Rational Engineering of a Thermostable α-Oxoamine Synthase Biocatalyst Expands the Substrate Scope and Synthetic Applicability

Rational Engineering of a Thermostable α-Oxoamine Synthase Biocatalyst Expands the Substrate Scope and Synthetic Applicability | 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 Article Rational Engineering of a Thermostable α-Oxoamine Synthase Biocatalyst Expands the Substrate Scope and Synthetic Applicability Dominic Campopiano, Ben Ashley, Yaoyi Zhu, Sam Mathew, Mariyah Sajjad, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4345858/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2025 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Abstract Carbon-carbon bond formation is one of the key pillars of organic synthesis. Green, selective and efficient biocatalytic methods for such are therefore highly desirable. The α-oxoamine synthases (AOSes) are a class of pyridoxal 5’-phosphate (PLP)-dependent, irreversible, carbon-carbon bond-forming enzymes, which have been limited previously by their narrow substrate specificity and requirement of acyl-CoA thioester substrates. We recently characterized a thermophilic enzyme from Thermus thermophilus ( Th AOS) with a much broader substrate scope and described its use in a chemo-biocatalytic cascade process to generate pyrroles in good yields and timescales. Herein, we report the structure-guided engineering of Th AOS to arrive at variants able to use a greatly expanded range of amino acid and simplified N-acetylcysteamine (SNAc) acyl-thioester substrates. The crystal structure of the improved Th AOS V79A mutant with a bound PLP:penicillamine external aldimine ligand, provides insight into the properties of the engineered biocatalyst. Biological sciences/Chemical biology/Biocatalysis Biological sciences/Chemical biology/Chemical libraries/Combinatorial libraries Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Biocatalysts continue to provide useful alternatives to traditional metal and organic catalysts for the synthesis numerous target molecules at small and industrial scales 1-6 . Once a natural enzyme has been characterised it can be further improved for bespoke applications by the methods of directed evolution and selection 7,8 . The α-oxoamine synthases (AOSs) are pyridoxal 5’-phosphate (PLP)-dependent enzymes which catalyse the irreversible, decarboxylative, Claisen-like condensation of an amino acid with an acyl-CoA thioester, to generate α-aminoketones (Fig. 1A) 9,10 . The AOS family are core enzymes in the biosynthesis of many important biological primary and secondary metabolites, including heme, biotin and sphingolipids, as well as complex natural products such as saxitoxin and the ketomemicins 11-14 . Many of the AOS enzymes bring together two primary metabolic building blocks, amino acids and fatty acids, but an understanding of the origin of their substrate specificity is still unclear. For this reason, their mechanism, structure and substrate specificity have been studied in great detail over the last seven decades 15-32 . Despite their potential as complexity-generating, C-C bond-forming biocatalysts, they have not been exploited for synthesis until recently. It appears they have been passed over for more tractable enzyme classes which are easier to engineer, have broader substrate scopes and have no requirement for expensive acyl-CoA substrates 33 . In recent years however, AOS enzymes have attracted greater attention as synthetically-useful biocatalysts since they generate α-aminoketones that can be elaborated by synthetic chemistry and/or other biocatalysts. We recently made use of the α-aminoketone products generated by the recombinant form of the Th AOS from the thermophilic microbe Thermus thermophilus 34 . The α-aminoketones were combined in a chemo-biocatalytic cascade with various b-keto esters in a Knorr pyrrole reaction (KPR), to generate pyrroles in excellent conversions over short timescales (>90% in <2 hours) (Fig. 1B) 34-36 . Also, the AOS enzyme Alb29 from the biosynthetic pathway of the Streptomyces albogriseolus natural product albogrisin was recently shown to be able to use L-glutamate and a small group of acyl-CoAs to form seven aminoketones 37 . Additionally, Narayan and colleagues have also exploited another AOS enzyme, SxtA from Microseira wollei to prepare α-deuterated amino acids in excellent yield and purity. There they used the ability of AOSs to deprotonate and re-protonate α-amino acids in the presence of D 2 O, (Fig. 1C) 38 . The same group have also made progress in understanding and circumventing the SxtA requirement for acyl-CoA thioester susbtrates 39 . However, the issues of a relatively strict substrate specificity and reliance on complex cofactor or cofactor-mimicking molecules remain. In this work, we show for the first time that an AOS biocatalyst ( Th AOS) can be rationally engineered to use an expanded range of natural and unnatural amino acid substrates. This was achieved by targeting a residue, valine 79 (V79), that is close to the Th AOS PLP binding site, but not thought to be involved directly in catalysis. Structural studies of AOSs have shown this residue is in a ~13 amino acid flexible loop that appears to control amino acid substrate binding. We found that substitution of the V79 side-chain opens up entry to a range of natural and unnatural amino acids. Spectroscopic analysis revealed that, in the absence of the second acyl-CoA substrate, the Th AOS V79 variants could also convert the bound PLP:amino acid external aldimine intermediate into reactive quinonoid, ready for C-C bond formation. Additionally, the turnover with simple and inexpensive acyl-CoA-mimicking N-acetylcysteamine (NAC or SNAc) thioesters was greatly enhanced with the use of engineered Th AOS variants. We used the most thermostable Th AOS variant to couple with the KPR removing the reliance on our previous acyl-CoASH recycling system (Fig. 1D). Furthermore, we determined the crystal structure of the Th AOS V79A variant in complex with a PLP:L-penicillamine external aldimine. The structure of this useful PLP enzyme inhibitor bound to the biocatalyst revealed very little differences when compared to the structure of the internal aldimine form of the wild type Th AOS. Our study suggests that very subtle changes near, but not in, the active site of an AOS biocatalyst influence the substrate binding and convert these conformationally-dynamic enzymes into a catalytically active state that promotes reaction with a range of non-natural substrates. Our findings should also encourage both rational engineering and random mutagenesis/selection of other members of the AOS family to deliver a range of synthetically useful biocatalysts. Results and Discussion Substrate binding to wild type ThAOS. The original paper that characterized Th AOS showed that it could accept Gly, L-Ala and L-Ser amino acids and acetyl-CoA, pimeloyl-CoA and palmitoyl-CoA acyl-thioester substrates 34 . It was also shown that Th AOS catalysed both KBL (Gly/acetyl-CoA) and AONS (L-Ala/pimeloyl-CoA) reactions. We used this information in our previous work to measure an expanded substrate scope of Th AOS using a relatively high-throughput kinetic assay which uses the thiol-reactive DTNB reagent (Ellman’s) to monitor CoASH release (Fig. 1A) 36 . The Th AOS enzyme was active with amino acids l -Ala, Gly, l -Ser and also l -Aba, and could catalyse condensation with acetyl-, propionyl-, butyl-, hexanoyl- and octanoyl-CoA thioester substrates. Therefore, we concluded that the Th AOS biocatalyst has unusually broad substrate scope which suggests it be further expanded. In this new study we wanted to further explore substrate binding so we took advantage of the inherent UV-vis properties of the PLP-dependent Th AOS. The PLP UV-vis spectrum gives insights into substrate binding, as well as the formation of key intermediates during the catalytic mechanism 40 . The most intensively studied members of the AOS family have been SPT, ALAS and AONS with a view to understand their substrate specificity within the biosynthetic pathways in which they operate 13,14,41 . These combined studies have led to a consensus catalytic mechanism of the AOS family with various evidence to support the structure and role(s) of key intermediates and active site residues (Fig. 2). The AOS enzymes exhibit characteristic PLP spectroscopic properties with a clear shift in the UV-vis spectrum upon binding of the amino acid substrate to form the PLP:amino acid external aldimine. The binding of the second, acyl-CoA substrate is thought to cause a conformational change (proposed by Dunathan 42 ) that allows deprotonation of the proton at C-a to generate the key quninonoid/carbanion intermediate. This species subsequently reacts in the C-C forming, Claisen-like condensation reaction to generate a b-keto acid intermediate which is readily decarboxylated to the amino ketone product. The observation of key intermediates, such as the quinonoid, is dependent on the specific AOS/substrate combination. We studied the binding of Th AOS with its amino acid substrates using UV-vis spectroscopy, according to the methods of Webster et al. and Raman et al that were used to study both E. coli AONS and S. paucimobilis SPT 9,43 . Recombinant, purified Th AOS displays a smaller, broad absorbance between 330–360 nm and a larger, broad absorbance between 380–480 nm corresponding to the tautomeric forms of the PLP-bound internal aldimines (Fig. S1 A and Fig. 2). Titration of the core substrate Gly (0–16 mM) led to a small decrease in the 330 nm peak and a small increase in the 430 nm peak (Fig. S1 B). A similar titration of L-Ser (0–16 mM) led to a small increase in the peak at 330 nm and a decrease in the 430 nm peak. With L-Ala (0–16 mM) there was little change in the 330 nm absorbance, a slight decrease and shift (by ~ 2-4nm to 426–428 nm) in the 430 nm peak and the observation of a small, broad peak between 490–505 nm. Taken together this spectroscopic data confirms that these substrates bind to the Th AOS to form PLP:amino acid external aldimines and that L-Ala binding leads to a small amount of PLP:L-Ala quinonoid formation. Having established amino acid binding, we then explored what happens upon addition of the second substrate, acetyl-CoA to the external aldimine forms of Th AOS. Little change was observed with Gly and L-Ser, but we were interested to observe the clear formation of a peak at 501 nm (with a shoulder at 472 nm) with L-Ala as the substrate, suggesting this substrate combination generates an intense quinonoid species (Fig. S2). The observation of such an intermediate suggests subtle differences in the way each substrate binds to and is stabilized by the enzyme. These results suggested to us that Th AOS could potentially bind a greater range of substrates than previously thought – either non-productively to form a Th AOS:PLP-amino acid external aldimine, or productively, to go on to product aminoketone formation in the presence of an acyl-CoA substrate (Fig. 2). Moreover, we hoped that the Th AOS could be engineered to further expand its substrate scope. Rational engineering of ThAOS for an expanded substrate scope. We previously determined the crystal structure of the Th AOS homo-dimer in the internal aldimine form with the PLP bound cofactor (PDBs: 7POA, 7POB and 7POC) 36 . In the absence of a substrate bound structure we modelled the PLP:Gly external aldimine from the crystal structure of Rc ALAS (PDB: 2BWP), a homologue from the AOS family. This model highlighted a close interaction (3.5 Å) between the proposed position of the substrate amino acid sidechain and that of Val79 from the other Th AOS monomer within the dimer structure (Fig. 3A). Thus, Val79 appeared to be an important site for amino acid substrate selectivity, and it was targeted for site-directed mutagenesis. This residue is conserved as a Val side chain in all KBL enzymes but replaced by a Ser, Leu and Thr in other AOS enzymes such as SPT, ALAS, AONS and the recently discovered Alb29 (Fig. 3B). This lack of conservation also suggests that changes at this residue might alter the substrate scope, but would not adversely impact the catalytic activity. Our initial hypothesis was that Th AOS V79 variants would display an expanded amino acid substrate scope. Activity screening of ThAOS V79 variants identifies improved biocatalysts. Initially, we aimed to reduce size of the Val79 side-chain, ideally permitting the entry of bulkier amino acids besides those already turned over by WT Th AOS. As proof of concept, we prepared the enzymes Th AOS V79A and V79G as initial variants to investigate their substrate scope. The mutant biocatalysts were expressed and purified as the wild type Th AOS (Fig. S5) and were screened against a panel of amino acids (16 mM) and acetyl-CoA (1 mM), by monitoring the reaction via the colorimetric DTNB thiol detection screen at 50°C 36,43 . In our screen we included all 20 proteinogenic amino acids, plus the unnatural amino acids (UAAs) l -Aba, dl- Alg, l -Cpa, l -Cpg, l -Hsr, l -Nle, l -Nva, l -Orn, l -Phg, l -Pra, l -Trl, L-Oas and a-Ile, (Fig. S3). Of the 33 amino acid substrates tested with acetyl-CoA as the acyl-thioester substrate, Th AOS V79A and Th AOS V79G displayed activity with 16 amino acids (Table 1). These split into 9 UAAs ( l -Aba, dl- Alg, l -Cpa, l -Cpg, l -Hsr, l -Nva, l -Pra and l -Oas) and 7 proteinogenic amino acids ( l -Ala, l -Asp, Gly, l -Ile, l -Ser, l -Thr and l -Val). Both variants were significantly faster than the wild type biocatalyst for the original set of four amino acid substrates ( l -Aba, l -Ala, Gly and l -Ser), and in general Th AOS V79G was faster than Th AOS V79A. These variants were kinetically characterised in more detail using the DTNB assay, confirming Th AOS V79G to be faster than Th AOS V79A in most cases (Fig. S4, Table S2). With the Th AOS V79A variant, the catalytic efficiency ( k cat / K M ) for all four original amino acid substrates was improved substantially (by 2.4x – 64.8x fold). Similarly, the Th AOS V79G variant showed improved catalytic efficiency with these four reactions by (2.7x – 57.5x fold), with both variants displaying the highest activity with l -Ser. Having identified Val79 as a critical residue involved in substrate binding, we attempted to obtain other useful and/or interesting Th AOS variants by performing saturation mutagenesis at this position (Table S3). To allow high throughput analysis this expanded set of V79 variants were prepared and expressed in a mini culture format, taking advantage of the thermostability of the biocatalysts. Under the standard culture conditions, no expression was detected for seven of the possible 17 new variants ( Th AOS V79D, F, H, N, P, Q and W). Two variants ( Th AOS V79K and V79Y) expressed but were insoluble, but eight ( Th AOS V79C, E, I, L, M, R, S and T) were obtained in soluble form (Table S3). These soluble variants were purified from the cell free extract via a heating step at 80°C for 30 minutes, prior to centrifugation (Fig. S5). The variants which were not stable to this treatment were purified by the full Ni 2+ affinity chromatography (IMAC) method. The purified Th AOS variants were screened against the broad range of amino acid substrates with acetyl-CoA and the release of CoASH observed using the DTNB assay. It was clear that the Th AOS V79S variant displayed increased activity relative to the wild type Th AOS with the core set of four amino acids ( l -Aba, l -Ala, Gly and l -Ser), but also accepted the UAAs, L-Cpg and L-Pra (Fig. 3). Of the other variants, Th AOS V79M and Th AOS V79R turned over only l -Ser to a lesser extent and Th AOS V79T was only active with l -Ala. Amongst the other soluble mutants, Th AOS V79C and Th AOS V79E displayed no detectable activity with the amino acid substrates tested. The Th AOS V79I variant showed the same substrate profile as wild type but with a decreased rate, whilst the Th AOS V79L turned over l -Cpg as well as the original four substrates, and at a slightly improved rate than the wild-type biocatalysts. The retention of activity observed with the Th AOS V79I/L variants is understandable since these are all similar, hydrophobic side-chains. We sought to understand the differences in the activities of the Th AOS V79 variants by simple consideration of the chemistry of each side chain (Fig. S6). It appears that the presence or absence of a substituent (e.g. methyl) at the β-position of the amino acid side chain influences not only the substrate scope but also the catalytic activity. Furthermore, this explains the relative lack of distinction between the Th AOS V79A and Th AOS V79G variants. It is clear that the residue at this position in the Th AOS structure is one of the important determinants of amino acid substrate selectivity. It is also notable that the Val79 residue is not conserved across other members of the AOS family that display differences in their amino acid selectivity e.g. in the well characterized S. paucimobilis SPT which uses L-serine as a substrate, this residue is Ser102 (Fig. 2B). The residues in this 13 amino stretch are part of a key flexible loop where the AOS enzymes display conformational flexibility in key residues involved in substrate binding and catalysis. The AOS enzymes have another flexible loop containing the conserved “PATP” motif near the C-terminus that also undergoes conformational change. Our work on S. paucimobilis SPT captured the key PLP:L-Ser external aldimine complex (PDB: 2W8J) showed that key arginine residues (R378 and R390) are involved in recognition of the carboxylate of L-Ser; the side-chain of Arg378 swings into place upon substrate binding and then “passes on” this carboxylate to Arg390 once the C16-CoA substrate binding has caused the conformational change that induces deprotonation at C-a (Fig. 2) 43 . The resulting PLP:L-Ser quinonoid intermediate is the nucleophile that forms the C-C bond in the b-keto acid intermediate. Mutagenesis of these Arg residues showed that the highly-conserved Arg378A was x40 fold less active than the wild type and this variant could not stabilize the quinonoid. It is interesting to note that KBL is the only AOS member that does not decarboxylate this unstable intermediate (2-amino-3-ketobutyrate, AKB) to give aminoacetone. It protects the AKB by forming a complex between KBL and L-threonine dehydrogenase (TDH). Together KBL and TDH are involved in a glycine/acetyl-CoA metabolic cycle with L-Thr via the AKB intermediate 16 . The fact that Th AOS in isolation can catalyse aminoketone formation with various substrates underscores its unusual, but useful, properties. Expanded acyl-CoA substrate scope of Th AOS V79 variants. Our next goal was to determine the impact, if any, of variations at Val79 on the acyl-CoA thioester substrate scope. In our previous study, we noted that wild-type Th AOS turned over all four of the amino acid substrates (L-Aba, L-Ala, Gly, L-Ser) with acetyl-, propionyl-, butyryl-, hexanoyl- and octanoyl-CoA 35 . Therefore, we used the DTNB assay to screen the three most promising variants ( Th AOS V79G, V79A and V79S) derived from the amino acid substrate screen with acyl-CoA thioesters of increasing acyl chain length (C2, C3, C4, C6, C8, C10 and C12), as well as benzoyl-CoA (Table S4-S6). We were pleased to observe that the acyl-CoA substrate range of Th AOS V79G, V79A and V79S was retained across the C2-C8 chain length when compared to wild type Th AOS. We noted that the Th AOS V79G variant was active with all four core amino acids and C10 and C12 acyl-CoA substrates and could also condense Gly and L-Ala with benzoyl-CoA albeit at very slow rates (Table S4). It was also pleasing to observe that two of the new UAA substrates (L-Cpg, L-Hsr) could also be condensed with acyl-CoA thioesters up to C 12 (Table S4). The Th AOS V79A and V79S variants also featured a slightly improved acyl-CoA substrate scope relative to the WT Th AOS (Tables S5 and S6). The Th AOS V79S is distinguishable in that it is the one variant that was able to catalyse condensation of the UAA substrate L-Pra with C2-C8 acyl-CoAs at modest rates. Furthermore, this variant displayed detectable activity with L-Ala, Gly, L-Ser and L-Cpg and benzoyl-CoA (Table S5). Acyl-CoA independent quinonoid formation in the Th AOS V79G variants. Since the Th AOS V79G variant displayed the broadest activity (Table 1) we have begun to probe how it behaves in the presence of a sub-set of substrates. Here we monitored formation of the key quinonoid intermediate between 490–510 nm to explore the Th AOS variants in terms of amino acid substrate binding alone, and in the presence of the acyl-CoA thioester. The Th AOS V79G variant was incubated with Gly, L-Ser and L-Ala. When compared with wild type Th AOS we noted similar changes to the UV-vis spectrum when the amino acid was added, although in each case there was a small, but clear absorbance between 480–520 nm indicating the formation of a ThAOS V79G PLP:amino acid quinonoid intermediate (Fig. S7 A-C). We also noted acetyl-CoA binding to the Th AOS V79G PLP:Gly complex led to enhancement of this peak (Fig. S7A). In contrast, addition of the acetyl-CoA to the Th AOS V79G PLP:L-Ser complex led to formation of a broad, intense quinonoid signal between 490–520 nm (Fig. S7B), with a shoulder at 468 nm. Moreover, addition of the acyl-CoA thioester substrate to the Th AOS V79G PLP:L-Ala external aldimine generated the most intense quinonoid signal between 490–520 nm (Fig. 7C), also with a shoulder 468 nm). Clearly, the removal of the Val79 side-chain has impacted the formation and stabilization of the Th AOS PLP:amino acid complex and binding of the acyl-CoA thioester substrate leads to deprotonation at C-a and quinonoid formation, supporting the overall proposed AOS catalytic mechanism (Fig. 2). Since the Th AOS V79G variant also accepted L-Asp, L-Thr and L-Val (Table 1) we looked at quinonoid formation with these three substrates and were pleased to observe the characteristic formation of the peak around 500 nm of varying intensities for each amino acid (Fig. S7 D-F). It is clear that this relatively easy assay is a useful tool to study not only substrate binding, but also formation of this key catalytic quinonoid intermediate. Quinonoid formation has been studied in only a few members of the AOS family. We have studied the E. coli AONS and provided supporting evidence for quinonoid formation using L-L-Ala methyl ester (L-Ala-OMe) as a substrate mimic 18 . This ligand bound to the enzyme to form the PLP:L-Ala-OMe external aldimine and upon addition of pimeloyl-CoA two new species were observed by UV-Vis spectroscopy; an intense absorption at 486 nm indicative of formation of the AONS: PLP:L-Ala-OMe quinonoid species and a novel peak at 454 nm which was due to accumulation of the β-ketoacid methyl ester aldimine complex that cannot undergo enzymatic decarboxylation. Taken together it suggests the clever use of the Me-ester substrate mimic prevents decarboxylation and is a useful probe of the AOS mechanism (Fig. 2). This Me-ester trick was also recently used by Chun and Narayan and colleagues to stall the SxtA AONS and, in the presence of D 2 O, allow selective a-deuteration of a panel of 24 natural and UAA a-amino ester substrates 38 . Another elegant example of quinonoid trapping is the study of S. paucimobilis SPT by Ikushiro and colleagues. They initially used an acyl-CoA thioether substrate mimic (S-(2-oxoheptadecyl)-CoA, a nonreactive thioester analogue of palmitoyl-CoA, to “trick” the SPT PLP:L-Ser complex 27 . This mimic bound to the enzyme and caused a conformational change which led to deprotonation at C-a and PLP:L-Ser quinonoid formation observed at 493 nm. Since the quinonoid could not react with the acyl-CoA thioether the authors used NMR to measure a rate acceleration of C-a deprotonation of more than 100 fold upon binding of the second substrate. This study also supported the over-arching AOS mechanism that we proposed based on our earlier work on the AOS enzyme E. coli AONS 9 .More in-depth kinetic analyses (both steady-state and stopped flow) of the ALAS enzyme that uses L-Ala and succinyl-CoA have also been reported by Ferreira and colleagues and from these combined studies the key conserved residues involved in AOS catalysis have been proposed 13 . The results presented here are the first to use knowledge of the catalytic mechanism to explore rational mutagenesis of an AOS enzyme and generate a biocatalyst with a much broader substrate specificity. In further studies, we aim to investigate formation, stability and breakdown of these quinonoid species which should provide more detailed insights into the substrate specificity across the whole AOS family. Thermostability and spectroscopic properties of selected ThAOS variants with expanded substrate scope. Having obtained a sub-set panel of four competent biocatalyst variants via saturation mutagenesis at Th AOS V79 (V79A, V79G, V79L, V79S), we set out to further investigate if the mutations had affected the thermostability of the biocatalyst. This was an important consideration in terms of selecting the best Th AOS variant that could be used for application in chemical synthesis. Since the Th AOS is from a thermophilic bacterium we decided to study their respective thermostabilities at elevated temperatures (Fig. S8). The wild type Th AOS was previously found to be stable to incubation for two hours at 70°C 21 . The Th AOS V70L variant displayed comparable residual activity (92.1%) to the WT Th AOS (88.6%), while the V79A variant (77.6%) and V79S (79.4%) mutants were slightly less stable. We then tested how stable the variants were when incubated at 70°C (Fig. S8A). Unfortunately, the Th AOS V79G variant which exhibited the biggest improvement in substrate scope during the screening process, displayed a significant drop in activity (retaining 56.0%) compared to the wild type biocatalyst. We then analysed how stable the variants were upon incubation at 90°C for 30–120 mins. The WT was to reduced ~ 15% activity after 120 mins and the V79S, V79G and V79L displayed varying retention of activity (45%, 5% and < 5% respectively). However, Th AOS V79A retained ~ 60% activity after incubation at this elevated temperature. By evaluating both the improved substrate scope and thermostability we then decided to take forward the Th AOS V79A variant for scaled-up synthesis of a Th AOS-derived pyrrole product in combination with the KPR. The Th AOS V79 variants are viable biocatalysts with alternative, inexpensive acyl N-acetylcysteamine (SNAc) substrates. Across the AOS family it has been shown that the acyl-CoA thioester substrates display low µM kinetic constants and high turnover rates that is ideal for synthesis. However, the high cost of acyl-CoAs prohibits their use in biocatalysis unless this issue can be overcome by inclusion of an acyl-CoA recycling system. This was achieved in our previous work with wild type Th AOS where we used acyl-CoA synthetase (ACS) to convert the CoASH product back into acyl-CoA (Ashley). Alternatively, a number of simple CoA substrate mimics have been reported that can be used in place of the acyl-CoA. 44 Of these, the most widely-used are thioesters of the low-molecular- weight CoA fragment, N-acetylcysteamine (SNAc, Fig. 5). Enzymes capable of using these simple acyl-SNAc thioester substrates in place of an acyl-CoA thioester or pantetheine thioester include thioesterases, ketosynthases, ACP synthases and many others, (reviewed in 44 ). Therefore replacing the expensive acyl-CoA substrates of AOS enzymes with SNAc-thioesters would greatly improve the synthetic potential of these biocatalysts in preparing valuable aminoketone products or being combined into chemo- and bio-catalytic cascades. The only issue that could prevent using these substrate mimics is a kinetic penalty, since the acyl-SNAc substrates display very high mM binding constants. An AOS homologue, M. wollei SxtA, has recently exhibited improved activity with acyl-SNAcs in the presence of a pantetheine-like auxiliary molecule, which mimics the presence of the acyl-CoA pantetheine arm 39 . This work prompted us to explore the use of acyl-SNAc in combination with the newly-discovered Th AOS V79 variants. Our hypothesis is based on the proposed AOS catalytic mechanism (Fig. 2), that only upon acyl-CoA binding will the enzyme catalyse formation of the nucleophilic quinonoid intermediate. However, since the Th AOS V79A and V79G variants generate a quinonoid in the presence of the amino acid substrate alone, we hoped that these biocatalysts would react with acyl-SNAc substrates, albeit at high concentrations. Acetyl-SNAc (N,S-diacetylcysteamine) was therefore prepared from cysteamine hydrochloride and acetic anhydride using a published method (see SI). The wild type Th AOS and three active variants (V79A, V79G and V79S) were screened for Claisen-condensation activity with acetyl-SNAc against a panel of three amino acids (L-Aba, L-Ala and Gly) with the DTNB assay at 50°C (Fig. S9). It is important to note that wild type Th AOS exhibited no detectable activity with any of the substrate combinations. In contrast, we were delighted to observe activity with all of the Th AOS variants. The Th AOS V79A displayed activity with acetyl-SNAc and L-Aba and Gly, the Th AOS V79G variant was active with L-Aba, L-Ala and Gly and the Th AOS V79S variant was also active with all three substrates with Gly displaying a turnover of 0.14 min -1 . The results of this screen allowed us to test the synthetic usefulness of the new reaction using combinations of Th AOS variants and amino acids with acetyl-SNAc. To do this we coupled the AOS biocatalytic reaction with the KPR (at elevated temperature) as we had done previously. The reaction was performed in the presence of methyl acetoacetate (MAA), with the aim of capturing the aminoketone product as a pyrrole, 1 (Fig. 5). The three biocatalysts were screened using high catalyst loadings (5 mgmL -1 ) with Gly, acetyl-SNAc and MAA (32 mM each) at a variety of elevated temperatures for 16 hours. Reactions were analysed by HPLC as described in our original paper 36 . The results showed that Th AOS V79A worked best with Gly and acetyl-SNAc at 60 o C with 35% analytical yield of pyrrole 1 (Fig. 5). We explored if the pyrrole yield could be increased with the higher loading of acetyl-SNAc (32–800 mM). However, no significant increase in yield was observed (Fig. S10). Since the Th AOS V79A variant was the most thermostable under long incubation times at elevated temperatures (Fig. S8) it was used in a preparative scale reaction (10 mL, 32 mM glycine, 32 mM acetyl-SNAc, 32 mM MAA, 15 mg/mL Th AOS V79A, 100 mM HEPES, pH 7.5, 16 hrs). The pyrrole product 1 was extracted and purified and its structure confirmed using NMR (Fig. S11 and S12). Taken together, this result shows that the rationally engineered Th AOS V79A variant could be used to prepare various products using inexpensive acyl-thioester starting materials. X-ray structure of the improved, engineered Th AOS V79A in complex with the inhibitor L-Pen. Inspired by the success of the engineering campaign to deliver improved variants of Th AOS at residue V79, we endeavored to crystallise the variants and solve their crystal structures with bound ligands. This would allow a comparison with the wild type Th AOS and might provide molecular insight into the origin of the broadened substrate scope of the engineered variant. Screening revealed that Th AOS V79A gave crystals that were not suitable for structural studies and we also screened unsuccessfully with various natural amino acid and UAA substrates that came from the screens (Fig. S3). As a final probe to study ligand binding we used the well known PLP enzyme inhibitor L-penicillamine (L-Pen) and monitored the reaction using UV-vis spectroscopy. We used L-Pen since we had studied the related AOS enzyme S. paucimobilis SPT described by Lowther et al . 45 . The D-Pen enantiomer is an FDA-approved drug used for the treatment of Wilson’s disease and also binds specifically to PLP enzymes 46 . Incubation of L-Pen (1 mM) to the wild type and both Th AOS V79A and V79G variants rapidly decolourised both reactions and led to the formation of a broad peak between 310–380 nm, with an absorbance maximum at 330 nm (Fig. S13 A, B and C). This data is consistent with the formation of a covalent Th AOS PLP:L-Pen ring-closed thiazolidine adduct and this was refined to an excellent fit and geometry. We subsequently used the L-Pen ligand in structural studies and when Th AOS V79A crystals were soaked with L-Pen, prior to flash cooling and data collection, clear ligand density was observed in each active site (Fig. 6A). The crystal structure was determined to 1.5 Å resolution in P1 space group using the wild type ThA OS structure (PDB ID: 7POA) as a molecular replacement model. The asymmetric unit contained two chains forming the stable dimer, and the final model refined to an Rwork of 0.16 and Rfree of 0.19 (Fig. S14). The data collection and refinement statistics are shown in Table S7. L-Pen was captured in the active site of Th AOS V79A in a conformation typical of amino acids co-crystallised in the active site of AOS enzymes. The L-Pen substrate amine is covalently linked to the PLP cofactor, and the substrate carboxylate is chelated by Arg366. The five-membered ring of the inhibitory thiazolidine intermediate is clearly resolved, and sits in the position of an amino acid substrate sidechain. Most interesting is the distinction between WT Th AOS and the engineered, more active variant, Th AOS V79A. For this reason, the structure of Th AOS internal aldimine was overlaid onto the structure of L-Pen-bound Th AOS V79A. Despite a long history of use as an inhibitor of PLP-dependent enzymes, this is the first published crystal structure of a PLP enzyme in complex with l -Pen. The two chains of the Th AOS V79A variant align with around 0.4 Å RMSD Cα to wild type Th AOS structure over the 397 residues. There is no significant structural change in the active site in the V79A variant with PLP:L-Pen bound when compared to the wild type PLP-bound Th AOS, with no shift in the loop around the altered residue. The distance between A79 and the thiazolidine methyl group and is 4Å and modelling the distance to the native V79 gives a distance of 3 Å to the thiazolidone (Fig. 6B). It appears that the removal of the valine side chain increases the volume of the ligand binding site without any large-scale remodelling of this region. Comparison of penicillamine inhibitor binding in structural homologues. Searching the PDB for the L- and D- forms of the penicillamine ligand identifies two unpublished structures of cysteine desulfurase (CSD) enzymes with structural homology to Th AOS with bound penicillamine ligand: NifS from Helicobacter pylori (PDB ID: 7XES); and SufS from Bacillus subtilis (PDD ID: 7XEN). There is also a structure of the SufS without penicillamine (PDB ID: 7XEN). The monomers of these two proteins align to ThAOS-V79A with overall RMSD Cα of 1.3 Å (7XES) and 1.2 Å (7XEN) (Fig. S15A). The primary structural difference between ThAOS and the cysteine desulfurase enzymes is in the position of the N-terminal region of these proteins and an extended beta-elbow that is present in the cysteine desulfurases (Fig. S15A). In Th AOS-V79A, the first 40 amino acids adopt an alpha helix and extended loop arrangement, forming a large interface with the partner chain (Fig. S15B); whereas, in the cysteine desulfurase enzymes the N-terminal region is shorter; and, in the case of the B. subtilis enzyme, it forms a twisted helix that mainly participates in interactions with its own chain (Fig. 15). These regions are shifted by a rotation of 110° between the cysteine desulfurase enzymes and Th AOS. The beta-elbow region in the cysteine desulfurase enzymes participates in the dimerisation interface and due to this additional region, the quaternary arrangement of these enzymes differs from Th AOS (Fig. 16A-B). It appears that it is the entrance to this cavity that has been engineered in the more active ThAOS V79 mutants. The active sites of NifS and SufS both have a bound penicillamine ligand captured in the external aldimine form between the penicillamine and PLP cofactor (Fig. S16A). There position of the PLP and penicillamine is well conserved between the three proteins other than the ring closure to form the thiazolidine in the Th AOS. There is a clear cavity at the dimer interface in each enzyme where the substrate can bind (Fig. 16B). There is only 20% sequence identity between Th AOS and the bacterial CSDs, and residue conservation in the active site is limited to histidine (H136), and arginine (R366); the active arginine (R243 in Th AOS) is conserved, but in different sequence positions in the NifS and SufS proteins (Fig. S17). The wider active site and ligand binding regions of the three proteins differ considerably, with distinct ligand binding tunnels in the three enzymes (Fig. S17). Conclusion There is a growing application for biocatalytic routes to both commodity chemicals, as well as high value intermediates and pharmaceuticals. 47 A key step in organic synthesis is C-C formation and a number of enzymes have been developed as biocatalysts. Databases such as RetroBioCat have allowed route developers to plan synthetic strategies using well characterized biocatalysts whose substrate specificity, catalytic rates and methodologies are well curated. 48,49 It is essential to continue to find new natural biocatalysts that can be enhanced by rational engineering and/or directed evolution/selection. Alternatively, protein scaffolds can be engineered to deliver biocatalysts with no known biological equivalent e.g. the Morita Baylis Hillman reaction 50 . The thermophilic PLP-dependent biocatalyst Th AOS catalyses an irreversible, Claisen-like, C-C bond forming reaction that yields useful aminoketone building blocks 36 . Studying the active site structure of this PLP-dependent biocatalyst, combined with knowledge of the enzyme mechanism, has permitted the generation of variant biocatalysts with significantly improved properties. A single active site residue V79 appears to control access to the active site and conversion to smaller residues such as Gly and Ala allows alternative amino acids and UAAs to bind, without compromising the catalytic activity. Saturation mutagenesis, complementing the original rational mutagenesis strategy, allowed variation at this residue to be fully explored. The best mutant catalyses a total of 71 unique condensations between various amino acid and acyl-thioester substrates, significantly greater than any other AOS previously reported. Furthermore, the ability of the hyperactive mutants to accept simple and affordable SNAc thioesters to a useful degree without the need for auxiliary CoA-mimicking compounds is also a first. We demonstrated the usefulness of this reaction by generating a pyrrole by combining the thermo-stable Th AOS variant in a KPR at elevated temperatures. A key determinant in AOS catalysis is the ability of the enzyme to catalyse the formation of a key PLP-bound external aldimine that subsequently generates the key PLP:amino acid quinonoid nucleophile in the present of the acyl-CoA substrate. A convenient UV-vis screen can rapidly identify hit variants from a library. The Th AOS V79 variants can generate this reactive species in the absence of the acyl-thioester and can also use truncated cysteamine-derived substrates. In essence we have changed the catalytic mechanism to being acyl-CoA independent. It is clear that this V79 residue, which is found on a dynamic loop, plays an important role and recent work on the related Alb29 AOS suggests other residues in this part of the enzyme could be modified to further expand the substrate scope of AOS 51 . The crystal structure of the Th AOS V79A variant with a PLP:L-Pen inhibitor bound displayed little change compared to the wild type enzyme so the enhanced properties must be due to subtle and dynamic changes to the structure and electronics of the PLP-bound transition states. This merits future study on the AOS family of enzymes by molecular dynamics and modelling. This work opens the door for the exploitation of members of the expanding AOS family as synthetically useful C-C bond-forming biocatalysts, in the same vein as aldolases, transaminases, racemases, and other widely-used PLP-dependent enzymes. Materials Materials Commercially available standards, solvents and reagents were purchased from Avanti Lipids, Fluorochem, Sigma Aldrich, Cambridge BioScience and Thermo Fisher Scientific and were used without any further purification. Enzyme Expression and Purification Expression of Th AOS constructs A single colony of E. coli BL21 (DE3) cells containing a pET28a plasmid encoding Th AOS wild type and V79 variants with a TEV-cleavable N-terminal His6 tag was used to inoculate a 5 mL overnight culture of L.B. containing 30 ug/mL kanamycin. After overnight shaking at 37 °C the culture was used to inoculate larger cultures of 1L L.B. media in 2L Erlenmeyer flasks, which were then grown with shaking at 180 rpm at 37 °C until the OD500 was 0.6-0.8. The culture was then induced with 0.25 mM IPTG overnight at 16 °C. Cells were harvested by centrifugation and cell pellets were stored at -20 °C. Heat Purification Cell pellets were resuspended in HEPES buffer (20 mM HEPES, 150 mM NaCl, 5% glycerol, pH 7.5, ~30 mL) before being sonicated for 15 minutes 30s on/30s off. Cell debris was pelleted with ~10,000xg centrifugation for 45 minutes. The clarified cell lysate was next heated in a water bath at 80 °C for 30 minutes with monitoring by SDS-PAGE. After all the E. coli proteins had precipitated, precipitate was pelleted once more with another centrifugation step. The solution was then filtered, protein concentration was determined using the Bradford assay and was used without further purification. Immobilsed metal affinity purification (IMAC) Ni 2+ purification of Th AOS Cell pellets were resuspended in HEPES buffer (~30 mL) before being sonicated for 15 minutes 30s on/30s off. Cell debris was pelleted with ~10,000xg centrifugation for 45 minutes. The mixture was then loaded onto a 5 mL G.E. Healthcare HisTrap FF column prior to a gradient elution/fractionation step with Nickel Elution Buffer (HEPES buffer + 500 mM imidazole). The purest fractions (determined by SDS-PAGE) were then buffer-exchanged back into HEPES buffer and enzyme concentration was determined using the Bradford assay. Full Purification for structural studies Cell pellets were treated as according to the Ni 2+ IMAC Purification, but after the IMAC step yellow fractions were pooled and loaded onto a pre-equilibrated Superdex S200 column, before elution and fractionation with 120 mL HEPES buffer, yielding highly pure mutant enzyme. Samples were concentrated to 20-50 mg/mL stocks, flash-frozen in liquid N 2 and stored at -80 °C. Enzymatic assay The activity assay of Th AOS and its variants were performed using DTNB assay. The reactions were performed in 60 mL scale using amino acid (16 mM), acetyl-CoA (1 mM), ThAOS/ThAOS variants (0.1-1 mg mL -1 ) and DTNB (1 mM) in HEPES buffer (20 mM, pH 7.5) at 50 °C. The UV-vis readings were recorded using a BioTek Synergy HT microplate reader. Chemo-biocatalytic Knorr Pyrrole Reaction (KPR) Cascades The Th AOS (0-3 mgmL -1 ) biocatalyst was mixed with amino acid (16-32 mM), methyl acetoacetate (MAA, 32 mM from a 3.2 M stock in MeCN), sodium benzoate (1 mM, internal standard) and acyl-CoA (0-2 mM) or acetyl-SNAc (0-32 mM from a 100× concentrated stock in MeCN) in HEPES buffer pH 7.5 buffer at a final volume of 200 µL in an Eppendorf tube. Reactions were performed in a Grant-Bio 24-well thermoshaker with shaking at 250 rpm at 30-90 °C for 0-24h. Reactions were initiated by addition of acyl-CoA. Reactions were terminated by addition of 1 volume of MeCN, and centrifugation for 10 minutes at 13,000 g . The supernatant was then analysed by HPLC. UV-Vis spectroscopy The UV-vis spectrum of the wild type Th AOS and V79 variants in the PLP bound state and in the presence of amino acid and acyl-CoA substrates were recorded on a Cary 50 UV vis spectrophotometer with 1 cm pathlength cuvettes. The spectrophotometer was blanked against buffer, and absorbance intensity was recorded between 250-600 nm on the Fast setting. For titrations, substrate was added to the cuvette from a 100 mM stock in buffer, mixed by pipetting and allowed to equilibrate for 30 seconds before recording of the new spectrum. The dilution was accounted for in the final spectra by amplifying the new spectra by the dilution factor caused by addition of the substrate. The whole spectrum was captured and changes in the absorbance maximum of the dominant peaks were plotted and analysed in OriginLab 2019. Protein Crystallisation Crystallisation of recombinant Th AOS V79 mutants was initially screened using commercial kits (Molecular Dimensions and Hampton Research). Protein concentration was 20-25 mg.mL -1 . The drops, comprising 0.1 or 0.2 µL of protein solution plus 0.1 µL of reservoir solution, were set up using a Mosquito crystallisation robot (SPT Labtech). The experiments were incubated at 20 °C. Initial hits were of good size, single and could be directly tested. Whilst hits were found in Index (Hampton Research), three conditions were found in Morpheus (A8, A12 and C8, Molecular Dimensions) to lead to alternative crystal space groups. Th AOS V79A crystallised in P1 (30 mM sodium nitrate, 30 mM sodium phosphate, 30 mM ammonium sulfate, 100 mM HEPES/MOPS pH 7.5, 12.5% (w/v) PEG1000 and 12.5% (w/v) PEG3350), P21 (30 mM magnesium chloride, 30 mM calcium chloride, 100 mM HEPES/MOPS pH 7.5, 12.5% (w/v) PEG1000 and 12.5% (w/v) PEG3350) and in P212121 (30 mM magnesium chloride, 30 mM calcium chloride, 100 mM Tris/bicine pH 8.5, 12.5% (v/v) MPD, 12.5% (w/v) PEG1000 and 12.5% (w/v) PEG3350). The samples did not require optimisation of additional cryo-protection. Synthesis of N,S-diacetylcysteamine (acetyl-SNAc). N,S-diacetylcysteamine (acetyl-SNAc) Cysteamine hydrochloride (2.11 g, 18.8 mmol) was dissolved in water (20 mL) at 0 °C, and the pH was adjusted to 8.0 with aqueous KOH (8 M). Acetic anhydride (5.72 g, 56.1 mmol) was next added dropwise, and the pH was adjusted to 7.0 with aqueous KOH (8 M). The solution was stirred at 0 °C for 90 minutes, until addition of a drop to a solution of Ellman’s reagent DTNB did not cause a colour change. The solution was then extracted with CH2Cl2 (3×). The organic phase was then washed with acidified water (3×), dried with magnesium sulfate and concentrated by rotary evaporation to afford the title compound as a viscous and colourless liquid (0.156 g). NMR The 1H and 13C NMR spectra were recorded on a Bruker Avance III 500 MHz or a Bruker CryoProbe Prodigy 500 MHz, and the solvent was CDCl 3 . 1H NMR (500 MHz, CDCl3): δH 5.95 (1H, broad s, NH), 3.46 (2H, t, J = 10 Hz, -NHCH2CH2S-), 3.04 (2H, t, J = 10 Hz, -NHCH2CH2S-), 2.37 (3H, s, -SC(O)CH3), 1.99 (3H, s, -NHC(O)CH3). 13C NMR (101 MHz, CDCl3): δC 196.3, 170.3, 39.6, 30.7, 28.9, 23.1. Data Collection, Structure Solution, Model Building, Refinement and Validation Diffraction data were collected at the synchrotron beamline I04 of Diamond light source (Didcot, U.K.) 07/07/2021 at a temperature of 100 K. The data set was integrated with XIA2 52 . using XDS 53 and scaled with Aimless 54 . The space group was confirmed with Pointless 55 . The phase problem was solved by molecular replacement with Phaser 56 using Th AOS structure as the search model (PDB: 7POA) 36 . The model was refined with refmac 57 . The PLP:L-Penicillamine ligand was generated using JLigand 58 and optimised with AceDRG 59 . Manual model building with COOT 60 was intercalated between refinement rounds. The models were validated using Coot and Molprobity 61 . Other software used were from CCP4 cloud 62 and CCP4 suite 63 . Figures were made with Chimerax 64 . Data collection processing and refinement statistics are presented in Table S7. Declarations Data availability Protein structure raw data files (MTZ and PDB) are available from the author. HPLC, NMR, kinetic and UV-vis data are also available upon request. Acknowledgements The authors thank the BBSRC for an EastBio (BB/J01446X/1) PhD studentship (B.A.) The BBSRC is thanked for grant funding awarded to D.J.C. (BB/T016841/1) to support S.M. The University of Edinburgh and the Derek Stewart Charitable Trust is thanked for PhD studentship funding (M.S.). Author contributions B.A. and D.J.C. designed the project with respect to engineering the Th AOS biocatalyst and isolating improved variants. B.A. designed, expressed and purified the Th AOS variants. B.A. also assayed the actitivities with the reported substrates. Y.Z. prepared and characterized Th AOS V79 variants. S.M. carried out characterization of the ThAOS V79 variant and scaled up reactions. M.S. prepared the acyl-SNAC substrate and carried out the isolation of pyrrole targets. A.B. isolated crystals of the Th AOS V79A PLP:L-Pen complex and acquired data from the Diamond Light Source. A.B. and J.M.-W. solved the structure of Th AOS V79APLP: L-Pen complex and deposited the structure in the Protein DataBank (PDB). B.A. wrote the initial manuscript. All authors made written contributions to the manuscript and prepared figures and tables. D.J.C. edited and wrote the final version of the paper. Competing Interests None. Additional Information Supplementary Information. 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Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010). Williams, C. J. et al. MolProbity: More and better reference data for improved all-atom structure validation. Prot. Sci. 27, 293–315 (2018). Krissinel, E. B., Uski, V., Lebedev, A. A., Winn, M. D. & Ballard, C. Distributed computing for macromolecular crystallography. Acta Crystallogr. D 74, 143–151 (2018). Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67, 235–242 (2011). Pettersen, E. F. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Prot. Sci. 30, 70–82 (2021). Tables Table 1 is available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files AshleyCommChem2024SuppInfo.docx Table1.docx Cite Share Download PDF Status: Published Journal Publication published 13 Mar, 2025 Read the published version in Communications Chemistry → Version 1 posted 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-4345858","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":297074964,"identity":"9ea7ffa7-fad4-4f3d-9cb6-f61810e64db2","order_by":0,"name":"Dominic Campopiano","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8573-6735","institution":"University of Edinburgh","correspondingAuthor":true,"prefix":"","firstName":"Dominic","middleName":"","lastName":"Campopiano","suffix":""},{"id":297074965,"identity":"9bc8f3c0-1e1c-4e62-a912-02651ddde63c","order_by":1,"name":"Ben Ashley","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Ben","middleName":"","lastName":"Ashley","suffix":""},{"id":297074966,"identity":"36850a9e-3d5c-4ca7-b62b-4dc9f67d3e44","order_by":2,"name":"Yaoyi Zhu","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Yaoyi","middleName":"","lastName":"Zhu","suffix":""},{"id":297074967,"identity":"a89ad5b5-777d-46fe-be8e-1ce8d365fbaa","order_by":3,"name":"Sam Mathew","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Sam","middleName":"","lastName":"Mathew","suffix":""},{"id":297074968,"identity":"2b728d91-ba70-4fd5-a020-50874732f0a3","order_by":4,"name":"Mariyah Sajjad","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Mariyah","middleName":"","lastName":"Sajjad","suffix":""},{"id":297074969,"identity":"d4c74396-0d90-48ec-a75f-f394e9305c1f","order_by":5,"name":"Arnaud Baslé","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Arnaud","middleName":"","lastName":"Baslé","suffix":""},{"id":297074970,"identity":"0f5e7d00-b6d0-4b90-ba82-fde121d2192e","order_by":6,"name":"Jon Marles-Wright","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jon","middleName":"","lastName":"Marles-Wright","suffix":""}],"badges":[],"createdAt":"2024-04-30 03:05:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4345858/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4345858/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42004-025-01448-8","type":"published","date":"2025-03-13T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56472503,"identity":"d87614c5-cfd5-486c-96e5-f1a17ea47173","added_by":"auto","created_at":"2024-05-14 16:25:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e The reaction catalysed by AOS enzymes. B) Previously presented work, using cofactor regeneration in an AOS-Knorr pyrrole reaction (KPR) chemo/bio-catalytic cascade reaction to generate pyrroles. C) The work of Narayan \u003cem\u003eet al\u003c/em\u003e generating activity with SNAc thioesters by addition of CoA-mimicking pantetheine. D) This work.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4345858/v1/98c6a287d4ee5b192d759294.png"},{"id":56472504,"identity":"e422d3bd-9a8d-4d3c-830f-703f1f680725","added_by":"auto","created_at":"2024-05-14 16:25:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63537,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe consensus mechanism of wild type and engineered AOS enzymes.\u003c/strong\u003e The enzyme resting state is that of an “internal aldimine” or Schiff base, in which the PLP cofactor is covalently bound to the enzyme via a conserved Lys residue (P\u003csub\u003ei\u003c/sub\u003e = phosphate). The first chemical step is reversible transimination of the internal aldimine by an amino-acid to generate a PLP:amino acid “external aldimine”. In the wild type AOS a conformational change is caused by binding of the acyl-CoA substrate that rotates the external aldimine into the “Dunathan conformation”. This permits the catalytic lysine to deprotonate the amino-acid at Cα and generate a reactive PLP:quinonoid intermediate (observed at 490-510 nm). The nucleophilic quninonoid reacts with the electrophilic acyl-CoA thioester in a Claisen-like condensation to form the C-C bond of a b-ketoacid intermediate and eliminates CoASH irreversibly (detected by the DTNB assay). The b-ketoacid decarboxylates to generate a PLP:a-aminoketone product external aldimine, which finally reacts with the Lys residue to release the a-aminoketone product and return the AOS to the internal aldimine. In blue the engineered \u003cem\u003eTh\u003c/em\u003eAOS V79 variants produced in this study have been shown to generate the PLP:quinonoid in the absence of the acyl-CoA\u003cem\u003e substrate\u003c/em\u003e. This expands the substrate scope to allow binding and reaction between a range of amino acids, acyl-CoAs and acyl-SNAc substrates.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4345858/v1/271cce6fd6756c6f9f78dcdf.png"},{"id":56472505,"identity":"965e7aeb-f919-45e7-80e6-4317969242e4","added_by":"auto","created_at":"2024-05-14 16:25:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":156437,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Interaction between Val79 and the sidechain of a Gly substrate in an overlay of the crystal structure of the PLP-bound form of \u003cem\u003eTh\u003c/em\u003eAOS (PDB: XXX) with the PLP:Gly external aldimine form of \u003cem\u003eR. capsulatus\u003c/em\u003e ALAS (PDB: 2BWP). The V79 sidechain of the opposite monomer is predicted to be in relatively close proximity (3.5 Å) to the C-a of the amino acid. (B) A sequence and structural alignment of various AOS biocatalysts highlighting the residues equivalent to position V79 of \u003cem\u003eTh\u003c/em\u003eAOS. The KBL enzyme from \u003cem\u003eE. coli\u003c/em\u003e (1FC4) retains the valine residue whereas the AONS enzyme from \u003cem\u003eE. coli\u003c/em\u003e and SPT enzyme from \u003cem\u003eS. paucimobilis \u003c/em\u003e(PDB: 2JG2) have a serine side chain. An Alb29 enzyme from \u003cem\u003eS. albogriseolus \u003c/em\u003e(8XHA) has a leucine and the ALAS enzyme from \u003cem\u003eR. capsulatus\u003c/em\u003e has a threonine at this equivalent position (PDB: 2BWP). The full alignment is Fig. S18.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4345858/v1/31ed90c780975304c53f24b1.png"},{"id":56472507,"identity":"9ec04b22-7723-4e43-835b-b0b34e276a12","added_by":"auto","created_at":"2024-05-14 16:25:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 5\u003c/strong\u003e. Analysis of the chemo/bio-catalytic AOS/KPR cascade using \u003cem\u003eTh\u003c/em\u003eAOS V79A, Gly (32 mM), acetyl-SNAc (32 mM), MAA (32 mM) in HEPES buffer (100 mM, pH 7.5) incubated overnight at 60, 70 and 80 \u003csup\u003eo\u003c/sup\u003eC. The product pyrrole (\u003cstrong\u003e1\u003c/strong\u003e), the benzoic acid internal standard (\u003cstrong\u003eI.S.\u003c/strong\u003e) and acetyl-SNAc substrate are shown.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4345858/v1/951d868501b9d902e43037b0.png"},{"id":56472502,"identity":"1ea7d446-b413-4dff-b8ce-49a26c56570f","added_by":"auto","created_at":"2024-05-14 16:25:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":317069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 6. Penicillamine binding in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTh\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAOS active site. A)\u003c/strong\u003e Experimental electron density map showing the PLP-penicillamine thiazolidine in the \u003cem\u003eTh\u003c/em\u003eAOS V79A variant. The two protein chains are coloured orange and blue, ligand is shown with grey carbon atoms and the 2mFo-DFc map is shown as a blue transparent surface at a level of 1s. \u003cstrong\u003eB)\u003c/strong\u003eComparison of the \u003cem\u003eTh\u003c/em\u003eAOSwild-type structure with the V79A variant. The loss of the valine side chain (shown in pink from the WT structure (7POA)), does not cause any gross structural changes at the active site. Removal of the valine side chain opens this region up by around 1 Å, to allow larger hydrophobic substrates to be accommodated. Figure created using ChimeraX version 1.6.1.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4345858/v1/8b14f334d57ae89ee159672f.png"},{"id":78502845,"identity":"288479f0-82ff-48f7-9127-3615471774f7","added_by":"auto","created_at":"2025-03-14 07:11:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1775463,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4345858/v1/c79808b7-576a-4aad-ab96-1a8ac78fcd4b.pdf"},{"id":56472508,"identity":"55cd4426-a1f3-446f-8f10-d645735e46df","added_by":"auto","created_at":"2024-05-14 16:26:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7889736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"AshleyCommChem2024SuppInfo.docx","url":"https://assets-eu.researchsquare.com/files/rs-4345858/v1/064112ebe5f2c4c1aba5e069.docx"},{"id":56472506,"identity":"ae19952b-bd8c-4d04-bd2d-797ae5cf510b","added_by":"auto","created_at":"2024-05-14 16:25:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31896,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4345858/v1/fd0dd5fbb89510f26708bef4.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Rational Engineering of a Thermostable α-Oxoamine Synthase Biocatalyst Expands the Substrate Scope and Synthetic Applicability","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiocatalysts continue to provide useful alternatives to traditional metal and organic catalysts for the synthesis numerous target molecules at small and industrial scales\u003csup\u003e1-6\u003c/sup\u003e. Once a natural enzyme has been characterised it can be further improved for bespoke applications by the methods of directed evolution and selection\u003csup\u003e7,8\u003c/sup\u003e. The \u0026alpha;-oxoamine synthases (AOSs) are pyridoxal 5\u0026rsquo;-phosphate (PLP)-dependent enzymes which catalyse the irreversible, decarboxylative, Claisen-like condensation of an amino acid with an acyl-CoA thioester, to generate \u0026alpha;-aminoketones (Fig. 1A)\u003csup\u003e9,10\u003c/sup\u003e. The AOS family are core enzymes in the biosynthesis of many important biological primary and secondary metabolites, including heme, biotin and sphingolipids, as well as complex natural products such as saxitoxin and the ketomemicins\u003csup\u003e11-14\u003c/sup\u003e. Many of the AOS enzymes bring together two primary metabolic building blocks, amino acids and fatty acids, but an understanding of the origin of their substrate specificity is still unclear. For this reason, their mechanism, structure and substrate specificity have been studied in great detail over the last seven decades\u003csup\u003e15-32\u003c/sup\u003e. Despite their potential as complexity-generating, C-C bond-forming biocatalysts, they have not been exploited for synthesis until recently. It appears they have been passed over for more tractable enzyme classes which are easier to engineer, have broader substrate scopes and have no requirement for expensive acyl-CoA substrates\u003csup\u003e33\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn recent years however, AOS enzymes have attracted greater attention as synthetically-useful biocatalysts since they generate \u0026alpha;-aminoketones that can be elaborated by synthetic chemistry and/or other biocatalysts. We recently made use of the \u0026alpha;-aminoketone products generated by the recombinant form of the \u003cem\u003eTh\u003c/em\u003eAOS from the thermophilic microbe \u003cem\u003eThermus thermophilus\u003c/em\u003e\u003csup\u003e34\u003c/sup\u003e. The \u0026alpha;-aminoketones were combined in a chemo-biocatalytic cascade with various\u0026nbsp;b-keto esters in a Knorr pyrrole reaction (KPR), to generate pyrroles in excellent conversions over short timescales (\u0026gt;90% in \u0026lt;2 hours) (Fig. 1B)\u003csup\u003e34-36\u003c/sup\u003e. Also, the AOS enzyme Alb29 from the biosynthetic pathway of the \u003cem\u003eStreptomyces albogriseolus\u003c/em\u003e natural product albogrisin was recently shown to be able to use L-glutamate and a small group of acyl-CoAs to form seven aminoketones\u003csup\u003e37\u003c/sup\u003e. Additionally, Narayan and colleagues have also exploited another AOS enzyme, SxtA from \u003cem\u003eMicroseira wollei\u003c/em\u003e to prepare \u0026alpha;-deuterated amino acids in excellent yield and purity. There they used the ability of AOSs to deprotonate and re-protonate \u0026alpha;-amino acids in the presence of D\u003csub\u003e2\u003c/sub\u003eO, (Fig. 1C)\u003csup\u003e38\u003c/sup\u003e. The same group have also made progress in understanding and circumventing the SxtA requirement for acyl-CoA thioester susbtrates\u003csup\u003e39\u003c/sup\u003e. However, the issues of a relatively strict substrate specificity and reliance on complex cofactor or cofactor-mimicking molecules remain.\u003c/p\u003e\n\u003cp\u003eIn this work, we show for the first time that an AOS biocatalyst (\u003cem\u003eTh\u003c/em\u003eAOS) can be rationally engineered to use an expanded range of natural and unnatural amino acid substrates. This was achieved by targeting a residue, valine 79 (V79), that is close to the \u003cem\u003eTh\u003c/em\u003eAOS PLP binding site, but not thought to be involved directly in catalysis. Structural studies of AOSs have shown this residue is in a ~13 amino acid flexible loop that appears to control amino acid substrate binding. We found that substitution of the V79 side-chain opens up entry to a range of natural and unnatural amino acids. Spectroscopic analysis revealed that, in the absence of the second acyl-CoA substrate, the \u003cem\u003eTh\u003c/em\u003eAOS V79 variants could also convert the bound PLP:amino acid external aldimine intermediate into reactive quinonoid, ready for C-C bond formation. Additionally, the turnover with simple and inexpensive acyl-CoA-mimicking N-acetylcysteamine (NAC or SNAc) thioesters was greatly enhanced with the use of engineered \u003cem\u003eTh\u003c/em\u003eAOS variants. We used the most thermostable \u003cem\u003eTh\u003c/em\u003eAOS variant to couple with the KPR removing the reliance on our previous acyl-CoASH recycling system (Fig. 1D). Furthermore, we determined the crystal structure of the \u003cem\u003eTh\u003c/em\u003eAOS V79A variant in complex with a PLP:L-penicillamine external aldimine. The structure of this useful PLP enzyme inhibitor bound to the biocatalyst revealed very little differences when compared to the structure of the internal aldimine form of the wild type \u003cem\u003eTh\u003c/em\u003eAOS. Our study suggests that very subtle changes near, but not in, the active site of an AOS biocatalyst influence the substrate binding and convert these conformationally-dynamic enzymes into a catalytically active state that promotes reaction with a range of non-natural substrates. Our findings should also encourage both rational engineering and random mutagenesis/selection of other members of the AOS family to deliver a range of synthetically useful biocatalysts.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cb\u003eSubstrate binding to wild type ThAOS.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe original paper that characterized \u003cem\u003eTh\u003c/em\u003eAOS showed that it could accept Gly, L-Ala and L-Ser amino acids and acetyl-CoA, pimeloyl-CoA and palmitoyl-CoA acyl-thioester substrates\u003csup\u003e34\u003c/sup\u003e. It was also shown that \u003cem\u003eTh\u003c/em\u003eAOS catalysed both KBL (Gly/acetyl-CoA) and AONS (L-Ala/pimeloyl-CoA) reactions. We used this information in our previous work to measure an expanded substrate scope of \u003cem\u003eTh\u003c/em\u003eAOS using a relatively high-throughput kinetic assay which uses the thiol-reactive DTNB reagent (Ellman\u0026rsquo;s) to monitor CoASH release (Fig.\u0026nbsp;1A)\u003csup\u003e36\u003c/sup\u003e. The \u003cem\u003eTh\u003c/em\u003eAOS enzyme was active with amino acids \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ala, Gly, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ser and also \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Aba, and could catalyse condensation with acetyl-, propionyl-, butyl-, hexanoyl- and octanoyl-CoA thioester substrates. Therefore, we concluded that the \u003cem\u003eTh\u003c/em\u003eAOS biocatalyst has unusually broad substrate scope which suggests it be further expanded.\u003c/p\u003e\u003cp\u003eIn this new study we wanted to further explore substrate binding so we took advantage of the inherent UV-vis properties of the PLP-dependent \u003cem\u003eTh\u003c/em\u003eAOS. The PLP UV-vis spectrum gives insights into substrate binding, as well as the formation of key intermediates during the catalytic mechanism\u003csup\u003e40\u003c/sup\u003e. The most intensively studied members of the AOS family have been SPT, ALAS and AONS with a view to understand their substrate specificity within the biosynthetic pathways in which they operate\u003csup\u003e13,14,41\u003c/sup\u003e. These combined studies have led to a consensus catalytic mechanism of the AOS family with various evidence to support the structure and role(s) of key intermediates and active site residues (Fig.\u0026nbsp;2). The AOS enzymes exhibit characteristic PLP spectroscopic properties with a clear shift in the UV-vis spectrum upon binding of the amino acid substrate to form the PLP:amino acid external aldimine. The binding of the second, acyl-CoA substrate is thought to cause a conformational change (proposed by Dunathan\u003csup\u003e42\u003c/sup\u003e) that allows deprotonation of the proton at C-a to generate the key quninonoid/carbanion intermediate. This species subsequently reacts in the C-C forming, Claisen-like condensation reaction to generate a b-keto acid intermediate which is readily decarboxylated to the amino ketone product. The observation of key intermediates, such as the quinonoid, is dependent on the specific AOS/substrate combination.\u003c/p\u003e\u003cp\u003eWe studied the binding of \u003cem\u003eTh\u003c/em\u003eAOS with its amino acid substrates using UV-vis spectroscopy, according to the methods of Webster \u003cem\u003eet al.\u003c/em\u003e and Raman \u003cem\u003eet al\u003c/em\u003e that were used to study both \u003cem\u003eE. coli\u003c/em\u003e AONS and \u003cem\u003eS. paucimobilis\u003c/em\u003e SPT\u003csup\u003e9,43\u003c/sup\u003e. Recombinant, purified \u003cem\u003eTh\u003c/em\u003eAOS displays a smaller, broad absorbance between 330\u0026ndash;360 nm and a larger, broad absorbance between 380\u0026ndash;480 nm corresponding to the tautomeric forms of the PLP-bound internal aldimines (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and Fig.\u0026nbsp;2). Titration of the core substrate Gly (0\u0026ndash;16 mM) led to a small decrease in the 330 nm peak and a small increase in the 430 nm peak (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). A similar titration of L-Ser (0\u0026ndash;16 mM) led to a small increase in the peak at 330 nm and a decrease in the 430 nm peak. With L-Ala (0\u0026ndash;16 mM) there was little change in the 330 nm absorbance, a slight decrease and shift (by ~\u0026thinsp;2-4nm to 426\u0026ndash;428 nm) in the 430 nm peak and the observation of a small, broad peak between 490\u0026ndash;505 nm. Taken together this spectroscopic data confirms that these substrates bind to the \u003cem\u003eTh\u003c/em\u003eAOS to form PLP:amino acid external aldimines and that L-Ala binding leads to a small amount of PLP:L-Ala quinonoid formation.\u003c/p\u003e\u003cp\u003eHaving established amino acid binding, we then explored what happens upon addition of the second substrate, acetyl-CoA to the external aldimine forms of \u003cem\u003eTh\u003c/em\u003eAOS. Little change was observed with Gly and L-Ser, but we were interested to observe the clear formation of a peak at 501 nm (with a shoulder at 472 nm) with L-Ala as the substrate, suggesting this substrate combination generates an intense quinonoid species (Fig. S2). The observation of such an intermediate suggests subtle differences in the way each substrate binds to and is stabilized by the enzyme. These results suggested to us that \u003cem\u003eTh\u003c/em\u003eAOS could potentially bind a greater range of substrates than previously thought \u0026ndash; either non-productively to form a \u003cem\u003eTh\u003c/em\u003eAOS:PLP-amino acid external aldimine, or productively, to go on to product aminoketone formation in the presence of an acyl-CoA substrate (Fig.\u0026nbsp;2). Moreover, we hoped that the \u003cem\u003eTh\u003c/em\u003eAOS could be engineered to further expand its substrate scope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRational engineering of ThAOS for an expanded substrate scope.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe previously determined the crystal structure of the \u003cem\u003eTh\u003c/em\u003eAOS homo-dimer in the internal aldimine form with the PLP bound cofactor (PDBs: 7POA, 7POB and 7POC)\u003csup\u003e36\u003c/sup\u003e. In the absence of a substrate bound structure we modelled the PLP:Gly external aldimine from the crystal structure of \u003cem\u003eRc\u003c/em\u003eALAS (PDB: 2BWP), a homologue from the AOS family. This model highlighted a close interaction (3.5 \u0026Aring;) between the proposed position of the substrate amino acid sidechain and that of Val79 from the other \u003cem\u003eTh\u003c/em\u003eAOS monomer within the dimer structure (Fig.\u0026nbsp;3A). Thus, Val79 appeared to be an important site for amino acid substrate selectivity, and it was targeted for site-directed mutagenesis. This residue is conserved as a Val side chain in all KBL enzymes but replaced by a Ser, Leu and Thr in other AOS enzymes such as SPT, ALAS, AONS and the recently discovered Alb29 (Fig.\u0026nbsp;3B). This lack of conservation also suggests that changes at this residue might alter the substrate scope, but would not adversely impact the catalytic activity. Our initial hypothesis was that \u003cem\u003eTh\u003c/em\u003eAOS V79 variants would display an expanded amino acid substrate scope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eActivity screening of ThAOS V79 variants identifies improved biocatalysts.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInitially, we aimed to reduce size of the Val79 side-chain, ideally permitting the entry of bulkier amino acids besides those already turned over by WT \u003cem\u003eTh\u003c/em\u003eAOS. As proof of concept, we prepared the enzymes \u003cem\u003eTh\u003c/em\u003eAOS V79A and V79G as initial variants to investigate their substrate scope. The mutant biocatalysts were expressed and purified as the wild type \u003cem\u003eTh\u003c/em\u003eAOS (Fig. S5) and were screened against a panel of amino acids (16 mM) and acetyl-CoA (1 mM), by monitoring the reaction \u003cem\u003evia\u003c/em\u003e the colorimetric DTNB thiol detection screen at 50\u0026deg;C\u003csup\u003e36,43\u003c/sup\u003e. In our screen we included all 20 proteinogenic amino acids, plus the unnatural amino acids (UAAs) \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Aba, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003edl-\u003c/span\u003eAlg, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Cpa, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Cpg, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Hsr, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Nle, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Nva, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Orn, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Phg, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Pra, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Trl, L-Oas and a-Ile, (Fig. S3).\u003c/p\u003e\u003cp\u003eOf the 33 amino acid substrates tested with acetyl-CoA as the acyl-thioester substrate, \u003cem\u003eTh\u003c/em\u003eAOS V79A and \u003cem\u003eTh\u003c/em\u003eAOS V79G displayed activity with 16 amino acids (Table\u0026nbsp;1). These split into 9 UAAs (\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Aba, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003edl-\u003c/span\u003eAlg, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Cpa, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Cpg, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Hsr, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Nva, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Pra and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Oas) and 7 proteinogenic amino acids (\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ala, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Asp, Gly, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ile, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ser, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Thr and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Val). Both variants were significantly faster than the wild type biocatalyst for the original set of four amino acid substrates (\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Aba, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ala, Gly and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ser), and in general \u003cem\u003eTh\u003c/em\u003eAOS V79G was faster than \u003cem\u003eTh\u003c/em\u003eAOS V79A. These variants were kinetically characterised in more detail using the DTNB assay, confirming \u003cem\u003eTh\u003c/em\u003eAOS V79G to be faster than \u003cem\u003eTh\u003c/em\u003eAOS V79A in most cases (Fig. S4, Table S2). With the \u003cem\u003eTh\u003c/em\u003eAOS V79A variant, the catalytic efficiency (\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e) for all four original amino acid substrates was improved substantially (by 2.4x \u0026ndash; 64.8x fold). Similarly, the \u003cem\u003eTh\u003c/em\u003eAOS V79G variant showed improved catalytic efficiency with these four reactions by (2.7x \u0026ndash; 57.5x fold), with both variants displaying the highest activity with \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ser. Having identified Val79 as a critical residue involved in substrate binding, we attempted to obtain other useful and/or interesting \u003cem\u003eTh\u003c/em\u003eAOS variants by performing saturation mutagenesis at this position (Table S3). To allow high throughput analysis this expanded set of V79 variants were prepared and expressed in a mini culture format, taking advantage of the thermostability of the biocatalysts.\u003c/p\u003e\u003cp\u003eUnder the standard culture conditions, no expression was detected for seven of the possible 17 new variants (\u003cem\u003eTh\u003c/em\u003eAOS V79D, F, H, N, P, Q and W). Two variants (\u003cem\u003eTh\u003c/em\u003eAOS V79K and V79Y) expressed but were insoluble, but eight (\u003cem\u003eTh\u003c/em\u003eAOS V79C, E, I, L, M, R, S and T) were obtained in soluble form (Table S3). These soluble variants were purified from the cell free extract \u003cem\u003evia\u003c/em\u003e a heating step at 80\u0026deg;C for 30 minutes, prior to centrifugation (Fig. S5). The variants which were not stable to this treatment were purified by the full Ni\u003csup\u003e2+\u003c/sup\u003e affinity chromatography (IMAC) method. The purified \u003cem\u003eTh\u003c/em\u003eAOS variants were screened against the broad range of amino acid substrates with acetyl-CoA and the release of CoASH observed using the DTNB assay. It was clear that the \u003cem\u003eTh\u003c/em\u003eAOS V79S variant displayed increased activity relative to the wild type \u003cem\u003eTh\u003c/em\u003eAOS with the core set of four amino acids (\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Aba, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ala, Gly and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ser), but also accepted the UAAs, L-Cpg and L-Pra (Fig.\u0026nbsp;3). Of the other variants, \u003cem\u003eTh\u003c/em\u003eAOS V79M and \u003cem\u003eTh\u003c/em\u003eAOS V79R turned over only \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ser to a lesser extent and \u003cem\u003eTh\u003c/em\u003eAOS V79T was only active with \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ala. Amongst the other soluble mutants, \u003cem\u003eTh\u003c/em\u003eAOS V79C and \u003cem\u003eTh\u003c/em\u003eAOS V79E displayed no detectable activity with the amino acid substrates tested. The \u003cem\u003eTh\u003c/em\u003eAOS V79I variant showed the same substrate profile as wild type but with a decreased rate, whilst the \u003cem\u003eTh\u003c/em\u003eAOS V79L turned over \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Cpg as well as the original four substrates, and at a slightly improved rate than the wild-type biocatalysts. The retention of activity observed with the \u003cem\u003eTh\u003c/em\u003eAOS V79I/L variants is understandable since these are all similar, hydrophobic side-chains.\u003c/p\u003e\u003cp\u003eWe sought to understand the differences in the activities of the \u003cem\u003eTh\u003c/em\u003eAOS V79 variants by simple consideration of the chemistry of each side chain (Fig. S6). It appears that the presence or absence of a substituent (e.g. methyl) at the β-position of the amino acid side chain influences not only the substrate scope but also the catalytic activity. Furthermore, this explains the relative lack of distinction between the \u003cem\u003eTh\u003c/em\u003eAOS V79A and \u003cem\u003eTh\u003c/em\u003eAOS V79G variants. It is clear that the residue at this position in the \u003cem\u003eTh\u003c/em\u003eAOS structure is one of the important determinants of amino acid substrate selectivity. It is also notable that the Val79 residue is not conserved across other members of the AOS family that display differences in their amino acid selectivity e.g. in the well characterized \u003cem\u003eS. paucimobilis\u003c/em\u003e SPT which uses L-serine as a substrate, this residue is Ser102 (Fig.\u0026nbsp;2B). The residues in this 13 amino stretch are part of a key flexible loop where the AOS enzymes display conformational flexibility in key residues involved in substrate binding and catalysis. The AOS enzymes have another flexible loop containing the conserved \u0026ldquo;PATP\u0026rdquo; motif near the C-terminus that also undergoes conformational change. Our work on \u003cem\u003eS. paucimobilis\u003c/em\u003e SPT captured the key PLP:L-Ser external aldimine complex (PDB: 2W8J) showed that key arginine residues (R378 and R390) are involved in recognition of the carboxylate of L-Ser; the side-chain of Arg378 swings into place upon substrate binding and then \u0026ldquo;passes on\u0026rdquo; this carboxylate to Arg390 once the C16-CoA substrate binding has caused the conformational change that induces deprotonation at C-a (Fig.\u0026nbsp;2)\u003csup\u003e43\u003c/sup\u003e. The resulting PLP:L-Ser quinonoid intermediate is the nucleophile that forms the C-C bond in the b-keto acid intermediate. Mutagenesis of these Arg residues showed that the highly-conserved Arg378A was x40 fold less active than the wild type and this variant could not stabilize the quinonoid. It is interesting to note that KBL is the only AOS member that does not decarboxylate this unstable intermediate (2-amino-3-ketobutyrate, AKB) to give aminoacetone. It protects the AKB by forming a complex between KBL and L-threonine dehydrogenase (TDH). Together KBL and TDH are involved in a glycine/acetyl-CoA metabolic cycle with L-Thr via the AKB intermediate\u003csup\u003e16\u003c/sup\u003e. The fact that \u003cem\u003eTh\u003c/em\u003eAOS in isolation can catalyse aminoketone formation with various substrates underscores its unusual, but useful, properties.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpanded acyl-CoA substrate scope of\u003c/b\u003e \u003cb\u003eTh\u003c/b\u003e\u003cb\u003eAOS V79 variants.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur next goal was to determine the impact, if any, of variations at Val79 on the acyl-CoA thioester substrate scope. In our previous study, we noted that wild-type \u003cem\u003eTh\u003c/em\u003eAOS turned over all four of the amino acid substrates (L-Aba, L-Ala, Gly, L-Ser) with acetyl-, propionyl-, butyryl-, hexanoyl- and octanoyl-CoA\u003csup\u003e35\u003c/sup\u003e. Therefore, we used the DTNB assay to screen the three most promising variants (\u003cem\u003eTh\u003c/em\u003eAOS V79G, V79A and V79S) derived from the amino acid substrate screen with acyl-CoA thioesters of increasing acyl chain length (C2, C3, C4, C6, C8, C10 and C12), as well as benzoyl-CoA (Table S4-S6).\u003c/p\u003e\u003cp\u003eWe were pleased to observe that the acyl-CoA substrate range of \u003cem\u003eTh\u003c/em\u003eAOS V79G, V79A and V79S was retained across the C2-C8 chain length when compared to wild type \u003cem\u003eTh\u003c/em\u003eAOS. We noted that the \u003cem\u003eTh\u003c/em\u003eAOS V79G variant was active with all four core amino acids and C10 and C12 acyl-CoA substrates and could also condense Gly and L-Ala with benzoyl-CoA albeit at very slow rates (Table S4). It was also pleasing to observe that two of the new UAA substrates (L-Cpg, L-Hsr) could also be condensed with acyl-CoA thioesters up to C\u003csub\u003e12\u003c/sub\u003e (Table S4). The \u003cem\u003eTh\u003c/em\u003eAOS V79A and V79S variants also featured a slightly improved acyl-CoA substrate scope relative to the WT \u003cem\u003eTh\u003c/em\u003eAOS (Tables S5 and S6). The \u003cem\u003eTh\u003c/em\u003eAOS V79S is distinguishable in that it is the one variant that was able to catalyse condensation of the UAA substrate L-Pra with C2-C8 acyl-CoAs at modest rates. Furthermore, this variant displayed detectable activity with L-Ala, Gly, L-Ser and L-Cpg and benzoyl-CoA (Table S5).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAcyl-CoA independent quinonoid formation in the\u003c/b\u003e \u003cb\u003eTh\u003c/b\u003e\u003cb\u003eAOS V79G variants.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSince the \u003cem\u003eTh\u003c/em\u003eAOS V79G variant displayed the broadest activity (Table\u0026nbsp;1) we have begun to probe how it behaves in the presence of a sub-set of substrates. Here we monitored formation of the key quinonoid intermediate between 490\u0026ndash;510 nm to explore the \u003cem\u003eTh\u003c/em\u003eAOS variants in terms of amino acid substrate binding alone, and in the presence of the acyl-CoA thioester. The \u003cem\u003eTh\u003c/em\u003eAOS V79G variant was incubated with Gly, L-Ser and L-Ala. When compared with wild type \u003cem\u003eTh\u003c/em\u003eAOS we noted similar changes to the UV-vis spectrum when the amino acid was added, although in each case there was a small, but clear absorbance between 480\u0026ndash;520 nm indicating the formation of a ThAOS V79G PLP:amino acid quinonoid intermediate (Fig. S7 A-C). We also noted acetyl-CoA binding to the \u003cem\u003eTh\u003c/em\u003eAOS V79G PLP:Gly complex led to enhancement of this peak (Fig. S7A). In contrast, addition of the acetyl-CoA to the \u003cem\u003eTh\u003c/em\u003eAOS V79G PLP:L-Ser complex led to formation of a broad, intense quinonoid signal between 490\u0026ndash;520 nm (Fig. S7B), with a shoulder at 468 nm. Moreover, addition of the acyl-CoA thioester substrate to the \u003cem\u003eTh\u003c/em\u003eAOS V79G PLP:L-Ala external aldimine generated the most intense quinonoid signal between 490\u0026ndash;520 nm (Fig.\u0026nbsp;7C), also with a shoulder 468 nm). Clearly, the removal of the Val79 side-chain has impacted the formation and stabilization of the \u003cem\u003eTh\u003c/em\u003eAOS PLP:amino acid complex and binding of the acyl-CoA thioester substrate leads to deprotonation at C-a and quinonoid formation, supporting the overall proposed AOS catalytic mechanism (Fig.\u0026nbsp;2). Since the \u003cem\u003eTh\u003c/em\u003eAOS V79G variant also accepted L-Asp, L-Thr and L-Val (Table\u0026nbsp;1) we looked at quinonoid formation with these three substrates and were pleased to observe the characteristic formation of the peak around 500 nm of varying intensities for each amino acid (Fig. S7 D-F). It is clear that this relatively easy assay is a useful tool to study not only substrate binding, but also formation of this key catalytic quinonoid intermediate.\u003c/p\u003e\u003cp\u003eQuinonoid formation has been studied in only a few members of the AOS family. We have studied the \u003cem\u003eE. coli\u003c/em\u003e AONS and provided supporting evidence for quinonoid formation using L-L-Ala methyl ester (L-Ala-OMe) as a substrate mimic\u003csup\u003e18\u003c/sup\u003e. This ligand bound to the enzyme to form the PLP:L-Ala-OMe external aldimine and upon addition of pimeloyl-CoA two new species were observed by UV-Vis spectroscopy; an intense absorption at 486 nm indicative of formation of the AONS: PLP:L-Ala-OMe quinonoid species and a novel peak at 454 nm which was due to accumulation of the β-ketoacid methyl ester aldimine complex that cannot undergo enzymatic decarboxylation. Taken together it suggests the clever use of the Me-ester substrate mimic prevents decarboxylation and is a useful probe of the AOS mechanism (Fig.\u0026nbsp;2). This Me-ester trick was also recently used by Chun and Narayan and colleagues to stall the SxtA AONS and, in the presence of D\u003csub\u003e2\u003c/sub\u003eO, allow selective a-deuteration of a panel of 24 natural and UAA a-amino ester substrates\u003csup\u003e38\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAnother elegant example of quinonoid trapping is the study of \u003cem\u003eS. paucimobilis\u003c/em\u003e SPT by Ikushiro and colleagues. They initially used an acyl-CoA thioether substrate mimic (S-(2-oxoheptadecyl)-CoA, a nonreactive thioester analogue of palmitoyl-CoA, to \u0026ldquo;trick\u0026rdquo; the SPT PLP:L-Ser complex\u003csup\u003e27\u003c/sup\u003e. This mimic bound to the enzyme and caused a conformational change which led to deprotonation at C-a and PLP:L-Ser quinonoid formation observed at 493 nm. Since the quinonoid could not react with the acyl-CoA thioether the authors used NMR to measure a rate acceleration of C-a deprotonation of more than 100 fold upon binding of the second substrate. This study also supported the over-arching AOS mechanism that we proposed based on our earlier work on the AOS enzyme \u003cem\u003eE. coli\u003c/em\u003e AONS\u003csup\u003e9\u003c/sup\u003e .More in-depth kinetic analyses (both steady-state and stopped flow) of the ALAS enzyme that uses L-Ala and succinyl-CoA have also been reported by Ferreira and colleagues and from these combined studies the key conserved residues involved in AOS catalysis have been proposed\u003csup\u003e13\u003c/sup\u003e. The results presented here are the first to use knowledge of the catalytic mechanism to explore rational mutagenesis of an AOS enzyme and generate a biocatalyst with a much broader substrate specificity. In further studies, we aim to investigate formation, stability and breakdown of these quinonoid species which should provide more detailed insights into the substrate specificity across the whole AOS family.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThermostability and spectroscopic properties of selected ThAOS variants with expanded substrate scope.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHaving obtained a sub-set panel of four competent biocatalyst variants \u003cem\u003evia\u003c/em\u003e saturation mutagenesis at \u003cem\u003eTh\u003c/em\u003eAOS V79 (V79A, V79G, V79L, V79S), we set out to further investigate if the mutations had affected the thermostability of the biocatalyst. This was an important consideration in terms of selecting the best \u003cem\u003eTh\u003c/em\u003eAOS variant that could be used for application in chemical synthesis. Since the \u003cem\u003eTh\u003c/em\u003eAOS is from a thermophilic bacterium we decided to study their respective thermostabilities at elevated temperatures (Fig. S8). The wild type \u003cem\u003eTh\u003c/em\u003eAOS was previously found to be stable to incubation for two hours at 70\u0026deg;C\u003csup\u003e21\u003c/sup\u003e. The \u003cem\u003eTh\u003c/em\u003eAOS V70L variant displayed comparable residual activity (92.1%) to the WT \u003cem\u003eTh\u003c/em\u003eAOS (88.6%), while the V79A variant (77.6%) and V79S (79.4%) mutants were slightly less stable. We then tested how stable the variants were when incubated at 70\u0026deg;C (Fig. S8A). Unfortunately, the \u003cem\u003eTh\u003c/em\u003eAOS V79G variant which exhibited the biggest improvement in substrate scope during the screening process, displayed a significant drop in activity (retaining 56.0%) compared to the wild type biocatalyst. We then analysed how stable the variants were upon incubation at 90\u0026deg;C for 30\u0026ndash;120 mins. The WT was to reduced\u0026thinsp;~\u0026thinsp;15% activity after 120 mins and the V79S, V79G and V79L displayed varying retention of activity (45%, 5% and \u0026lt;\u0026thinsp;5% respectively). However, \u003cem\u003eTh\u003c/em\u003eAOS V79A retained\u0026thinsp;~\u0026thinsp;60% activity after incubation at this elevated temperature. By evaluating both the improved substrate scope and thermostability we then decided to take forward the \u003cem\u003eTh\u003c/em\u003eAOS V79A variant for scaled-up synthesis of a \u003cem\u003eTh\u003c/em\u003eAOS-derived pyrrole product in combination with the KPR.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe\u003c/b\u003e \u003cb\u003eTh\u003c/b\u003e\u003cb\u003eAOS V79 variants are viable biocatalysts with alternative, inexpensive acyl N-acetylcysteamine (SNAc) substrates.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAcross the AOS family it has been shown that the acyl-CoA thioester substrates display low \u0026micro;M kinetic constants and high turnover rates that is ideal for synthesis. However, the high cost of acyl-CoAs prohibits their use in biocatalysis unless this issue can be overcome by inclusion of an acyl-CoA recycling system. This was achieved in our previous work with wild type \u003cem\u003eTh\u003c/em\u003eAOS where we used acyl-CoA synthetase (ACS) to convert the CoASH product back into acyl-CoA (Ashley). Alternatively, a number of simple CoA substrate mimics have been reported that can be used in place of the acyl-CoA.\u003csup\u003e44\u003c/sup\u003e Of these, the most widely-used are thioesters of the low-molecular- weight CoA fragment, N-acetylcysteamine (SNAc, Fig.\u0026nbsp;5). Enzymes capable of using these simple acyl-SNAc thioester substrates in place of an acyl-CoA thioester or pantetheine thioester include thioesterases, ketosynthases, ACP synthases and many others, (reviewed in \u003csup\u003e44\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eTherefore replacing the expensive acyl-CoA substrates of AOS enzymes with SNAc-thioesters would greatly improve the synthetic potential of these biocatalysts in preparing valuable aminoketone products or being combined into chemo- and bio-catalytic cascades. The only issue that could prevent using these substrate mimics is a kinetic penalty, since the acyl-SNAc substrates display very high mM binding constants. An AOS homologue, \u003cem\u003eM. wollei\u003c/em\u003e SxtA, has recently exhibited improved activity with acyl-SNAcs in the presence of a pantetheine-like auxiliary molecule, which mimics the presence of the acyl-CoA pantetheine arm\u003csup\u003e39\u003c/sup\u003e. This work prompted us to explore the use of acyl-SNAc in combination with the newly-discovered \u003cem\u003eTh\u003c/em\u003eAOS V79 variants.\u003c/p\u003e\u003cp\u003eOur hypothesis is based on the proposed AOS catalytic mechanism (Fig.\u0026nbsp;2), that only upon acyl-CoA binding will the enzyme catalyse formation of the nucleophilic quinonoid intermediate. However, since the \u003cem\u003eTh\u003c/em\u003eAOS V79A and V79G variants generate a quinonoid in the presence of the amino acid substrate alone, we hoped that these biocatalysts would react with acyl-SNAc substrates, albeit at high concentrations. Acetyl-SNAc (N,S-diacetylcysteamine) was therefore prepared from cysteamine hydrochloride and acetic anhydride using a published method (see SI). The wild type \u003cem\u003eTh\u003c/em\u003eAOS and three active variants (V79A, V79G and V79S) were screened for Claisen-condensation activity with acetyl-SNAc against a panel of three amino acids (L-Aba, L-Ala and Gly) with the DTNB assay at 50\u0026deg;C (Fig. S9). It is important to note that wild type \u003cem\u003eTh\u003c/em\u003eAOS exhibited no detectable activity with any of the substrate combinations. In contrast, we were delighted to observe activity with all of the \u003cem\u003eTh\u003c/em\u003eAOS variants. The \u003cem\u003eTh\u003c/em\u003eAOS V79A displayed activity with acetyl-SNAc and L-Aba and Gly, the \u003cem\u003eTh\u003c/em\u003eAOS V79G variant was active with L-Aba, L-Ala and Gly and the \u003cem\u003eTh\u003c/em\u003eAOS V79S variant was also active with all three substrates with Gly displaying a turnover of 0.14 min\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe results of this screen allowed us to test the synthetic usefulness of the new reaction using combinations of \u003cem\u003eTh\u003c/em\u003eAOS variants and amino acids with acetyl-SNAc. To do this we coupled the AOS biocatalytic reaction with the KPR (at elevated temperature) as we had done previously. The reaction was performed in the presence of methyl acetoacetate (MAA), with the aim of capturing the aminoketone product as a pyrrole, \u003cb\u003e1\u003c/b\u003e (Fig.\u0026nbsp;5). The three biocatalysts were screened using high catalyst loadings (5 mgmL\u003csup\u003e-1\u003c/sup\u003e) with Gly, acetyl-SNAc and MAA (32 mM each) at a variety of elevated temperatures for 16 hours. Reactions were analysed by HPLC as described in our original paper\u003csup\u003e36\u003c/sup\u003e. The results showed that \u003cem\u003eTh\u003c/em\u003eAOS V79A worked best with Gly and acetyl-SNAc at 60 \u003csup\u003eo\u003c/sup\u003eC with 35% analytical yield of pyrrole 1 (Fig.\u0026nbsp;5). We explored if the pyrrole yield could be increased with the higher loading of acetyl-SNAc (32\u0026ndash;800 mM). However, no significant increase in yield was observed (Fig. S10).\u003c/p\u003e\u003cp\u003eSince the \u003cem\u003eTh\u003c/em\u003eAOS V79A variant was the most thermostable under long incubation times at elevated temperatures (Fig. S8) it was used in a preparative scale reaction (10 mL, 32 mM glycine, 32 mM acetyl-SNAc, 32 mM MAA, 15 mg/mL \u003cem\u003eTh\u003c/em\u003eAOS V79A, 100 mM HEPES, pH 7.5, 16 hrs). The pyrrole product \u003cb\u003e1\u003c/b\u003e was extracted and purified and its structure confirmed using NMR (Fig. S11 and S12). Taken together, this result shows that the rationally engineered \u003cem\u003eTh\u003c/em\u003eAOS V79A variant could be used to prepare various products using inexpensive acyl-thioester starting materials.\u003c/p\u003e\u003cp\u003e\u003cb\u003eX-ray structure of the improved, engineered\u003c/b\u003e \u003cb\u003eTh\u003c/b\u003e\u003cb\u003eAOS V79A in complex with the inhibitor L-Pen.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInspired by the success of the engineering campaign to deliver improved variants of \u003cem\u003eTh\u003c/em\u003eAOS at residue V79, we endeavored to crystallise the variants and solve their crystal structures with bound ligands. This would allow a comparison with the wild type \u003cem\u003eTh\u003c/em\u003eAOS and might provide molecular insight into the origin of the broadened substrate scope of the engineered variant. Screening revealed that \u003cem\u003eTh\u003c/em\u003eAOS V79A gave crystals that were not suitable for structural studies and we also screened unsuccessfully with various natural amino acid and UAA substrates that came from the screens (Fig. S3).\u003c/p\u003e\u003cp\u003eAs a final probe to study ligand binding we used the well known PLP enzyme inhibitor L-penicillamine (L-Pen) and monitored the reaction using UV-vis spectroscopy. We used L-Pen since we had studied the related AOS enzyme \u003cem\u003eS. paucimobilis\u003c/em\u003e SPT described by Lowther \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e45\u003c/sup\u003e. The D-Pen enantiomer is an FDA-approved drug used for the treatment of Wilson\u0026rsquo;s disease and also binds specifically to PLP enzymes\u003csup\u003e46\u003c/sup\u003e. Incubation of L-Pen (1 mM) to the wild type and both \u003cem\u003eTh\u003c/em\u003eAOS V79A and V79G variants rapidly decolourised both reactions and led to the formation of a broad peak between 310\u0026ndash;380 nm, with an absorbance maximum at 330 nm (Fig. S13 A, B and C). This data is consistent with the formation of a covalent \u003cem\u003eTh\u003c/em\u003eAOS PLP:L-Pen ring-closed thiazolidine adduct and this was refined to an excellent fit and geometry. We subsequently used the L-Pen ligand in structural studies and when \u003cem\u003eTh\u003c/em\u003eAOS V79A crystals were soaked with L-Pen, prior to flash cooling and data collection, clear ligand density was observed in each active site (Fig.\u0026nbsp;6A). The crystal structure was determined to 1.5 \u0026Aring; resolution in P1 space group using the wild type \u003cem\u003eThA\u003c/em\u003eOS structure (PDB ID: 7POA) as a molecular replacement model. The asymmetric unit contained two chains forming the stable dimer, and the final model refined to an Rwork of 0.16 and Rfree of 0.19 (Fig. S14). The data collection and refinement statistics are shown in Table S7.\u003c/p\u003e\u003cp\u003eL-Pen was captured in the active site of \u003cem\u003eTh\u003c/em\u003eAOS V79A in a conformation typical of amino acids co-crystallised in the active site of AOS enzymes. The L-Pen substrate amine is covalently linked to the PLP cofactor, and the substrate carboxylate is chelated by Arg366. The five-membered ring of the inhibitory thiazolidine intermediate is clearly resolved, and sits in the position of an amino acid substrate sidechain. Most interesting is the distinction between WT \u003cem\u003eTh\u003c/em\u003eAOS and the engineered, more active variant, \u003cem\u003eTh\u003c/em\u003eAOS V79A. For this reason, the structure of \u003cem\u003eTh\u003c/em\u003eAOS internal aldimine was overlaid onto the structure of L-Pen-bound \u003cem\u003eTh\u003c/em\u003eAOS V79A. Despite a long history of use as an inhibitor of PLP-dependent enzymes, this is the first published crystal structure of a PLP enzyme in complex with \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Pen.\u003c/p\u003e\u003cp\u003eThe two chains of the \u003cem\u003eTh\u003c/em\u003eAOS V79A variant align with around 0.4 \u0026Aring; RMSD Cα to wild type \u003cem\u003eTh\u003c/em\u003eAOS structure over the 397 residues. There is no significant structural change in the active site in the V79A variant with PLP:L-Pen bound when compared to the wild type PLP-bound \u003cem\u003eTh\u003c/em\u003eAOS, with no shift in the loop around the altered residue. The distance between A79 and the thiazolidine methyl group and is 4\u0026Aring; and modelling the distance to the native V79 gives a distance of 3 \u0026Aring; to the thiazolidone (Fig.\u0026nbsp;6B). It appears that the removal of the valine side chain increases the volume of the ligand binding site without any large-scale remodelling of this region.\u003c/p\u003e\u003cp\u003e\u003cb\u003eComparison of penicillamine inhibitor binding in structural homologues.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSearching the PDB for the L- and D- forms of the penicillamine ligand identifies two unpublished structures of cysteine desulfurase (CSD) enzymes with structural homology to \u003cem\u003eTh\u003c/em\u003eAOS with bound penicillamine ligand: NifS from \u003cem\u003eHelicobacter pylori\u003c/em\u003e (PDB ID: 7XES); and SufS from \u003cem\u003eBacillus subtilis\u003c/em\u003e (PDD ID: 7XEN). There is also a structure of the SufS without penicillamine (PDB ID: 7XEN). The monomers of these two proteins align to ThAOS-V79A with overall RMSD Cα of 1.3 \u0026Aring; (7XES) and 1.2 \u0026Aring; (7XEN) (Fig. S15A). The primary structural difference between ThAOS and the cysteine desulfurase enzymes is in the position of the N-terminal region of these proteins and an extended beta-elbow that is present in the cysteine desulfurases (Fig. S15A). In \u003cem\u003eTh\u003c/em\u003eAOS-V79A, the first 40 amino acids adopt an alpha helix and extended loop arrangement, forming a large interface with the partner chain (Fig. S15B); whereas, in the cysteine desulfurase enzymes the N-terminal region is shorter; and, in the case of the \u003cem\u003eB. subtilis\u003c/em\u003e enzyme, it forms a twisted helix that mainly participates in interactions with its own chain (Fig.\u0026nbsp;15). These regions are shifted by a rotation of 110\u0026deg; between the cysteine desulfurase enzymes and \u003cem\u003eTh\u003c/em\u003eAOS. The beta-elbow region in the cysteine desulfurase enzymes participates in the dimerisation interface and due to this additional region, the quaternary arrangement of these enzymes differs from \u003cem\u003eTh\u003c/em\u003eAOS (Fig.\u0026nbsp;16A-B). It appears that it is the entrance to this cavity that has been engineered in the more active ThAOS V79 mutants.\u003c/p\u003e\u003cp\u003eThe active sites of NifS and SufS both have a bound penicillamine ligand captured in the external aldimine form between the penicillamine and PLP cofactor (Fig. S16A). There position of the PLP and penicillamine is well conserved between the three proteins other than the ring closure to form the thiazolidine in the \u003cem\u003eTh\u003c/em\u003eAOS. There is a clear cavity at the dimer interface in each enzyme where the substrate can bind (Fig.\u0026nbsp;16B). There is only 20% sequence identity between \u003cem\u003eTh\u003c/em\u003eAOS and the bacterial CSDs, and residue conservation in the active site is limited to histidine (H136), and arginine (R366); the active arginine (R243 in \u003cem\u003eTh\u003c/em\u003eAOS) is conserved, but in different sequence positions in the NifS and SufS proteins (Fig. S17). The wider active site and ligand binding regions of the three proteins differ considerably, with distinct ligand binding tunnels in the three enzymes (Fig. S17).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThere is a growing application for biocatalytic routes to both commodity chemicals, as well as high value intermediates and pharmaceuticals.\u003csup\u003e47\u003c/sup\u003e A key step in organic synthesis is C-C formation and a number of enzymes have been developed as biocatalysts. Databases such as RetroBioCat have allowed route developers to plan synthetic strategies using well characterized biocatalysts whose substrate specificity, catalytic rates and methodologies are well curated.\u003csup\u003e48,49\u003c/sup\u003e It is essential to continue to find new natural biocatalysts that can be enhanced by rational engineering and/or directed evolution/selection. Alternatively, protein scaffolds can be engineered to deliver biocatalysts with no known biological equivalent e.g. the Morita Baylis Hillman reaction\u003csup\u003e50\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe thermophilic PLP-dependent biocatalyst \u003cem\u003eTh\u003c/em\u003eAOS catalyses an irreversible, Claisen-like, C-C bond forming reaction that yields useful aminoketone building blocks\u003csup\u003e36\u003c/sup\u003e. Studying the active site structure of this PLP-dependent biocatalyst, combined with knowledge of the enzyme mechanism, has permitted the generation of variant biocatalysts with significantly improved properties. A single active site residue V79 appears to control access to the active site and conversion to smaller residues such as Gly and Ala allows alternative amino acids and UAAs to bind, without compromising the catalytic activity. Saturation mutagenesis, complementing the original rational mutagenesis strategy, allowed variation at this residue to be fully explored. The best mutant catalyses a total of 71 unique condensations between various amino acid and acyl-thioester substrates, significantly greater than any other AOS previously reported. Furthermore, the ability of the hyperactive mutants to accept simple and affordable SNAc thioesters to a useful degree without the need for auxiliary CoA-mimicking compounds is also a first. We demonstrated the usefulness of this reaction by generating a pyrrole by combining the thermo-stable \u003cem\u003eTh\u003c/em\u003eAOS variant in a KPR at elevated temperatures. A key determinant in AOS catalysis is the ability of the enzyme to catalyse the formation of a key PLP-bound external aldimine that subsequently generates the key PLP:amino acid quinonoid nucleophile in the present of the acyl-CoA substrate. A convenient UV-vis screen can rapidly identify hit variants from a library. The \u003cem\u003eTh\u003c/em\u003eAOS V79 variants can generate this reactive species in the absence of the acyl-thioester and can also use truncated cysteamine-derived substrates. In essence we have changed the catalytic mechanism to being acyl-CoA independent. It is clear that this V79 residue, which is found on a dynamic loop, plays an important role and recent work on the related Alb29 AOS suggests other residues in this part of the enzyme could be modified to further expand the substrate scope of AOS\u003csup\u003e51\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe crystal structure of the \u003cem\u003eTh\u003c/em\u003eAOS V79A variant with a PLP:L-Pen inhibitor bound displayed little change compared to the wild type enzyme so the enhanced properties must be due to subtle and dynamic changes to the structure and electronics of the PLP-bound transition states. This merits future study on the AOS family of enzymes by molecular dynamics and modelling. This work opens the door for the exploitation of members of the expanding AOS family as synthetically useful C-C bond-forming biocatalysts, in the same vein as aldolases, transaminases, racemases, and other widely-used PLP-dependent enzymes.\u003c/p\u003e"},{"header":"Materials","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCommercially available standards, solvents and reagents were purchased from Avanti Lipids, Fluorochem, Sigma Aldrich, Cambridge BioScience and Thermo Fisher Scientific and were used without any further purification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme Expression and Purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of \u003cem\u003eTh\u003c/em\u003eAOS constructs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA single colony of \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) cells containing a pET28a plasmid encoding \u003cem\u003eTh\u003c/em\u003eAOS wild type and V79 variants with a TEV-cleavable N-terminal His6 tag was used to inoculate a 5 mL overnight culture of L.B. containing 30 ug/mL kanamycin. After overnight shaking at 37 \u0026deg;C the culture was used to inoculate larger cultures of 1L L.B. media in 2L Erlenmeyer flasks, which were then grown with shaking at 180 rpm at 37 \u0026deg;C until the OD500 was 0.6-0.8. The culture was then induced with 0.25 mM IPTG overnight at 16 \u0026deg;C. Cells were harvested by centrifugation and cell pellets were stored at -20 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHeat Purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell pellets were resuspended in HEPES buffer (20 mM HEPES, 150 mM NaCl, 5% glycerol, pH 7.5, ~30 mL) before being sonicated for 15 minutes 30s on/30s off. Cell debris was pelleted with ~10,000xg centrifugation for 45 minutes. The clarified cell lysate was next heated in a water bath at 80 \u0026deg;C for 30 minutes with monitoring by SDS-PAGE. After all the \u003cem\u003eE. coli\u003c/em\u003e proteins had precipitated, precipitate was pelleted once more with another centrifugation step. The solution was then filtered, protein concentration was determined using the Bradford assay and was used without further purification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmobilsed metal affinity purification (IMAC) Ni\u003csup\u003e2+\u003c/sup\u003e purification of \u003cem\u003eTh\u003c/em\u003eAOS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell pellets were resuspended in HEPES buffer (~30 mL) before being sonicated for 15 minutes 30s on/30s off. Cell debris was pelleted with ~10,000xg centrifugation for 45 minutes. The mixture was then loaded onto a 5 mL G.E. Healthcare HisTrap FF column prior to a gradient elution/fractionation step with Nickel Elution Buffer (HEPES buffer + 500 mM imidazole). The purest fractions (determined by SDS-PAGE) were then buffer-exchanged back into HEPES buffer and enzyme concentration was determined using the Bradford assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFull Purification for structural studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell pellets were treated as according to the Ni\u003csup\u003e2+\u003c/sup\u003e IMAC Purification, but after the IMAC step yellow fractions were pooled and loaded onto a pre-equilibrated Superdex S200 column, before elution and fractionation with 120 mL HEPES buffer, yielding highly pure mutant enzyme. Samples were concentrated to 20-50 mg/mL stocks, flash-frozen in liquid N\u003csub\u003e2\u003c/sub\u003e and stored at -80 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzymatic assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe activity assay of \u003cem\u003eTh\u003c/em\u003eAOS and its variants were performed using DTNB assay. The reactions were performed in 60\u0026nbsp;mL scale using amino acid (16 mM), acetyl-CoA (1 mM), ThAOS/ThAOS variants (0.1-1 mg mL\u003csup\u003e-1\u003c/sup\u003e) and DTNB (1 mM) in HEPES buffer (20 mM, pH 7.5) at 50 \u0026deg;C. The UV-vis readings were recorded using a BioTek Synergy HT microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemo-biocatalytic Knorr Pyrrole Reaction (KPR) Cascades\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eTh\u003c/em\u003eAOS (0-3 mgmL\u003csup\u003e-1\u003c/sup\u003e) biocatalyst was mixed with amino acid (16-32 mM), methyl acetoacetate (MAA, 32 mM from a 3.2 M stock in MeCN), sodium benzoate (1 mM, internal standard) and acyl-CoA (0-2 mM) or acetyl-SNAc (0-32 mM from a 100\u0026times; concentrated stock in MeCN) in HEPES buffer pH 7.5 buffer at a final volume of 200 \u0026micro;L in an Eppendorf tube. Reactions were performed in a Grant-Bio 24-well thermoshaker with shaking at 250 rpm at 30-90 \u0026deg;C for 0-24h. Reactions were initiated by addition of acyl-CoA. Reactions were terminated by addition of 1 volume of MeCN, and centrifugation for 10 minutes at 13,000 \u003cem\u003eg\u003c/em\u003e. The supernatant was then analysed by HPLC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUV-Vis spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe UV-vis spectrum of the wild type \u003cem\u003eTh\u003c/em\u003eAOS and V79 variants in the PLP bound state and in the presence of amino acid and acyl-CoA substrates were recorded on a Cary 50 UV vis spectrophotometer with 1 cm pathlength cuvettes. The spectrophotometer was blanked against buffer, and absorbance intensity was recorded between 250-600 nm on the Fast setting. For titrations, substrate was added to the cuvette from a 100 mM stock in buffer, mixed by pipetting and allowed to equilibrate for 30 seconds before recording of the new spectrum. The dilution was accounted for in the final spectra by amplifying the new spectra by the dilution factor caused by addition of the substrate. The whole spectrum was captured and changes in the absorbance maximum of the dominant peaks were plotted and analysed in OriginLab 2019.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein Crystallisation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCrystallisation of recombinant \u003cem\u003eTh\u003c/em\u003eAOS V79 mutants was initially screened using commercial kits (Molecular Dimensions and Hampton Research). Protein concentration was 20-25 mg.mL\u003csup\u003e-1\u003c/sup\u003e. The drops, comprising 0.1 or 0.2 \u0026micro;L of protein solution plus 0.1 \u0026micro;L of reservoir solution, were set up using a Mosquito crystallisation robot (SPT Labtech). The experiments were incubated at 20 \u0026deg;C. Initial hits were of good size, single and could be directly tested. Whilst hits were found in Index (Hampton Research), three conditions were found in Morpheus (A8, A12 and C8, Molecular Dimensions) to lead to alternative crystal space groups. \u003cem\u003eTh\u003c/em\u003eAOS V79A crystallised in P1 (30 mM sodium nitrate, 30 mM sodium phosphate, 30 mM ammonium sulfate, 100 mM HEPES/MOPS pH 7.5, 12.5% (w/v) PEG1000 and 12.5% (w/v) PEG3350), P21 (30 mM magnesium chloride, 30 mM calcium chloride, 100 mM HEPES/MOPS pH 7.5, 12.5% (w/v) PEG1000 and 12.5% (w/v) PEG3350) and in P212121 (30 mM magnesium chloride, 30 mM calcium chloride, 100 mM Tris/bicine pH 8.5, 12.5% (v/v) MPD, 12.5% (w/v) PEG1000 and 12.5% (w/v) PEG3350). The samples did not require optimisation of additional cryo-protection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of N,S-diacetylcysteamine (acetyl-SNAc). N,S-diacetylcysteamine (acetyl-SNAc)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCysteamine hydrochloride (2.11 g, 18.8 mmol) was dissolved in water (20 mL) at 0 \u0026deg;C, and the pH was adjusted to 8.0 with aqueous KOH (8 M). Acetic anhydride (5.72 g, 56.1 mmol) was next added dropwise, and the pH was adjusted to 7.0 with aqueous KOH (8 M). The solution was stirred at 0 \u0026deg;C for 90 minutes, until addition of a drop to a solution of Ellman\u0026rsquo;s reagent DTNB did not cause a colour change. The solution was then extracted with CH2Cl2 (3\u0026times;). The organic phase was then washed with acidified water (3\u0026times;), dried with magnesium sulfate and concentrated by rotary evaporation to afford the title compound as a viscous and colourless liquid (0.156 g).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNMR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 1H and 13C NMR spectra were recorded on a Bruker Avance III 500 MHz or a Bruker CryoProbe Prodigy 500 MHz, and the solvent was CDCl\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1H NMR\u003c/strong\u003e (500 MHz, CDCl3): \u0026delta;H 5.95 (1H, broad s, NH), 3.46 (2H, t, J = 10 Hz, -NHCH2CH2S-), 3.04 (2H, t, J = 10 Hz, -NHCH2CH2S-), 2.37 (3H, s, -SC(O)CH3), 1.99 (3H, s, -NHC(O)CH3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e13C NMR\u003c/strong\u003e (101 MHz, CDCl3): \u0026delta;C 196.3, 170.3, 39.6, 30.7, 28.9, 23.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Collection, Structure Solution, Model Building, Refinement and Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiffraction data were collected at the synchrotron beamline I04 of Diamond light source (Didcot, U.K.) 07/07/2021 at a temperature of 100 K.\u0026nbsp;The data set was integrated with XIA2\u003csup\u003e52\u003c/sup\u003e. using XDS\u003csup\u003e53\u003c/sup\u003e and scaled with Aimless\u003csup\u003e54\u003c/sup\u003e.\u0026nbsp;The space group was confirmed with Pointless\u003csup\u003e55\u003c/sup\u003e. The phase problem was solved by molecular replacement with Phaser\u0026nbsp;\u003csup\u003e56\u003c/sup\u003e using \u003cem\u003eTh\u003c/em\u003eAOS structure\u0026nbsp;as the search model (PDB: 7POA)\u003csup\u003e36\u003c/sup\u003e.\u0026nbsp;The model was refined with refmac\u003csup\u003e57\u003c/sup\u003e. The PLP:L-Penicillamine ligand was generated using JLigand\u003csup\u003e58\u003c/sup\u003e and optimised with AceDRG\u003csup\u003e59\u003c/sup\u003e. Manual model building with COOT\u003csup\u003e60\u003c/sup\u003e was intercalated between refinement rounds.\u0026nbsp;The models were validated using Coot and Molprobity\u0026nbsp;\u003csup\u003e61\u003c/sup\u003e. Other software used were from CCP4 cloud\u003csup\u003e62\u003c/sup\u003e and CCP4 suite\u003csup\u003e63\u003c/sup\u003e. Figures were made with Chimerax\u003csup\u003e64\u003c/sup\u003e. Data collection processing and refinement statistics are presented in Table S7.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein structure raw data files (MTZ and PDB) are available from the author. HPLC, NMR, kinetic and UV-vis data are also available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the BBSRC for an EastBio (BB/J01446X/1) PhD studentship (B.A.) The BBSRC is thanked for grant funding awarded to D.J.C. (BB/T016841/1) to support S.M. The University of Edinburgh and the Derek Stewart Charitable Trust is thanked for PhD studentship funding (M.S.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB.A. and D.J.C. designed the project with respect to engineering the \u003cem\u003eTh\u003c/em\u003eAOS biocatalyst and isolating improved variants. B.A. designed, expressed and purified the \u003cem\u003eTh\u003c/em\u003eAOS variants. B.A. also assayed the actitivities with the reported substrates. Y.Z. prepared and characterized \u003cem\u003eTh\u003c/em\u003eAOS V79 variants. S.M. carried out characterization of the ThAOS V79 variant and scaled up reactions. M.S. prepared the acyl-SNAC substrate and carried out the isolation of pyrrole targets. A.B. isolated crystals of the \u003cem\u003eTh\u003c/em\u003eAOS V79A PLP:L-Pen complex and acquired data from the Diamond Light Source. A.B. and J.M.-W. solved the structure of \u003cem\u003eTh\u003c/em\u003eAOS V79APLP: L-Pen complex and deposited the structure in the Protein DataBank (PDB). B.A. wrote the initial manuscript. All authors made written contributions to the manuscript and prepared figures and tables. D.J.C. edited and wrote the final version of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information.\u003c/strong\u003e The online version contains supplementary material. Synthesis, sequences \u0026amp; mutagenesis, UV-vis absorption spectra, kinetic studies, ESI-MS spectra, enzyme mutant activity screen, and NMR data. \u003cem\u003eTh\u003c/em\u003eAOS_V79A structures are provided in the supplementary information file.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBell, E. L. \u003cem\u003eet al.\u003c/em\u003e Biocatalysis. Nature Reviews Methods Primers 1, 46 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuller, R. \u003cem\u003eet al.\u003c/em\u003e From nature to industry: Harnessing enzymes for biocatalysis. Science 382, eadh8615 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'Connell, A. \u003cem\u003eet al.\u003c/em\u003e Biocatalysis: landmark discoveries and applications in chemical synthesis. Chem Soc Rev 53, 2828\u0026ndash;2850 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSheldon, R. A. \u0026amp; Brady, D. 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Sci. 30, 70\u0026ndash;82 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4345858/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4345858/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCarbon-carbon bond formation is one of the key pillars of organic synthesis. Green, selective and efficient biocatalytic methods for such are therefore highly desirable. The α-oxoamine synthases (AOSes) are a class of pyridoxal 5’-phosphate (PLP)-dependent, irreversible, carbon-carbon bond-forming enzymes, which have been limited previously by their narrow substrate specificity and requirement of acyl-CoA thioester substrates. We recently characterized a thermophilic enzyme from \u003cem\u003eThermus thermophilus\u003c/em\u003e (\u003cem\u003eTh\u003c/em\u003eAOS) with a much broader substrate scope and described its use in a chemo-biocatalytic cascade process to generate pyrroles in good yields and timescales. Herein, we report the structure-guided engineering of \u003cem\u003eTh\u003c/em\u003eAOS to arrive at variants able to use a greatly expanded range of amino acid and simplified N-acetylcysteamine (SNAc) acyl-thioester substrates. The crystal structure of the improved \u003cem\u003eTh\u003c/em\u003eAOS V79A mutant with a bound PLP:penicillamine external aldimine ligand, provides insight into the properties of the engineered biocatalyst.\u003c/p\u003e","manuscriptTitle":"Rational Engineering of a Thermostable α-Oxoamine Synthase Biocatalyst Expands the Substrate Scope and Synthetic Applicability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-14 16:25:52","doi":"10.21203/rs.3.rs-4345858/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-chemistry","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commschem","sideBox":"Learn more about [Communications Chemistry](http://www.nature.com/commschem/)","snPcode":"","submissionUrl":"","title":"Communications Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7e7b4a53-99cf-4f67-9ceb-b7a30de19788","owner":[],"postedDate":"May 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31335274,"name":"Biological sciences/Chemical biology/Biocatalysis"},{"id":31335275,"name":"Biological sciences/Chemical biology/Chemical libraries/Combinatorial libraries"}],"tags":[],"updatedAt":"2025-03-14T07:11:46+00:00","versionOfRecord":{"articleIdentity":"rs-4345858","link":"https://doi.org/10.1038/s42004-025-01448-8","journal":{"identity":"communications-chemistry","isVorOnly":false,"title":"Communications Chemistry"},"publishedOn":"2025-03-13 04:00:00","publishedOnDateReadable":"March 13th, 2025"},"versionCreatedAt":"2024-05-14 16:25:52","video":"","vorDoi":"10.1038/s42004-025-01448-8","vorDoiUrl":"https://doi.org/10.1038/s42004-025-01448-8","workflowStages":[]},"version":"v1","identity":"rs-4345858","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4345858","identity":"rs-4345858","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}