehydes into its catalytic site, the strict conservation of N137 in ALDHs and the potential role of R145. \u003cstrong\u003e(b) \u003c/strong\u003eThe distances (Å) showing the proximity of the four amino acid residues in the active site of SALD\u003csub\u003eAP\u003c/sub\u003e proposed to be involved in catalysis. \u003cstrong\u003e(c) \u003c/strong\u003eThe electron density as seen in the active site of SALD\u003csub\u003eAP\u003c/sub\u003e after refinement using 2-naphthaldehyde as a ligand, in two conformations (yellow and cyan carbons with occupancies of 0.4 and 0.6, respectively).\u003c/p\u003e","description":"","filename":"Fig5abcCatalyticmechanism.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6604919/v1/75c622329d3fd49fb5010e2d.jpg"},{"id":95563844,"identity":"3862f9b2-3d27-4b61-8271-bbbafaf1c26f","added_by":"auto","created_at":"2025-11-10 15:58:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2267046,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6604919/v1/96b4d48f-6eff-48a5-891b-7b0741a539b4.pdf"},{"id":83031798,"identity":"be210e7c-8557-41c1-ab89-049fd18d5d81","added_by":"auto","created_at":"2025-05-19 09:16:20","extension":"docx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":1699030,"visible":true,"origin":"","legend":"","description":"","filename":"SALDAPSupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6604919/v1/34a6cb3bf20f22e248245f8e.docx"}],"financialInterests":"","formattedTitle":"A Novel Salicylaldehyde Dehydrogenase from Alpine Soil Metagenomes Reveals a Unique Catalytic Mechanism","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicrobial enzymes are key players in the degradation and detoxification of environmental pollutants, including persistent aromatic hydrocarbons such as polycyclic aromatic hydrocarbons (PAHs). Among these, aldehyde dehydrogenases (ALDHs) represent a structurally conserved but functionally diverse superfamily of NAD(P)\u003csup\u003e+\u003c/sup\u003e-dependent enzymes responsible for oxidising aldehydes to their corresponding carboxylic acids, a key step in microbial aromatic compound catabolism [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In the aerobic breakdown of PAHs by bacteria, salicylaldehyde (SAL) emerges as an important metabolic intermediate that links the upper and lower catabolic pathways [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSalicylaldehyde dehydrogenase (SALD; EC 1.2.1.65), a member of the ALDH superfamily, catalyses the NAD(P)\u003csup\u003e+\u003c/sup\u003e-dependent oxidation of SAL to salicylate, facilitating its entry into central carbon metabolism via the catechol or gentisate pathways [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. While ALDHs share a conserved domain and catalytic core, they exhibit remarkable diversity in substrate range, oligomeric state, and cofactor preference [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the central importance of SALDs in aromatic hydrocarbon metabolism, only a handful have been structurally or biochemically characterised to date, mainly from \u003cem\u003ePseudomonas\u003c/em\u003e and related genera. The \u003cem\u003ein vivo\u003c/em\u003e activity of SALD has been documented in several PAH-degrading microorganisms [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], but detailed \u003cem\u003ein vitro\u003c/em\u003e biochemical characterisation remains limited. Zhao and colleagues [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] purified and partially characterised two SALDs (NahV and NahF) from \u003cem\u003eP. putida\u003c/em\u003e ND6. Additional studies have examined SALDs from \u003cem\u003ePseudomonas\u003c/em\u003e sp. strain C6 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and \u003cem\u003eAlteromonas naphthalenivorans\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Notably, Coitinho and colleagues reported the first crystal structure and kinetic analysis of a broad-substrate specificity SALD (NahF) from \u003cem\u003eP. putida\u003c/em\u003e G7 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. More recently, we determined the crystal structure of the first metagenome-derived SALD [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], which shares only 43% amino acid identity with the well-characterised \u003cem\u003eNahF\u003c/em\u003e from \u003cem\u003eP. putida\u003c/em\u003e G7, highlighting the sequence diversity within this enzyme family.\u003c/p\u003e \u003cp\u003eThe rise of metagenomic sequencing has enabled access to novel enzyme diversity from extreme or underexplored environments. Alpine soils, shaped by glaciation and harsh climatic conditions, represent unique reservoirs of new microbial enzymes with potentially distinct biochemical properties. While recent metagenomic studies have begun to reveal the functional diversity of such environments [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], no SALD from a metagenomic source has been fully characterised until now.\u003c/p\u003e \u003cp\u003eIn this study, we report the comprehensive characterisation of a novel salicylaldehyde dehydrogenase (SALD\u003csub\u003eAP\u003c/sub\u003e) identified from an alpine soil metagenome. Through integrated phylogenetic, structural, kinetic, ligand-binding, and molecular docking analyses, we show that SALD\u003csub\u003eAP\u003c/sub\u003e is the first experimentally characterised alphaproteobacterial SALD and exhibits a unique catalytic mechanism involving a strictly conserved asparagine residue. SALD\u003csub\u003eAP\u003c/sub\u003e displays strict NAD\u003csup\u003e+\u003c/sup\u003e dependence, high catalytic efficiency for aromatic aldehydes, and is stabilised by substrate and cofactor. These findings expand the functional and mechanistic landscape of the ALDH superfamily and provide a foundation for engineering novel biocatalysts for environmental and synthetic biology applications.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSoil Sample Collection and Metagenomic Library Construction\u003c/h2\u003e \u003cp\u003eThis procedure has been described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In brief, soil samples were aseptically collected at mid-depth from different horizons (Ah, Bw, Cox, Cu) in glacial moraines from the Guil and Po River valleys of the French and Italian Alps, respectively. Samples were transported on ice and stored at -20 \u003csup\u003eo\u003c/sup\u003eC. Total DNA was extracted using the PowerSoil DNA extraction kit (Mo Bio) according to the manufacturer\u0026rsquo;s instructions, with minor modifications. DNA concentrations were determined using a Quantus Fluorometer (Promega). DNA libraries were prepared and sequenced in paired-end mode on an Illumina MiSeq at the University of Cambridge DNA sequencing facility. Raw metagenome data are available in the NCBI Sequence Read Archive (Bioproject number: PRJNA490486).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGene Mining, Assembly, and Phylogenetic Analysis\u003c/h3\u003e\n\u003cp\u003eTo identify aldehyde dehydrogenases (ALDHs), a vanillin dehydrogenase from \u003cem\u003ePseudomonas putida\u003c/em\u003e KT2440 (NP_745497.1) served as a reference for BLAST searches against merged Illumina datasets. Sequence data from different soil horizons and nearby sample sites were combined to maximise coverage. Discontinuous MagaBLAST (dc-megablast) was used with an E-value threshold of 1.0e-5 and a minimum 70% identity. Hit sequences were extracted and used as seeds for gene-targeted assembly using the PRICE \u003cem\u003ede novo\u003c/em\u003e assembler [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], which builds longer contigs from paired-end reads. PRICE was run for ten assembly cycles, with only contigs matching initial BLAST hits retained. Contigs longer than the expected gene length were analysed for open reading frames (ORFs) with NCBI\u0026rsquo;s ORF Finder (bacterial code). The quality and novelty of each ORF were confirmed by BLAST. The assembled SALD\u003csub\u003eAP\u003c/sub\u003e sequence is available in NCBI with the GenBank accession PV600639.\u003c/p\u003e \u003cp\u003eProtein sequences were aligned with Clustal Omega [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and poorly aligned regions were trimmed with trimAl [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. A phylogenetic tree was constructed using FastTree [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and visualised in iTOL [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCloning, Expression, and Purification of SALD\u003c/h3\u003e\n\u003cp\u003ePrimers designed based on the assembled SALD\u003csub\u003eAP\u003c/sub\u003e sequence were used to amplify the gene from alpine soil metagenomic DNA. The PCR product was cloned into the pLATE51 vector and expressed as previously described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The recombinant plasmid (pLATE51-SALD\u003csub\u003eAP\u003c/sub\u003e) was transformed into chemically competent \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) cells. Cultures were grown to OD₆₀₀ \u0026asymp; 0.6 at 30\u0026deg;C, induced with 1 mM IPTG, and incubated for 6 hours. Cells were harvested, resuspended in lysis buffer, and lysed by sonication. The soluble fraction was purified by Co\u0026sup2;⁺-affinity chromatography and dialysed. The recombinant protein was expressed with an N-terminal 6xHis tag, and all experiments were performed with the 6xHis-SALD\u003csub\u003eAP\u003c/sub\u003e.\u003c/p\u003e\n\u003ch3\u003eEnzyme activity assay\u003c/h3\u003e\n\u003cp\u003eSALD\u003csub\u003eAP\u003c/sub\u003e activity was measured as described by Coitinho et al. (2016). Reactions were performed at 25\u0026deg;C, monitoring NAD⁺ reduction to NADH at 340 nm. The assay mixture contained 200 \u0026micro;M salicylaldehyde (SAL), 200 \u0026micro;M NAD⁺, and 1\u0026ndash;2 \u0026micro;M purified enzyme in 1 mL of sodium phosphate buffer (pH 8.0). NADH formation was confirmed by colorimetric detection of salicylate. Initial reaction rates were calculated from the linear region of the absorbance curve using linear regression. The NADH extinction coefficient (6,220 M⁻\u0026sup1; cm⁻\u0026sup1;) was used for concentration conversion [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eSubstrate Specificity and Steady-State Kinetics\u003c/h3\u003e\n\u003cp\u003eOptimal NAD⁺ and enzyme concentrations were established by varying NAD⁺ concentrations (0\u0026ndash;1000 \u0026micro;M) with 1 \u0026micro;M enzyme and 200 \u0026micro;M salicylaldehyde, then varying enzyme (0\u0026ndash;1 \u0026micro;M) at the optimal NAD\u003csup\u003e+\u003c/sup\u003e concentration. Steady-state kinetics were performed at 25\u0026deg;C with 50 \u0026micro;M NAD⁺ and varying substrate (2\u0026ndash;1500 \u0026micro;M) in 100 mM Tricine buffer (pH 8.0, 1 mL volume). For phenolic and long-chain aldehydes, 1.7% DMSO was included as a solvent carrier, a concentration shown to have minimal effect on ALDH activity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].​ Initial rates were calculated as described above. kinetic parameters (Kₘ, K\u003csub\u003ei\u003c/sub\u003e, Vₘₐₓ) were obtained by fitting rate data to the Michaelis-Menten and substrate inhibition models (equations 1 and 2) using GraphPad Prism 9.0. Turnover number (k\u003csub\u003ecat\u003c/sub\u003e) was calculated using Eq.\u0026nbsp;3 below, where [E\u003csub\u003eo\u003c/sub\u003e] is the total enzyme concentration [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:v=\\frac{{V}_{max}\\left[S\\right]}{{K}_{m}+\\left[S\\right]}\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(1\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:v=\\frac{{V}_{max}\\left[S\\right]}{{K}_{m}+\\left[S\\right]+\\frac{{\\left[S\\right]}^{2}}{{K}_{i}}}\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(2\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:{k}_{cat}=\\frac{{V}_{max}}{{\\left[E\\right]}_{o}}\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(3\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e is the maximum velocity, \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e is the Michaelis-Menten constant, \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the substrate inhibition constant, and [\u003cem\u003eS\u003c/em\u003e] is the substrate concentration.\u003c/p\u003e\n\u003ch3\u003eDifferential Scanning Fluorimetry (DSF)\u003c/h3\u003e\n\u003cp\u003eDSF was performed with SYPRO Orange dye to assess thermal stability and ligand effects. Enzyme (5\u0026ndash;7 \u0026micro;M) in 50 mM HEPES buffer (pH 7.4) was mixed with substrates, NAD⁺, and Ca\u0026sup2;⁺ as required. Substrates were prepared in DMSO, kept below 1%. SYPRO Orange was added immediately before measurement. Triplicate reactions were run in 0.2 mL PCR tubes using a Rotor-Gene Q cycler (Qiagen), with temperature ramped from 25\u0026deg;C to 95\u0026deg;C at 1\u0026deg;C/5 s. Fluorescence (excitation 460 nm, emission 510 nm) was used to monitor protein unfolding. The melting temperature (Tₘ) was determined from the first derivative of the fluorescence (ΔF/ΔT).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Docking\u003c/h2\u003e \u003cp\u003eDocking was performed with AutoDock Vina 1.1.2 with UCSF Chimera 1.12 as the interface. Crystal structures of SALD\u003csub\u003eAP\u003c/sub\u003e (PDB ID: 6QHN) and \u003cem\u003eP. putida\u003c/em\u003e G7 NahF (PDB ID: 4JZ6) were prepared by removing non-standard residues and ligands, adding hydrogens, and assigning Gasteiger charges.​ Ligands were built in Chimera from PubChem SMILES, energy-minimised, and converted to .pdbqt format. Docking used grid boxes centred on residues within 6 \u0026Aring; of the co-crystallised ligands. Default parameters were used for docking. Binding interactions and hydrogen bonds were visualised in Chimera and PyMOL 2.6. Dissociation constants (K\u003csub\u003ed\u003c/sub\u003e) were calculated from binding affinities (ΔG) using:\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:{K}_{d}={e}^{\\left(\\frac{\\varDelta\\:G}{RT}\\right)}\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(4\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere R is the gas constant (1.987 cal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e K\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and T is the temperature (298.15 K) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].​\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSequence and Phylogenetic Analysis of Assembled Salicylaldehyde Dehydrogenase (SALD\u003csub\u003eAP\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003eAfter ten assembly cycles, 215 contigs were recovered, with only five exceeding 1,000 nucleotides. Analysis revealed two major ORFs (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and only the second contig encoded a full-length gene with both start and stop codons. BLAST analysis showed this gene shares just 82% nucleotide identity with known sequences in NCBI and contains an aldehyde dehydrogenase superfamily (ALDH-SF) and a salicylaldehyde dehydrogenase DoxF-like (ALDH SaliADH) domains (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), confirming it as a bona fide SALD enzyme.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis placed SALD\u003csub\u003eAP\u003c/sub\u003e within a distinct alphaproteobacteria cluster (including \u003cem\u003eCroceicoccus, Erythrobacter, Kordiimonas\u003c/em\u003e and \u003cem\u003eNovosphingobium\u003c/em\u003e), separate from clusters of beta- (\u003cem\u003eRalstonia, Paraburkholderia\u003c/em\u003e and \u003cem\u003ePolaromonas\u003c/em\u003e) and \u003cem\u003eGammaproteobacteria\u003c/em\u003e (\u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eAlteromonas\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This distinct phylogenetic position highlights SALD\u003csub\u003eAP\u003c/sub\u003e as a novel representative of the SALD family. Previous survey[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] showed most bacterial SALDs are distributed among \u003cem\u003eBetaproteobacteria\u003c/em\u003e (31.33%), \u003cem\u003eGammaproteobacteria\u003c/em\u003e (21.58%), and \u003cem\u003eAlphaproteobacteria\u003c/em\u003e (16.80%); however, to our knowledge, no alphaproteobacterial SALD has yet been characterised, motivating our study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMultiple sequence alignment revealed that SALD\u003csub\u003eAP\u003c/sub\u003e, NahF, and NahV share 40% amino acid identity, with key conservation at catalytic and cofactor-binding domains. Notably, the cofactor binding domain, which conforms to the Rossmann fold (residues 128\u0026ndash;221) contains a glycine-rich motif (GSTXVG, residues 216\u0026ndash;221), analogous to the G\u003csub\u003e1\u003c/sub\u003eXXXXG\u003csub\u003e2\u003c/sub\u003e motif in other NAD\u003csup\u003e+\u003c/sup\u003e-dependent dehydrogenases (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The invariant catalytic cysteine (Cys272), as well as substrate-binding residues Asn137, Arg145, Glu238 and Leu239, are conserved. Aromatic residues which may be implicated in substrate recognition (Trp83, Phe87 and Phe262) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] are also preserved.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of SALD\u003csub\u003eAP\u003c/sub\u003e from Soil Metagenome and Recombinant Production\u003c/h2\u003e \u003cp\u003eSpecific primers designed from the SALD\u003csub\u003eAP\u003c/sub\u003e sequence enabled its targeted amplification from alpine metagenomic DNA (Figure S2a), where it was present in all samples tested. The gene was cloned and optimally expressed, yielding soluble protein purified to homogeneity with Co\u003csup\u003e2+\u003c/sup\u003e-affinity resin (Figure S2b). From 1 L of culture, approximately 2 mg/mL (40 \u0026micro;M) purified protein was obtained. SDS-PAGE analysis indicated a monomeric molecular weight of 49.6 kDa, determined by measuring and comparing the distances travelled by the protein band and the size markers (Figure S2c).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSALD\u003csub\u003eAP\u003c/sub\u003e is a NAD\u003csup\u003e+\u003c/sup\u003e-dependent Aldehyde Dehydrogenase\u003c/h2\u003e \u003cp\u003eThe purified enzyme\u0026rsquo;s activity was assayed using SAL as substrate, with NAD\u003csup\u003e+\u003c/sup\u003e and NADP\u003csup\u003e+\u003c/sup\u003e as possible cofactors. SALD\u003csub\u003eAP\u003c/sub\u003e showed strong activity with NAD\u003csup\u003e+\u003c/sup\u003e but none with NADP\u003csup\u003e+\u003c/sup\u003e. This selectivity reflects a common pattern among ALDHs, where NAD\u003csup\u003e+\u003c/sup\u003e is typically used in oxidative degradation, while NADP\u003csup\u003e+\u003c/sup\u003e is reserved for reductive biosynthesis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSequence analysis revealed that, unlike the well-characterised \u003cem\u003ePseudomonas putida\u003c/em\u003e NahF enzyme, which accepts both cofactors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] SALD\u003csub\u003eAP\u003c/sub\u003e contains a glycine in place of an arginine in the nucleotide binding site (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). While this substitution may contribute to cofactor selectivity, broader studies suggest that cofactor discrimination depends on the overall architecture of the binding pocket, rather than individual residues [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSALD\u003csub\u003eAP\u003c/sub\u003e has an Alkaline pH Optimum and Is Stable Over a Wide Temperature Range\u003c/h2\u003e \u003cp\u003eThe optimal pH for recombinant 6xHis-SALD\u003csub\u003eAP\u003c/sub\u003e was pH 8.0, with a bell-shaped activity profile across pH 5.3\u0026ndash;11.1. Activity dropped by over 30% with a one-unit shift from the optimum, indicating high sensitivity to pH changes. At least 40% and 35% activity loss was observed at pH 7.0 and 9.0, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This behaviour is consistent with classical SALDs from \u003cem\u003ePseudomonas\u003c/em\u003e species [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and other reported ALDHs [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThermostability studies showed that 6xHis-SALD\u003csub\u003eAP\u003c/sub\u003e retained more than 60% activity after 1 h at 40 \u003csup\u003eo\u003c/sup\u003eC, but was quickly inactivated at higher temperatures, where it showed marginal activity at 50 \u003csup\u003eo\u003c/sup\u003eC after 1 h, and complete inactivation after 30 mins at 60 \u003csup\u003eo\u003c/sup\u003eC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Compared to NahF from \u003cem\u003eP. putida\u003c/em\u003e, which remains active longer at elevated temperatures [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], SALD\u003csub\u003eAP\u003c/sub\u003e is less thermostable but remains robust under moderate conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Metal Ions, Organic Solvents, Detergents, and Inhibitors on SALD\u003csub\u003eAP\u003c/sub\u003e Activity\u003c/h2\u003e \u003cp\u003eThe activity of SALD\u003csub\u003eAP\u003c/sub\u003e was affected by several metal ions, solvents, detergents, and inhibitors. At 1 mM, Fe\u003csup\u003e2+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e increased activity by 31% and 17%, respectively, while K\u0026thinsp;+\u0026thinsp;and La3\u0026thinsp;+\u0026thinsp;had negligible effects. In contrast, Zn\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Rb\u003csup\u003e+\u003c/sup\u003e, Co\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, and Mg\u003csup\u003e2+\u003c/sup\u003e inhibited activity to varying extents, with Zn\u003csup\u003e2+\u003c/sup\u003e causing more than 67% inhibition. At 50 mM, most metal ions except Na\u003csup\u003e+\u003c/sup\u003e caused substantial inhibition. Notably, the inhibitory effect depended on the salt type; for example, CuCl\u003csub\u003e2\u003c/sub\u003e caused total inactivation, while CuSO\u003csub\u003e4\u003c/sub\u003e retained 15% activity. These results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These findings differ somewhat from previous reports on \u003cem\u003ePseudomonas\u003c/em\u003e SALDs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], which showed different metal ion sensitivities.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of metal ions on the activity of recombinant 6xHis-SALD\u003csub\u003eAP\u003c/sub\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMetal Ions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMetal Salts\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eRelative activity (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 mM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50 mM\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCalcium chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePotassium chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMg\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMagnesium chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNa\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSodium chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCopper (II) sulphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCopper (II) chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFe\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFerrous sulfate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLa\u003csup\u003e3+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLanthanum (III) chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCo\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCobalt chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRb\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRubidium chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMn\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eManganese (II) chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNi\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNickel (II) chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZn\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZinc sulphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe activity of SALD\u003csub\u003eAP\u003c/sub\u003e was assayed using 100 mM Tricine buffer (pH 8.0), 50 \u0026micro;M NAD\u003csup\u003e+\u003c/sup\u003e, 70 \u0026micro;M salicylaldehyde and 1 \u0026micro;M enzyme. Values reported are relative to the activity of the control (set as 100%).\u003c/p\u003e \u003cp\u003eSALD\u003csub\u003eAP\u003c/sub\u003e activity was also affected by organic solvents (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At low concentrations, ethanol and DMSO enhanced activity by \u0026sim;35% and 18%, respectively, while methanol and isopropanol had no effect. At moderate concentrations (10% v/v), activity decreased for all solvents, but higher concentrations (50% v/v) of solvents inhibited the enzyme, except DMSO, which retained more than 30% activity. These results are consistent with evidence that low concentrations of DMSO can stabilise protein structure and function [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of organic solvents, detergents and potentially inhibitory compounds on SALD\u003csub\u003eAP\u003c/sub\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFinal Conc.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRelative activity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFinal Conc.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRelative activity (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganic solvents (% v/v)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e99.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEthanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e134.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e68.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e86.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsopropanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e99.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDMSO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e118.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e82.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e97.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e33.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetergents (% w/v)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriton X-100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e107.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTween-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e102.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e68.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e71.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnzyme inhibitors\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEDTA (\u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e115.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDTT (mM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e102.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e114.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e101.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e111.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e96.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e106.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e95.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e104.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e93.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e90.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e77.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe activity of SALD\u003csub\u003eAP\u003c/sub\u003e was assayed using 100 mM Tricine buffer (pH 8.0), 50 \u0026micro;M NAD\u003csup\u003e+\u003c/sup\u003e, 70 \u0026micro;M salicylaldehyde and 1 \u0026micro;M enzyme. Values reported are relative to the activity of the control (set as 100%) and are from triplicate measurements presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003cp\u003eDetergents (Tween-20, Triton X-100) \u0026agrave;nd reducing/chelating agents had minimal effects except at their highest concentrations, where the detergents showed 30% reduction in activity while EDTA and DTT showed 10 and 23% reduction, respectively. The inability of EDTA to inhibit SALD\u003csub\u003eAP\u003c/sub\u003e indicates the absence of metal ions that are reversibly coordinated within the active site of the enzyme. However, very high concentrations (50 mM) of chelators (EDTA and EGTA) have been shown to reduce the activity of SALDH [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCollectively, these findings demonstrate that SALD\u003csub\u003eAP\u003c/sub\u003e is generally tolerant to a broad range of chemical environments but is inhibited by high levels of certain metals or solvents. This pattern likely reflects the chaotropic or kosmotropic effects of these compounds on protein structure and stability [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe SALD\u003csub\u003eAP\u003c/sub\u003e has Broad Substrate Specificity and High Catalytic Efficiency for Aromatic Aldehydes\u003c/h2\u003e \u003cp\u003eSALD\u003csub\u003eAP\u003c/sub\u003e displayed activity towards both aliphatic and aromatic aldehydes, with notably higher activity against aromatic substrates (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Figure S3). Catalytic efficiency, assessed by specificity constant (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e/\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e), was greatest for aromatic aldehydes, with benzaldehyde being the preferred substrate. The k\u003csub\u003em\u003c/sub\u003e values for aromatics were in the low micromolar range (0.76\u0026ndash;13.14 \u0026micro;M), indicating high affinity, whereas k\u003csub\u003em\u003c/sub\u003e values for aliphatic aldehydes were substantially higher (230\u0026ndash;5697 \u0026micro;M). SALD\u003csub\u003eAP\u003c/sub\u003e thus binds and processes aromatic aldehydes much more efficiently than aliphatic ones.\u003c/p\u003e \u003cp\u003eThe substrate preference (benzaldehyde\u0026thinsp;\u0026gt;\u0026thinsp;3-hydroxybenzaldehyde\u0026thinsp;\u0026gt;\u0026thinsp;salicylaldehyde) matches previous reports on \u003cem\u003ePseudomonas\u003c/em\u003e SALDs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The activity of an enzyme towards an aromatic substrate depends on the size and the position of the substituent(s) attached to the aromatic ring, which in turn affects several factors that play crucial roles in activity. These factors include hydrogen bonding, hydrophobicity, electronic and steric effects [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTurnover number (k\u003csub\u003ecat\u003c/sub\u003e) was highest for benzaldehyde (120.5 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), while short-chain aliphatic aldehydes, propionaldehyde and crotonaldehyde exhibited much lower rates of 5.9 and 5.6 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. This represents \u0026sim;20-fold lower \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cem\u003eap\u003c/em\u003e\u003c/sup\u003e than benzaldehyde. Long-chain aliphatic substrates showed intermediate turnover. For comparison, although \u003cem\u003ePseudomonas\u003c/em\u003e NahF and SALDH have higher affinity for benzaldehyde, their highest k\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cem\u003eap\u003c/em\u003e\u003c/sup\u003e values were observed with salicylaldehyde [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSubstrate specificities and steady state kinetic parameters of 6xHis-SALD\u003csub\u003eAP\u003c/sub\u003e for aromatic and aliphatic aldehydes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e (\u0026micro;M s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e\u003csup\u003eap\u003c/sup\u003e (s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e\u003csup\u003eap\u003c/sup\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e 10\u003csup\u003e3\u003c/sup\u003e (M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-Hydroxy-benzaldehyde (Salicylaldehyde)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e48.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3700\u0026thinsp;\u0026plusmn;\u0026thinsp;430\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3-Hydroxy-benzaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e94.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e95200\u0026thinsp;\u0026plusmn;\u0026thinsp;27300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBenzaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e120.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e158500\u0026thinsp;\u0026plusmn;\u0026thinsp;63700\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyclohexane carboxaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e62.15\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e42.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e700\u0026thinsp;\u0026plusmn;\u0026thinsp;80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePropionaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5685\u0026thinsp;\u0026plusmn;\u0026thinsp;937.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eButyraldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4473\u0026thinsp;\u0026plusmn;\u0026thinsp;411.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e6.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsobutyraldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5697\u0026thinsp;\u0026plusmn;\u0026thinsp;367.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrotonaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3319\u0026thinsp;\u0026plusmn;\u0026thinsp;524.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValeraldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1450\u0026thinsp;\u0026plusmn;\u0026thinsp;118.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e18.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHexaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e230.4\u0026thinsp;\u0026plusmn;\u0026thinsp;57.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e92.5\u0026thinsp;\u0026plusmn;\u0026thinsp;22.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeptaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e504.6\u0026thinsp;\u0026plusmn;\u0026thinsp;62.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e53.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOctaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e469.1\u0026thinsp;\u0026plusmn;\u0026thinsp;51.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e51.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e110\u0026thinsp;\u0026plusmn;\u0026thinsp;12.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDecanaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1224\u0026thinsp;\u0026plusmn;\u0026thinsp;298.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e53.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e43.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe activity of SALD\u003csub\u003eAP\u003c/sub\u003e was assayed using varied enzyme and substrate concentrations in 100 mM Tricine buffer (pH 8.0) and 50 \u0026micro;M NAD\u003csup\u003e+\u003c/sup\u003e at 25 \u003csup\u003eo\u003c/sup\u003eC. Values of Kinetic parameters reported are those returned from non-linear fitting using Eq.\u0026nbsp;1 and are shown as \u0026plusmn;\u0026thinsp;standard errors derived from this process. Refer to Table S2 and Figure S3 for additional kinetic data and individual plots, respectively.\u003c/p\u003e \u003cp\u003eOverall, SALD\u003csub\u003eAP\u003c/sub\u003e catalysed aromatic substrate oxidation with catalytic efficiencies (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cem\u003eap\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/k\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e) exceeding 10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, markedly higher than those for aliphatic substrates. The enzyme\u0026rsquo;s high catalytic efficiency for aromatics and broad specificity suggest adaptation for aromatic compound metabolism in soil environments. To better visualise the catalytic trends, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cem\u003eap\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cem\u003eap\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/K\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e for SALD\u003csub\u003eAP\u003c/sub\u003e listed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e are presented graphically in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubstrate inhibition was observed for some substrates (Table S2), especially benzaldehyde and 3-hydroxybenzaldehyde, a phenomenon also reported for other ALDHs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This may serve regulatory functions in metabolic pathways [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], though its mechanism remains unclear.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLigands Stabilise SALD\u003csub\u003eAP\u003c/sub\u003e Against Heat Denaturation\u003c/h2\u003e \u003cp\u003eDifferential scanning fluorimetry (DSF) was used to assess the thermal stability of SALD\u003csub\u003eAP\u003c/sub\u003e and the effect of ligands (substrates and cofactors) on its melting temperature (T\u003csub\u003em\u003c/sub\u003e). The untreated enzyme exhibited a T\u003csub\u003em\u003c/sub\u003e of \u0026sim;68 \u003csup\u003eo\u003c/sup\u003eC, with higher stability observed in the presence of both substrate and cofactor (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Aromatic aldehydes and NAD\u003csup\u003e+\u003c/sup\u003e, especially when combined, significantly increased the T\u003csub\u003em\u003c/sub\u003e (up to 71 \u003csup\u003eo\u003c/sup\u003eC), indicating that ligand binding stabilises SALD\u003csub\u003eAP\u003c/sub\u003e. The enzymes T\u003csub\u003em\u003c/sub\u003e were higher than those reported for yeast ALDHs [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], suggesting greater intrinsic stability.\u003c/p\u003e \u003cp\u003eNeither NAD⁺ nor salicylaldehyde alone significantly stabilised the enzyme, highlighting that both are required for maximal thermal stability. Enhanced stability was also more pronounced with aromatic aldehydes and longer-chain aliphatic substrates; short-chain aliphatic aldehydes provided little or no stabilisation, paralleling trends in catalytic efficiency (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Figure S3). Substrates that did not stabilise SALD\u003csub\u003eAP\u003c/sub\u003e (propionaldehyde, crotonaldehyde) also showed low activity and weak binding.\u003c/p\u003e \u003cp\u003eCalcium, previously shown to enhance activity, did not affect the enzyme\u0026rsquo;s thermal stability, either alone or in combination with NAD⁺ and/or substrate. Low concentrations of DMSO (1%) significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decreased T\u003csub\u003em\u003c/sub\u003e by about 1.2\u0026deg;C; this destabilisation was partially reversed by calcium and completely masked by NAD⁺ or substrate. This is consistent with reports that DMSO can destabilise certain protein\u0026ndash;ligand complexes [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDSF melting curves revealed a biphasic pattern with two peaks, corresponding to ligand-free and ligand-bound states (Figure S4). The rightward peak (higher T\u003csub\u003em\u003c/sub\u003e) was favoured when both substrate and cofactor were present, while DMSO favoured the lower T\u003csub\u003em\u003c/sub\u003e. Biphasic melting behaviour, similar to that observed for coenzyme A binding to WcbI from \u003cem\u003eBurkholderia pseudomallei\u003c/em\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] may reflect multiple oligomeric or binding states of SALD\u003csub\u003eAP\u003c/sub\u003e. In contrast, yeast ALDHs exhibit monophasic melting under comparable conditions [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], possibly due to differences in subunit organisation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThermal stability of 6xHis-SALD\u003csub\u003eAP\u003c/sub\u003e showing its melting temperatures (T\u003csub\u003em\u003c/sub\u003e \u003csup\u003eo\u003c/sup\u003eC) upon interaction with different ligands.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAromatic Substrate/Cofactor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003csub\u003em\u003c/sub\u003e \u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAliphatic Substrate/Cofactor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003csub\u003em\u003c/sub\u003e \u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLigand/Cofactor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eT\u003csub\u003em\u003c/sub\u003e \u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUntreated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e67.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePropionaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e68.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSalicylaldehyde only\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e67.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNAD\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e68.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCrotonaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e68.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCalcium only\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e67.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBenzaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e69.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eButyraldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e68.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNAD\u003csup\u003e+\u003c/sup\u003e + Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e68.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalicylaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e70.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIsobutyraldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e69.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNAD\u003csup\u003e+\u003c/sup\u003e + Sal\u0026thinsp;+\u0026thinsp;Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e69.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3-hydroxybenzaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e70.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eValeraldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e69.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDMSO only\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e66.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-hydroxybenzaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e70.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHexaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e70.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDMSO\u0026thinsp;+\u0026thinsp;Calcium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e67.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3,4-dihydroxybenzaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e71.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHeptaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e70.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNAD\u003csup\u003e+\u003c/sup\u003e + DMSO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e68.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyclohexane carboxaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e71.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOctaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e69.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNAD\u003csup\u003e+\u003c/sup\u003e + DMSO\u0026thinsp;+\u0026thinsp;Calcium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e68.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVanillin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e69.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDecanaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e69.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNAD\u003csup\u003e+\u003c/sup\u003e + DMSO\u0026thinsp;+\u0026thinsp;Ca\u003csup\u003e2+\u003c/sup\u003e + Sal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e69.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe T\u003csub\u003em\u003c/sub\u003e \u003csup\u003eo\u003c/sup\u003eC of SALD\u003csub\u003eAP\u003c/sub\u003e bound to aldehyde substrates and cofactor were measured in the presence of 2 mM substrates and 1.5 mM NAD\u003csup\u003e+\u003c/sup\u003e. The T\u003csub\u003em\u003c/sub\u003e \u003csup\u003eo\u003c/sup\u003eC of the enzyme bound to cofactor only was measured in the presence of 1.5 mM NAD\u003csup\u003e+\u003c/sup\u003e. All experiments were carried out with 6 \u0026micro;M enzyme. The values indicate means of triplicate measurements\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. All results were compared with the T\u003csub\u003em\u003c/sub\u003e \u003csup\u003eo\u003c/sup\u003eC of the untreated enzyme (control) for statistical significance using one-way ANOVA and Dunnett\u0026rsquo;s multiple comparison post-test. (*) indicates a statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the test and the control.\u003c/p\u003e \u003cp\u003eOverall, the SALD\u003csub\u003eAP\u003c/sub\u003e melting pattern shows that when its cofactor and/or substrates bind, the protein favours the peak to the right, signifying denaturation at the ligand-bound melting temperature (Figures S4a, b, c, and d), conferring significant thermal stability to SALD\u003csub\u003eAP\u003c/sub\u003e. Conversely, in the presence of inhibitors such as DMSO, the ligand-free melting temperature to the left is favoured (Figure S4e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Docking Elucidates Substrate Specificity and Catalytic Mechanism in SALD\u003csub\u003eAP\u003c/sub\u003e and its Structural Analogue NahF\u003c/h2\u003e \u003cp\u003eThe crystal structure of SALD\u003csub\u003eAP\u003c/sub\u003e, previously resolved by our group, revealed high structural similarity to \u003cem\u003ePseudomonas putida\u003c/em\u003e NahF, despite low sequence identity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Being the only two SALD crystal structures in the Protein Data Bank (PDB), we performed molecular docking with 17 aldehyde substrates (aromatic and aliphatic) to elucidate substrate interactions and specificity.\u003c/p\u003e \u003cp\u003eDocking results (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) showed that aromatic substrates bind more tightly to both SALD\u003csub\u003eAP\u003c/sub\u003e and NahF, as reflected by lower binding energies (more negative ΔG) and dissociation constants (K\u003csub\u003ed\u003c/sub\u003e), compared to short-chain aliphatic substrates. Binding affinity for aliphatic substrates improved with increasing chain length. NahF exhibited generally higher affinity for aromatic substrates than SALD\u003csub\u003eAP\u003c/sub\u003e, while affinities for aliphatic substrates were comparable between the two enzymes.\u003c/p\u003e \u003cp\u003eInterestingly, for most substrates, SALD\u003csub\u003eAP\u003c/sub\u003e formed a key hydrogen bond between the aldehyde group and ASN137, whereas NahF typically interacted via CYS284. The different positioning of substrate relative to active-site residues suggests distinct mechanistic strategies, despite structural similarity (see Figure S5).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of \u003cem\u003ein silico\u003c/em\u003e docking results with the crystal structures of SALD\u003csub\u003eAP\u003c/sub\u003e and NahF.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eSALD\u003csub\u003eAP\u003c/sub\u003e (PDB: 6QHN)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eNahF (PDB: (PDB: 4JZ6)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eΔG (Kcal/mol)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInteracting residue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eH-bond (\u0026Aring;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eΔG (Kcal/mol)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInteracting residue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eH-bond (\u0026Aring;)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBenzaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e154.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e130.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCYS284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalicylaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e110.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCYS284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3-hydroxybenzaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e154.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e33.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCYS284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-hydroxybenzaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e154.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e66.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCYS284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3,4-dihydroxybenzaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e182.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e66.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCYS284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVanillin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCYS284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-naphthaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eARG157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyclohexane carboxaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e216.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e154.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSER208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePropionaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7482.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7482.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCYS284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eButyraldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3809.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3217.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eARG157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsobutyraldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3217.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3217.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrotonaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3809.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2295.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCYS284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValeraldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1939.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1637.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCYS284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHexaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e987.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e987.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCYS284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeptaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e594.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e704.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCYS284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOctanaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-4.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e358.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-4.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e358.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eARG157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDecanaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e182.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eASN137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e216.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCYS284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eΔG is the binding energy.\u003c/p\u003e \u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e is the substrate dissociation constant, and\u003c/p\u003e \u003cp\u003eH-bond is the hydrogen bond distance between the substrate substituent and the interacting residue.\u003c/p\u003e \u003cp\u003eStructural features underlying substrate specificity include the presence of an \u0026ldquo;aromatic box\u0026rdquo; [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In SALD\u003csub\u003eAP\u003c/sub\u003e, Ala138 and Phe433 fulfil this role, which is more consistent with a catalytic than a binding function. Additionally, the presence of a less bulky residue (Ile141 in SALD\u003csub\u003eAP\u003c/sub\u003e, Val153 in NahF) at the position analogous to Met177 in \u003cem\u003eS. cerevisiae\u003c/em\u003e Ald6p likely enables accommodation and turnover of aromatic and cyclic substrates, as supported by mutagenesis studies in yeast ALDHs [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOverall, the docking analysis demonstrates that both SALD\u003csub\u003eAP\u003c/sub\u003e and NahF preferentially bind and catalyse aromatic substrates. However, they use different active site residues to engage substrates, potentially reflecting mechanistic divergence within this enzyme family.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eProposed Catalytic Mechanism of SALD\u003csub\u003eAP\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eThe catalytic mechanism of ALDHs typically involves hydride transfer from the substrate aldehyde to NAD\u003csup\u003e+\u003c/sup\u003e, via a tetrahedral intermediate stabilised by an invariant catalytic cysteine. Upon cofactor binding, this cysteine forms a thiohemiacetal intermediate with the substrate. Hydride transfer yields a thioacyl-enzyme and NADH, after which a conserved glutamate activates a water molecule to hydrolyse the thioester, regenerating the cysteine and releasing the carboxylic acid product [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Numerous mutagenesis studies support the central role of the invariant cysteine as the catalytic thiol [\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn NahF, the proposed mechanism follows this canonical pathway, with CYS-284 acting as the catalytic thiol and ARG-157 aiding in the positioning of the nucleophilic water molecule [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Our docking studies support this model for NahF. However, for SALD\u003csub\u003eAP\u003c/sub\u003e, docking and structural analysis reveal that substrate binding is primarily mediated by ASN-137, a strictly conserved residue which lies close to the catalytic cysteine and NAD\u003csup\u003e+\u003c/sup\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on our data, we propose a catalytic mechanism for SALD\u003csub\u003eAP\u003c/sub\u003e involving \u003cb\u003eASN-137\u003c/b\u003e, ARG-145, \u003cb\u003eGLU-238\u003c/b\u003e and \u003cb\u003eCYS-272\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb); the bolded residues are strictly conserved across ALDHs. Electron density maps and docking place the substrate carbonyl oxygen near ASN-137, supporting its central role in catalysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon cofactor binding, the catalytic cysteine becomes activated. Furthermore, aldehyde substrate binding reduces active site solvation, thereby increasing catalytic thiol nucleophilicity [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The \u003cb\u003ethiol group attacks\u003c/b\u003e the substrate carbonyl (\u003cb\u003eI\u003c/b\u003e), forming a transient tetrahedral intermediate. Stabilised by H-bonding between the ASN-137 side chain amide and the substrate\u0026rsquo;s carbonyl oxygen. \u003cb\u003eA hydride transfer\u003c/b\u003e to NAD\u003csup\u003e+\u003c/sup\u003e occurs (\u003cb\u003eII\u003c/b\u003e), forming NADH and a thioester intermediate (\u003cb\u003eIII\u003c/b\u003e). The thioester is positioned and ready for \u003cb\u003ebase catalysis\u003c/b\u003e. GLU-238 activates a water molecule for nucleophilic attack on the thioester, generating a second, transient tetrahedral intermediate (\u003cb\u003eIV\u003c/b\u003e). The nucleophilic water molecule is well-positioned by ARG-145, as clearly seen in the electron density map (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The weak intermediate collapses to \u003cb\u003eyield the product\u003c/b\u003e and regenerate the thiolate (\u003cb\u003eV\u003c/b\u003e). Once the product dissociates, the regenerated enzyme becomes ready for another turnover.\u003c/p\u003e \u003cp\u003eWhile broadly consistent with the established ALDH paradigm, this mechanism uniquely highlights ASN-137\u0026rsquo;s key role in substrate binding and stabilisation and identifies ARG-145 as critical for water activation and positioning\u0026mdash;roles not emphasised in previous models [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The spatial arrangement and proximity of these four active-site residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) provide a strong mechanistic framework for SALD\u003csub\u003eAP\u003c/sub\u003e catalysis, though further studies will be required for experimental validation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we have identified and comprehensively characterised a novel salicylaldehyde dehydrogenase (SALD\u003csub\u003eAP\u003c/sub\u003e) from an alpine soil metagenome. This enzyme expands the known diversity of the ALDH superfamily by representing the first experimentally characterised alphaproteobacterial SALD, distinguished by high affinity and catalytic efficiency for aromatic aldehydes. Detailed structural, biochemical, and phylogenetic analyses reveal that SALD\u003csub\u003eAP\u003c/sub\u003e exhibits a unique substrate-binding mode, and a distinct catalytic mechanism centred on a strictly conserved asparagine residue, ASN-137, in concert with ARG-145, GLU-238, and CYS-272.\u003c/p\u003e \u003cp\u003eUnlike previously described SALDs, SALD\u003csub\u003eAP\u003c/sub\u003e displays pronounced specificity for NAD⁺ and broad tolerance to various chemical environments but is uniquely stabilised by the combined presence of substrate and cofactor. Molecular docking and DSF studies confirm the central role of key active-site residues in substrate recognition and enzyme stability. The catalytic mechanism proposed for SALD\u003csub\u003eAP\u003c/sub\u003e differs from classical paradigms by emphasising the dual roles of ASN-137 and ARG-145 in catalysis and water activation.\u003c/p\u003e \u003cp\u003eOverall, this work not only enriches our understanding of ALDH family evolution and function but also introduces a new mechanistic paradigm for aldehyde oxidation. These findings may inform future engineering of ALDHs for biocatalysis, environmental detoxification, and synthetic biology applications.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003eAuthors Contributions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSUD:\u003c/strong\u003e Conceptualisation; Methodology; formal analysis and investigation; writing-original draft preparation; writing - review and editing; Funding acquisition. \u003cstrong\u003eAID:\u003c/strong\u003e Methodology; writing \u0026ndash; review and editing. \u003cstrong\u003eDK:\u003c/strong\u003e Supervision; writing \u0026ndash; review and editing. \u003cstrong\u003eCCRA:\u003c/strong\u003e Conceptualisation; resources; supervision; funding acquisition; writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe Commonwealth Scholarships Commission UK supported this work with a PhD studentship (NGCA-2014-78) awarded to S.U.D.\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eAll the experimental data and other results that support the findings of this study are presented in the manuscript and supplementary files. Metagenomes and assembled sequences are available in the NCBI database under the Bioproject code and accession number specified in the manuscript.\u003c/p\u003e\n\u003cp\u003eEthical Approval\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eConsent to Participate\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eConsent to Publish\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCoitinho, J. B., Pereira, M. S., Costa, D. M. A., Guimar\u0026atilde;es, S. L., Ara\u0026uacute;jo, S. S., Hengge, A. C., \u0026hellip; Nagem, R. A. P. (2016). Structural and kinetic properties of the aldehyde dehydrogenase NahF, a broad substrate specificity enzyme for aldehyde oxidation. \u003cem\u003eBiochemistry\u003c/em\u003e, \u003cem\u003e55\u003c/em\u003e(38), 5453\u0026ndash;5463. https://doi.org/10.1021/ACS.BIOCHEM.6B00614/ASSET/IMAGES/LARGE/BI-2016-00614J_0007.JPEG\u003c/li\u003e\n\u003cli\u003eWang, W., Wang, L., \u0026amp; Shao, Z. (2018). Polycyclic aromatic hydrocarbon (PAH) degradation pathways of the obligate marine PAH degrader Cycloclasticus sp. strain P1. \u003cem\u003eApplied and Environmental Microbiology\u003c/em\u003e, \u003cem\u003e84\u003c/em\u003e(21). https://doi.org/10.1128/AEM.01261-18/SUPPL_FILE/ZAM021188824S1.PDF\u003c/li\u003e\n\u003cli\u003eGhosal, D., Ghosh, S., Dutta, T. K., \u0026amp; Ahn, Y. (2016). Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons (PAHs): A review. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(AUG), 208111. https://doi.org/10.3389/FMICB.2016.01369/PDF\u003c/li\u003e\n\u003cli\u003eMallick, S., Chakraborty, J., \u0026amp; Dutta, T. K. (2011). Role of oxygenases in guiding diverse metabolic pathways in the bacterial degradation of low-molecular-weight polycyclic aromatic hydrocarbons: A review. \u003cem\u003eCritical Reviews in Microbiology\u003c/em\u003e, \u003cem\u003e37\u003c/em\u003e(1), 64\u0026ndash;90. https://doi.org/10.3109/1040841X.2010.512268\u003c/li\u003e\n\u003cli\u003eLiu, Z. J., Sun, Y. J., Rose, J., Chung, Y. J., Hsiao, C. D., Chang, W. R., \u0026hellip; Wang, B. C. (1997). The first structure of an aldehyde dehydrogenase reveals novel interactions between NAD and the Rossmann fold. \u003cem\u003eNature structural biology\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(4), 317\u0026ndash;326. https://doi.org/10.1038/NSB0497-317\u003c/li\u003e\n\u003cli\u003ePerozich, J., Nicholas, H., Wang, B., Lindahl, R., \u0026amp; Hempel, J. (1999). Relationships within the aldehyde dehydrogenase extended family. \u003cem\u003eProtein Science : A Publication of the Protein Society\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e(1), 137. https://doi.org/10.1110/PS.8.1.137\u003c/li\u003e\n\u003cli\u003eDavies, J. I., \u0026amp; Evans, W. C. (1964). Oxidative metabolism of naphthalene by soil pseudomonads. The ring-fission mechanism. \u003cem\u003eBiochemical Journal\u003c/em\u003e, \u003cem\u003e91\u003c/em\u003e(2), 251. https://doi.org/10.1042/BJ0910251\u003c/li\u003e\n\u003cli\u003eShamsuzzaman, K. M., \u0026amp; Barnsley, E. A. (1974). The regulation of naphthalene metabolism in pseudomonads. \u003cem\u003eBiochemical and Biophysical Research Communications\u003c/em\u003e, \u003cem\u003e60\u003c/em\u003e(2), 582\u0026ndash;589. https://doi.org/10.1016/0006-291X(74)90280-0\u003c/li\u003e\n\u003cli\u003eSchell, M. A. (1983). Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7. \u003cem\u003eJournal of bacteriology\u003c/em\u003e, \u003cem\u003e153\u003c/em\u003e(2), 822\u0026ndash;829. https://doi.org/10.1128/JB.153.2.822-829.1983\u003c/li\u003e\n\u003cli\u003eGrund, E., Denecke, B., \u0026amp; Eichenlaub, R. (1992). Naphthalene degradation via salicylate and gentisate by Rhodococcus sp. strain B4. \u003cem\u003eApplied and environmental microbiology\u003c/em\u003e, \u003cem\u003e58\u003c/em\u003e(6), 1874\u0026ndash;1877. https://doi.org/10.1128/AEM.58.6.1874-1877.1992\u003c/li\u003e\n\u003cli\u003eRossello-Mora, R. A., Lalucat, J., \u0026amp; Garcia-Valdes, E. (1994). Comparative biochemical and genetic analysis of naphthalene degradation among Pseudomonas stutzeri strains. \u003cem\u003eApplied and Environmental Microbiology\u003c/em\u003e, \u003cem\u003e60\u003c/em\u003e(3), 966. https://doi.org/10.1128/AEM.60.3.966-972.1994\u003c/li\u003e\n\u003cli\u003eZhao, H. B., Li, Y. J., Chen, W., \u0026amp; Cai, B. L. (2007). A novel salicylaldehyde dehydrogenase-NahV involved in catabolism of naphthalene from Pseudomonas putida ND6. \u003cem\u003eChinese Science Bulletin\u003c/em\u003e, \u003cem\u003e52\u003c/em\u003e(14), 1942\u0026ndash;1948. https://doi.org/10.1007/S11434-007-0296-8/METRICS\u003c/li\u003e\n\u003cli\u003eSingh, R., Trivedi, V. D., \u0026amp; Phale, P. S. (2014). Purification and characterization of NAD+-dependent salicylaldehyde dehydrogenase from carbaryl-degrading pseudomonas sp. Strain C6. \u003cem\u003eApplied Biochemistry and Biotechnology\u003c/em\u003e, \u003cem\u003e172\u003c/em\u003e(2), 806\u0026ndash;819. https://doi.org/10.1007/S12010-013-0581-8/TABLES/3\u003c/li\u003e\n\u003cli\u003eJia, B., Jia, X., Hyun Kim, K., Ji Pu, Z., Kang, M. S., \u0026amp; Ok Jeon, C. (2017). Evolutionary, computational, and biochemical studies of the salicylaldehyde dehydrogenases in the naphthalene degradation pathway. \u003cem\u003eScientific Reports 2017 7:1\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(1), 1\u0026ndash;13. https://doi.org/10.1038/srep43489\u003c/li\u003e\n\u003cli\u003eDandare, S. U., Ha kansson, M., Anders Svensson, L., Timson, D. J., \u0026amp; Allen, C. C. R. (2022). Expression, purification and crystallization of a novel metagenome-derived salicylaldehyde dehydrogenase from Alpine soil. \u003cem\u003eActa crystallographica. Section F, Structural biology communications\u003c/em\u003e, \u003cem\u003e78\u003c/em\u003e(Pt 4), 161\u0026ndash;169. https://doi.org/10.1107/S2053230X22002345\u003c/li\u003e\n\u003cli\u003eYoung, J. M., Skvortsov, T., Kelleher, B. P., Mahaney, W. C., Somelar, P., \u0026amp; Allen, C. C. R. (2019). Effect of soil horizon stratigraphy on the microbial ecology of alpine paleosols. \u003cem\u003eScience of The Total Environment\u003c/em\u003e, \u003cem\u003e657\u003c/em\u003e, 1183\u0026ndash;1193. https://doi.org/10.1016/J.SCITOTENV.2018.11.442\u003c/li\u003e\n\u003cli\u003eDandare, S. U., Young, J. M., Kelleher, B. P., \u0026amp; Allen, C. C. R. (2019). The distribution of novel bacterial laccases in alpine paleosols is directly related to soil stratigraphy. \u003cem\u003eThe Science of the total environment\u003c/em\u003e, \u003cem\u003e671\u003c/em\u003e, 19\u0026ndash;27. https://doi.org/10.1016/J.SCITOTENV.2019.03.250\u003c/li\u003e\n\u003cli\u003eRuby, J. G., Bellare, P., \u0026amp; DeRisi, J. L. (2013). PRICE: Software for the targeted assembly of components of (Meta) genomic sequence data. \u003cem\u003eG3: Genes, Genomes, Genetics\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(5), 865\u0026ndash;880. https://doi.org/10.1534/G3.113.005967/-/DC1\u003c/li\u003e\n\u003cli\u003eSievers, F., \u0026amp; Higgins, D. G. (2018). Clustal Omega for making accurate alignments of many protein sequences. \u003cem\u003eProtein Science\u003c/em\u003e, \u003cem\u003e27\u003c/em\u003e(1), 135\u0026ndash;145. https://doi.org/10.1002/PRO.3290\u003c/li\u003e\n\u003cli\u003eCapella-Guti\u0026eacute;rrez, S., Silla-Mart\u0026iacute;nez, J. M., \u0026amp; Gabald\u0026oacute;n, T. (2009). trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. \u003cem\u003eBioinformatics\u003c/em\u003e, \u003cem\u003e25\u003c/em\u003e(15), 1972\u0026ndash;1973. https://doi.org/10.1093/BIOINFORMATICS/BTP348\u003c/li\u003e\n\u003cli\u003ePrice, M. N., Dehal, P. S., \u0026amp; Arkin, A. P. (2010). FastTree 2 \u0026ndash; Approximately Maximum-Likelihood Trees for Large Alignments. \u003cem\u003ePLOS ONE\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(3), e9490. https://doi.org/10.1371/JOURNAL.PONE.0009490\u003c/li\u003e\n\u003cli\u003eLetunic, I., \u0026amp; Bork, P. (2021). Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. \u003cem\u003eNucleic Acids Research\u003c/em\u003e, \u003cem\u003e49\u003c/em\u003e(W1), W293\u0026ndash;W296. https://doi.org/10.1093/NAR/GKAB301\u003c/li\u003e\n\u003cli\u003eHorecker, B. L., \u0026amp; Kornberg, A. (1948). THE EXTINCTION COEFFICIENTS OF THE REDUCED BAND OF PYRIDINE NUCLEOTIDES. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e, \u003cem\u003e175\u003c/em\u003e(1), 385\u0026ndash;390. https://doi.org/10.1016/S0021-9258(18)57268-9\u003c/li\u003e\n\u003cli\u003eParajuli, B., Kimble-Hill, A. C., Khanna, M., Ivanova, Y., Meroueh, S., \u0026amp; Hurley, T. D. (2011). Discovery of Novel Regulators of Aldehyde Dehydrogenase Isoenzymes. \u003cem\u003eChemico-biological interactions\u003c/em\u003e, \u003cem\u003e191\u003c/em\u003e(1\u0026ndash;3), 153. https://doi.org/10.1016/J.CBI.2011.02.018\u003c/li\u003e\n\u003cli\u003eCopeland, R. a, \u0026amp; Wiley, a J. (2000). ENZYMES A Practical Introduction. \u003cem\u003eThe Communications of the TeX Users Group\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(2), 103\u0026ndash;145. Retrieved from http://pubs.acs.org/doi/abs/10.1021/jm020467f\u003c/li\u003e\n\u003cli\u003eJohnson, K. A., \u0026amp; Goody, R. S. (2011). The Original Michaelis Constant: Translation of the 1913 Michaelis-Menten Paper. \u003cem\u003eBiochemistry\u003c/em\u003e, \u003cem\u003e50\u003c/em\u003e(39), 8264. https://doi.org/10.1021/BI201284U\u003c/li\u003e\n\u003cli\u003eAtkins, P. W. (1989). Physical chemistry (p. 263). Oxford: Oxford University Press.\u003c/li\u003e\n\u003cli\u003eHempel, J., Nicholas, H., \u0026amp; Lindahl, R. (1993). Aldehyde dehydrogenases: widespread structural and functional diversity within a shared framework. \u003cem\u003eProtein science : a publication of the Protein Society\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e(11), 1890\u0026ndash;1900. https://doi.org/10.1002/PRO.5560021111\u003c/li\u003e\n\u003cli\u003eCarugo, O., \u0026amp; Argos, P. (1997). NADP-dependent enzymes. I: Conserved stereochemistry of cofactor binding - PubMed. \u003cem\u003eProteins\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e(1), 10\u0026ndash;28.\u003c/li\u003e\n\u003cli\u003eRaj, S. M., Rathnasingh, C., Jung, W. C., Selvakumar, E., \u0026amp; Park, S. (2010). A novel NAD+-dependent aldehyde dehydrogenase encoded by the puuC gene of klebsiella pneumoniae DSM 2026that utilizes 3- hydroxypropionaldehyde as a substrate. \u003cem\u003eBiotechnology and Bioprocess Engineering\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(1), 131\u0026ndash;138. https://doi.org/10.1007/S12257-010-0030-2/METRICS\u003c/li\u003e\n\u003cli\u003eLi, X., Li, Y., Wei, D., Li, P., Wang, L., \u0026amp; Feng, L. (2010). Characterization of a broad-range aldehyde dehydrogenase involved in alkane degradation in Geobacillus thermodenitrificans NG80-2. \u003cem\u003eMicrobiological research\u003c/em\u003e, \u003cem\u003e165\u003c/em\u003e(8), 706\u0026ndash;712. https://doi.org/10.1016/J.MICRES.2010.01.006\u003c/li\u003e\n\u003cli\u003eAlam, M. F., Laskar, A. A., Choudhary, H. H., \u0026amp; Younus, H. (2016). Human Salivary Aldehyde Dehydrogenase: Purification, Kinetic Characterization and Effect of Ethanol, Hydrogen Peroxide and Sodium Dodecyl Sulfate on the Activity of the Enzyme. \u003cem\u003eCell biochemistry and biophysics\u003c/em\u003e, \u003cem\u003e74\u003c/em\u003e(3), 307\u0026ndash;315. https://doi.org/10.1007/S12013-016-0742-9\u003c/li\u003e\n\u003cli\u003eLandreh, M., Alvelius, G., Johansson, J., \u0026amp; J\u0026ouml;rnvall, H. (2014). Protective effects of dimethyl sulfoxide on labile protein interactions during electrospray ionization. \u003cem\u003eAnalytical Chemistry\u003c/em\u003e, \u003cem\u003e86\u003c/em\u003e(9), 4135\u0026ndash;4139. https://doi.org/10.1021/AC500879C/SUPPL_FILE/AC500879C_SI_001.PDF\u003c/li\u003e\n\u003cli\u003eCray, J. A., Russell, J. T., Timson, D. J., Singhal, R. S., \u0026amp; Hallsworth, J. E. (2013). A universal measure of chaotropicity and kosmotropicity. \u003cem\u003eEnvironmental Microbiology\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(1), 287\u0026ndash;296. https://doi.org/10.1111/1462-2920.12018\u003c/li\u003e\n\u003cli\u003eMacKintosh, R. W., \u0026amp; Fewson, C. A. (1988). Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Substrate specificities and inhibition studies. \u003cem\u003eBiochemical Journal\u003c/em\u003e, \u003cem\u003e255\u003c/em\u003e(2), 653. Retrieved from https://pmc.ncbi.nlm.nih.gov/articles/PMC1135276/\u003c/li\u003e\n\u003cli\u003eRietveld, E. C., de Zwart, M., Cox, P. G. F., \u0026amp; Seutter-Berlage, F. (1987). Substituent effects during the rat liver aldehyde dehydrogenase catalyzed oxidation of aromatic aldehydes. \u003cem\u003eBiochimica et biophysica acta\u003c/em\u003e, \u003cem\u003e914\u003c/em\u003e(2), 162\u0026ndash;169. https://doi.org/10.1016/0167-4838(87)90059-8\u003c/li\u003e\n\u003cli\u003eShrivastava, R., Basu, A., \u0026amp; Phale, P. S. (2011). Purification and characterization of benzyl alcohol- and benzaldehyde- dehydrogenase from Pseudomonas putida CSV86. \u003cem\u003eArchives of microbiology\u003c/em\u003e, \u003cem\u003e193\u003c/em\u003e(8), 553\u0026ndash;563. https://doi.org/10.1007/S00203-011-0697-6\u003c/li\u003e\n\u003cli\u003eReed, M. C., Lieb, A., \u0026amp; Nijhout, H. F. (2010). The biological significance of substrate inhibition: A mechanism with diverse functions. \u003cem\u003eBioEssays\u003c/em\u003e, \u003cem\u003e32\u003c/em\u003e(5), 422\u0026ndash;429. https://doi.org/10.1002/BIES.200900167\u003c/li\u003e\n\u003cli\u003eDatta, S., Annapure, U. S., \u0026amp; Timson, D. J. (2016). Characterization of Cd36_03230p, a putative vanillin dehydrogenase from Candida dubliniensis. \u003cem\u003eRSC Advances\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(102), 99774\u0026ndash;99780. https://doi.org/10.1039/C6RA22209A\u003c/li\u003e\n\u003cli\u003eDatta, S., Annapure, U. S., \u0026amp; Timson, D. J. (2017). Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii. \u003cem\u003eBioscience reports\u003c/em\u003e, \u003cem\u003e37\u003c/em\u003e(2). https://doi.org/10.1042/BSR20160529\u003c/li\u003e\n\u003cli\u003eCubrilovic, D., \u0026amp; Zenobi, R. (2013). Influence of dimehylsulfoxide on protein-ligand binding affinities. \u003cem\u003eAnalytical Chemistry\u003c/em\u003e, \u003cem\u003e85\u003c/em\u003e(5), 2724\u0026ndash;2730. https://doi.org/10.1021/AC303197P/SUPPL_FILE/AC303197P_SI_001.PDF\u003c/li\u003e\n\u003cli\u003eVivoli, M., Novak, H. R., Littlechild, J. A., \u0026amp; Harmer, N. J. (2014). Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry. \u003cem\u003eJournal of Visualized Experiments : JoVE\u003c/em\u003e, (91), 51809. https://doi.org/10.3791/51809\u003c/li\u003e\n\u003cli\u003eRiveros-Rosas, H., Gonz\u0026aacute;lez-Segura, L., Juli\u0026aacute;n-S\u0026aacute;nchez, A., D\u0026iacute;az-S\u0026aacute;nchez, \u0026Aacute;. G., \u0026amp; Mu\u0026ntilde;oz-Clares, R. A. (2013). Structural determinants of substrate specificity in aldehyde dehydrogenases. \u003cem\u003eChemico-biological interactions\u003c/em\u003e, \u003cem\u003e202\u003c/em\u003e(1\u0026ndash;3), 51\u0026ndash;61. https://doi.org/10.1016/J.CBI.2012.11.015\u003c/li\u003e\n\u003cli\u003eWang, M. F., Han, C. L., \u0026amp; Yin, S. J. (2009). Substrate specificity of human and yeast aldehyde dehydrogenases. \u003cem\u003eChemico-biological interactions\u003c/em\u003e, \u003cem\u003e178\u003c/em\u003e(1\u0026ndash;3), 36\u0026ndash;39. https://doi.org/10.1016/J.CBI.2008.10.002\u003c/li\u003e\n\u003cli\u003eWenzl, M. V., Beretta, M., Griesberger, M., Russwurm, M., Koesling, D., Schmidt, K., \u0026hellip; Gorren, A. C. F. (2011). Site-Directed Mutagenesis of Aldehyde Dehydrogenase-2 Suggests Three Distinct Pathways of Nitroglycerin Biotransformation. \u003cem\u003eMolecular Pharmacology\u003c/em\u003e, \u003cem\u003e80\u003c/em\u003e(2), 258\u0026ndash;266. https://doi.org/10.1124/MOL.111.071704\u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez-Segura, L., M\u0026uacute;jica-Jim\u0026eacute;nez, C., \u0026amp; Mu\u0026ntilde;oz-Clares, R. A. (2009). Reaction of the catalytic cysteine of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa with arsenite-BAL and phenylarsine oxide. \u003cem\u003eChemico-Biological Interactions\u003c/em\u003e, \u003cem\u003e178\u003c/em\u003e(1\u0026ndash;3), 64\u0026ndash;69. https://doi.org/10.1016/J.CBI.2008.10.049\u003c/li\u003e\n\u003cli\u003eFarres, J., Wang, T. T. Y., Cunningham, S. J., \u0026amp; Weiner, H. (1995). Investigation of the Active Site Cysteine Residue of Rat Liver Mitochondrial Aldehyde Dehydrogenase by Site-Directed Mutagenesis\u0026rsquo;. \u003cem\u003eBiochemistry\u003c/em\u003e, \u003cem\u003e34\u003c/em\u003e, 2592\u0026ndash;2598. Retrieved from https://pubs.acs.org/sharingguidelines\u003c/li\u003e\n\u003cli\u003eKitson, T. M., Hill, J. P., \u0026amp; Midwinter, G. G. (1991). Identification of a catalytically essential nucleophilic residue in sheep liver cytoplasmic aldehyde dehydrogenase. \u003cem\u003eThe Biochemical journal\u003c/em\u003e, \u003cem\u003e275 ( Pt 1)\u003c/em\u003e(Pt 1), 207\u0026ndash;210. https://doi.org/10.1042/BJ2750207\u003c/li\u003e\n\u003cli\u003eHempel, J., Perozich, J., Chapman, T., Rose, J., Boesch, J. S., Liu, Z. J., \u0026hellip; Wang, B. C. (1999). Aldehyde Dehydrogenase Catalytic Mechanism. \u003cem\u003eAdvances in Experimental Medicine and Biology\u003c/em\u003e, \u003cem\u003e463\u003c/em\u003e, 53\u0026ndash;59. https://doi.org/10.1007/978-1-4615-4735-8_7\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Metagenomics, Alpine soil microbiome, Aldehyde dehydrogenase, Aromatic aldehydes, Catalytic mechanism","lastPublishedDoi":"10.21203/rs.3.rs-6604919/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6604919/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMetagenomic approaches have revolutionised the discovery of novel enzymes with biotechnological potential from unexplored environments. Here, we report the identification and comprehensive characterisation of a novel salicylaldehyde dehydrogenase (SALD\u003csub\u003eAP\u003c/sub\u003e) from an alpine soil metagenome. Phylogenetic analysis revealed that SALD\u003csub\u003eAP\u003c/sub\u003e is the first experimentally characterised alphaproteobacterial SALD, forming a distinct evolutionary clade among known bacterial SALDs. The recombinant enzyme showed strict specificity for NAD⁺ and exceptional catalytic efficiency toward aromatic aldehydes, with benzaldehyde as the preferred substrate. SALD\u003csub\u003eAP\u003c/sub\u003e was most active under mildly alkaline conditions (optimum pH 8.0) and tolerated a range of chemical environments, though high concentrations of certain metal ions and solvents were inhibitory. Kinetic analysis demonstrated that SALD\u003csub\u003eAP\u003c/sub\u003e binds and oxidises aromatic substrates much more efficiently than aliphatic aldehydes, with catalytic efficiencies exceeding 10⁶ M⁻\u0026sup1; s⁻\u0026sup1; for aromatics. The enzyme was stabilised by the simultaneous presence of substrate and cofactor, as shown by differential scanning fluorimetry. Molecular docking with the crystal structures of SALD\u003csub\u003eAP\u003c/sub\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e NahF revealed that SALD\u003csub\u003eAP\u003c/sub\u003e utilises a unique arrangement of active site residues (ASN-137, ARG-145, GLU-238, and CYS-272) to mediate catalysis. Based on structural and docking data, we propose a distinct catalytic mechanism for SALD\u003csub\u003eAP\u003c/sub\u003e, in which ASN-137 plays a central role in substrate binding and stabilisation. This study expands the functional diversity of the ALDH superfamily. It establishes a new paradigm for aromatic aldehyde oxidation, providing valuable insights for the engineering of ALDHs for environmental bioremediation and synthetic biology applications.\u003c/p\u003e","manuscriptTitle":"A Novel Salicylaldehyde Dehydrogenase from Alpine Soil Metagenomes Reveals a Unique Catalytic Mechanism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-19 09:16:15","doi":"10.21203/rs.3.rs-6604919/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept with revisions","date":"2025-07-28T02:49:32+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-05-15T01:49:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-15T01:47:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Applied Biochemistry and Biotechnology","date":"2025-05-12T15:44:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Applied Biochemistry and Biotechnology","date":"2025-05-09T05:12:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dadce69a-e621-4b6b-967e-cd0cb41c6555","owner":[],"postedDate":"May 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T15:58:22+00:00","versionOfRecord":{"articleIdentity":"rs-6604919","link":"https://doi.org/10.1007/s12010-025-05445-4","journal":{"identity":"applied-biochemistry-and-biotechnology","isVorOnly":false,"title":"Applied Biochemistry and Biotechnology"},"publishedOn":"2025-11-03 15:56:50","publishedOnDateReadable":"November 3rd, 2025"},"versionCreatedAt":"2025-05-19 09:16:15","video":"","vorDoi":"10.1007/s12010-025-05445-4","vorDoiUrl":"https://doi.org/10.1007/s12010-025-05445-4","workflowStages":[]},"version":"v1","identity":"rs-6604919","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6604919","identity":"rs-6604919","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}