Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa

Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa | 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 Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa Marco Pedretti, Carmen Fernández-Rodríguez, Carolina Conter, Iker Oyenarte, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3869461/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Apr, 2024 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract The escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (H 2 S), via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa ( Pa CGL). In addition to a canonical L-cystathionine hydrolysis, purified Pa CGL can catalyze the production of H 2 S using L-cysteine and/or L-homocysteine as alternative substrates. Comparative analysis with counterparts in other pathogens and humans revealed distinct structural features within the primary enzyme cavities, including a differently folded entrance loop to the catalytic site, potentially influencing substrate and/or inhibitor access. These findings offer opportunities for developing specific inhibitors to limit or eliminate bacterial H 2 S synthesis, weakening a defense barrier against the host immune system. Health sciences/Diseases/Infectious diseases Health sciences/Diseases/Infectious diseases/Bacterial infection Biological sciences/Biochemistry Biological sciences/Biophysics Biological sciences/Structural biology Biological sciences/Structural biology/X ray crystallography Pseudomonas aeruginosa Cystathionine γ-lyase Hydrogen sulfide Multidrug resistant bacteria Catalytic specificity Crystal structure. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION The diseases caused by bacteria, fungi, or parasites pose a growing problem for today's healthcare system. Infectious and parasitic diseases are among the top ten global causes of mortality, as identified by the World Health Organization [ 1 ]. Of particular concern is the opportunistic Gram-negative bacterium Pseudomonas aeruginosa , which has been designated as a critical priority for study, discovery, and the development of new antibiotics. Infections caused by P. aeruginosa can progress to extensive colonization and be more severe with a higher mortality rate, especially in cystic fibrosis patients and immunocompromised hospitalized individuals [ 2 ]. At present, treating P. aeruginosa infections effectively poses a substantial challenge owing to the bacterium's increasing resistance to numerous clinically available antibiotics. Recent studies have shown that a defense mechanism of bacteria against reactive oxygen species (ROS) and antibiotic-induced oxidative damage relies on the endogenous production of hydrogen sulfide (H 2 S) [ 3 – 6 ]. Based on these findings, inhibiting the endogenous generation of this gasotransmitter has been proposed as a strategy to combat these pathogens [ 3 , 4 , 7 – 12 ], although it is still a matter of debate whether this defensive role applies equally to all bacterial species [ 13 , 14 ]. Therefore, the detailed study and characterization of the enzymes involved in the potential production of H 2 S in these organisms becomes particularly relevant for selecting suitable targets upon which to design new antibiotics. The synthesis of H 2 S in bacteria varies across species and depends on substrate availability and presence of the specific enzymes. Under anaerobic conditions, the primary route for H 2 S production is the sulfate reduction pathway (SRP) involving the reduction of inorganic sulfate (SO 4 2− ) to H 2 S [ 15 ] (Fig. 1 A). However, under aerobic conditions, the production of H 2 S by the SRP is quite inefficient compared to specialized sulfate-reducing bacteria. Therefore, some bacteria can synthetize H 2 S through 3-mercaptopyruvate sulfurtransferase (3-MST) under conditions of sulfur limitation [ 3 ] (Fig. 1 A). The 3-MST enzyme is part of a larger pathway involved in the biosynthesis of cysteine, which also includes other enzymes such as cysteine synthase and O-acetylserine sulfhydrylase. Finally, the third known bacterial mechanism to produce H 2 S is the reverse transsulfuration pathway (RTP), which involves the conversion of L-homocysteine (L-Hcys) to L-cysteine (L-Cys) through two consecutive steps catalyzed by two distinct pyridoxal-5’-phosphate (PLP)-dependent enzymes, the cystathionine b-synthase (CBS) and the cystathionine g-lyase (CGL) (Fig. 1 A). CBS catalyzes a β-replacement reaction in which the hydroxyl group of L-serine (L-Ser) is replaced by L-Hcys, yielding L-cystathionine (L-Cth) and H 2 O (reviewed in [ 16 ]). Next, CGL catalyzes the α,γ-elimination of L-Cth into L-Cys, α-ketobutyrate, and ammonia (Fig. 1 B, reaction 1 ). In addition, many CGL can catalyze β-elimination of L-Cth as a side reaction, producing L-Hcys, pyruvate, and ammonia (Fig. 1 B, reaction 2 ). This reaction is referred to as the β-lyase activity of CGL. Besides these canonical reactions, both CBS and CGL can catalyze the synthesis of H 2 S using L-Cys and L-Hcys as substrates exploiting alternative reactivity (Fig. 1 B, reactions 3 to 7 ). It has recently been demonstrated that inhibitors of human CGL ( Hs CGL) require much higher concentration to achieve inhibition or may entirely lack any efficacy on homolog enzymes encoded by different bacteria [ 4 ]. This is likely due to structural and physico-chemical differences within each CGL enzyme, which could be exploited to develop new antibiotics and/or adjuvants to treat recurrent infections caused by the specific pathogen. Recent studies have revealed the presence of the Cse gene within P. aeruginosa genome encoding for the CGL ( Pa CGL) and that inactivation of the Cse gene leads to a significant reduction of H 2 S production in clinical isolates of P. aeruginosa [ 3 , 4 ]. In this study we expressed, purified, and biochemically characterized Pa CGL as well as solved its crystal structure. We found that Pa CGL can hydrolyze L-Cth via g- as well as β-elimination mechanism (Fig. 1 B, reactions 1,2 ) and, in addition, can catalyze the generation of H 2 S from L-Cys or/and L-Hcys (Fig. 1 B, reactions 3 – 6 ). Structural comparisons to other CGL enzymes and a complementary analysis of Pa CGL using deep learning predictions by AlphaFold2 (AF2) revealed significant structural diversity within the main cavities of the enzymes as well as a differently organized entrance loop that may direct the access of substrates and/or inhibitors into the PLP-containing catalytic center. These findings open new avenues for the design of more selective inhibitors of Pa CGL and provide significant insights on the structural evolution of CGL enzymes across different pathogenic organisms. RESULTS Production and biochemical properties of Pa CGL Pa CGL was overexpressed in E. coli and purified to homogeneity using Ni-NTA chromatography ( Fig. S1 A ). Gel filtration showed a molecular mass of 173 kDa for Pa CGL, suggesting that it adopts a tetrameric structure in solution, in accordance with a monomer molecular mass of ~ 44 kDa ( Fig. S1 B) . The UV-Vis absorption spectrum of Pa CGL at pH 8.0 exhibited, in addition to the protein band centered at 278 nm, a peak at 425 nm that is typical of the ketoenamine tautomer of the internal aldimine (protein-bound PLP) ( Fig. S1 C) . Pa CGL binds ~ 1 mol of PLP/mol of monomer with a K d value for PLP of 0.15 ± 0.01 µM, as calculated by fluorescence titrations of apo- Pa CGL with PLP ( Fig. S1 D) . The apo- Pa CGL displayed substantially decreased thermal stability with a melting temperature (T m ) of 57°C compared to the PLP-loaded holo-enzyme yielding T m of 68°C ( Fig. S1 E) . Enzymatic properties of Pa CGL enzyme Canonical reactions The reaction catalyzed by CGL in the transsulfuration pathway involves elimination at the γ-carbon of L-Cth. However, it is well established [ 17 – 19 ] that CGLs, owing to the chemistry of the catalyzed reaction, exhibit an appreciable cystathionine β-lyase (CBL)-like activity, i.e. , they can cleave both the C–γ–S and C–β–S bonds of L-Cth producing L-Cys or L-Hcys, respectively (Fig. 1 B, reaction 1–2 ). In our first approach to distinguish between these types of elimination reactions catalyzed by Pa CGL (γ- or β-elimination), the reaction products were analyzed by applying LC-MS/MS. As shown in Fig. 2 A, when L-Cth ( m / z = 223) was used as a substrate, both L-Cys ( m / z = 122) and L-Hcys ( m / z = 136) products were observed, consistent with the ability of the enzyme to catalyse both γ-elimination (reaction 1 ) and β-elimination (reaction 2 ) of L-Cth. The kinetics of L-Cth hydrolysis by Pa CGL were then characterized using the DTNB assay. Firstly, we determined the optimal pH and temperature for Pa CGL by measuring the CGL activity at various conditions. Pa CGL displayed its highest enzymatic activity at pH 8.0 and around 42°C ( Fig. S2 ) . However, considering the physiological temperature of 37°C, we opted to conduct enzymatic characterization of Pa CGL at pH 8.0 and 37°C. The obtained enzymatic kinetics for the hydrolysis of L-Cth catalyzed by Pa CGL followed the Michaelis–Menten profile (Fig. 2 B) with k cat and K m values of 5.2 ± 0.2 s − 1 and 0.30 ± 0.04 mM, respectively (Table 1 ). The kinetics of pyruvate formation (reaction 2 ) from L-Cth was also characterized using the LDH assay (Fig. 2 C and Table 1 ). We also monitored the behavior of the enzyme-bound PLP in the presence of L-Cth by absorption and CD spectroscopy (Fig. 2 D, E ) . Addition of L-Cth to P aCGL resulted in significant changes in the spectrum of the enzyme. The most pronounced difference is the presence of two chromophores with absorption in the region of 440–480 nm with positive CD of their absorption bands. For PLP-dependent enzymes involved in γ- and β-elimination reactions, absorbance bands in the region of 440–480 nm usually are assigned to the α-aminocrotonate or α-aminoacrylate intermediates [ 20 , 21 ]. The absorption region of 320–340 nm is not considered because pyruvate produced during the turnover also contributes to this region. Alternative reactions The ability of Pa CGL to produce H 2 S was assessed by examining the various reactions outlined in Fig. 1 B (reactions 3–7 ). The use of LC-MS/MS for product analysis provided direct evidence for all the five potential H 2 S-generating reactions attributed to CGL. When L-Cys served as substrate, we observed the formation of both the L-Ser ( m / z = 106) and L-Lanthionine ( m / z = 209) products, confirming the enzyme's proficiency in catalyzing both L-Cys β-lyase (reaction 3 ) and L-Cys β-replacement (reaction 4 ) activities, respectively (Fig. 3 A). Similarly, in the presence of L-Hcys alone, L-Homoserine ( m / z = 120) and L-Homolanthionine ( m / z = 237) were detected, consistent with γ-elimination (reaction 4 ) and γ-replacement (reaction 5 ) reactions (Fig. 3 B). In the presence of L-Hcys and L-Cys, L-Cth ( m / z = 223) was detected, consistent with a replacement reaction (reaction 7 ) (Fig. 3 C). Next, we determined the kinetics of H 2 S generation from L-Cys or/and L-Hcys. The active site pocket of CGL has binding requirements for two amino acids, since the main substrate of the enzyme, which is L-Cth, is a condensation product of two amino acids, L-Ser and L-Hcys. In the H 2 S-generating reactions catalyzed by PaCGL (Fig. 1 B, reactions 3–7) , either one (reaction 3 and 5 ) or both (reactions 4 , 6 , and 7 ) amino acid binding pockets are occupied. We analyzed the kinetic parameters associated with the single substrate reaction ( i.e. ignoring H 2 O) or bimolecular reactions involving two amino acids, as outlined in the Methods section. The dependence of the rate of H 2 S formation on L-Cys concentration is markedly biphasic, simplifying the deconvolution of kinetic parameters related to two distinct phases corresponding to reactions 3 (β-elimination of L-Cys) and 4 (β- replacement of L-Cys). The enzyme kinetics of pyruvate (reaction 3 ) and of H 2 S (reactions 3 + 4 ) formation from L-Cys are shown in Fig. 3 D and the resulting kinetic parameters are detailed in Table 1 . Notably, Pa CGL exhibits a significant higher affinity for L-Cys binding at site 1 (0.43 ± 0.03 mM) compared to site 2 (33 ± 6 mM), with observed cooperativity in the binding of the second L-Cys molecule (n = 3 ± 1). Interestingly, like many other CGLs [ 19 , 22 – 24 ], Pa CGL also displayed L-Cys inhibition (Table 1 and Fig. 3 D). The dependence of the rate of H 2 S formation on L-Hcys concentration is not as markedly biphasic as observed with L-Cys, even though mass spectrometry data clearly indicate that both L-Homoserine (reaction 5 ) and L-Homolanthionine (reaction 6 ) are produced when L-Hcys serves as a substrate. We reported the kinetic parameters for the overall rate of H 2 S formation (reaction 5 + 6 ) without applying deconvolution analysis of the two phases associated with the single substrate reaction (reaction 5 ) or double substrate reaction (reaction 6 ). The kinetic data for H 2 S production are shown in Fig. 3 E and Table 1 . Analysis of reaction 7 (i.e., the condensation of L-Hcys and L-Cys) was done by monitoring the L-Cth formation through LC-MS/MS, showing that L-Cth increases with increasing L-Hcys concentration (Fig. 3 F). Table 1 Kinetic parameters determined for reactions catalyzed by Pa CGL. Values correspond to the means ± SEMs of at least three independent experiments. Reaction number (Fig. 1 B) k cat (s − 1 ) K m (mM) Ki (mM) k cat / K m (mM − 1 s − 1 ) Hydrolysis of L-Cth a 1 + 2 5.2 ± 0.2 0.30 ± 0.04 17 ± 3 Pyruvate generation from L-Cth b 2 2.2 ± 0.1 0.22 ± 0.01 10 ± 1 H 2 S generation from L-Cys c 3 0.31 ± 0.02 0.43 ± 0.03 16 ± 4 0.7 ± 0.1 4 0.6 ± 0.1 33 ± 6 0.02 ± 0.01 Pyruvate generation from L-Cys b 3 0.5 ± 0.1 3.2 ± 0.6 11 ± 3 0.16 ± 0.06 H 2 S generation from L-Hcys c 5 + 6 0.42 ± 0.04 1.7 ± 0.3 0.25 ± 0.07 a Activity was determined using the DTNB assay. b Activity was determined using the LDH assay. c Activity was determined using the lead acetate assay. Overall structure of Pa CGL The crystal structure of Pa CGL complexed with PLP was solved at 2.0 Å (Fig. 4 ). The few missing or disordered segments not visible in the crystals were predicted by AF2 [ 25 ]. As expected, the overall fold of Pa CGL is consistent with the type-I PLP-dependent enzymes, resembling human, yeast, and bacteria CGLs, as well as enzymes like cystathionine γ-synthase (CGS), or cystathionine β-lyase (CBL) [ 22 , 26 ]. Each Pa CGL subunit consists of 394 amino acids distributed in three domains: (i) an N-terminal domain (residues 1–62), (ii) a central PLP-binding domain (residues 63–260), and (iii) a C-terminal domain (residues 261–394). The N-terminal domain begins with an unstructured segment (residues 1–13), followed by a short α-helix (α0, residues 14–22) and a long-disordered loop (L23-60). However, residues 46–57 are not visible in three out of the four molecules in the asymmetric unit. The PLP-binding domain is built up of a seven-stranded, mostly parallel, β-sheet (↑β1↓β7↑β6↑β5↑β4↑β2↑β3), with strand β7 (residues 219–223) antiparallel to the rest. This domain additionally contains eight α-helices, split in two sets (α1, α2, α5, α6, α7 and α3, α4, α8, respectively) that flank the central β-sheet core at both sides. In our crystals, the short helix α1 (residues 55–60), inserted in the loop L23-60, is only visible in one protein subunit. The PLP-binding domain houses the enzyme’s catalytic center containing PLP covalently anchored to the enzyme via a conserved K208 residue and forming an internal aldimine as confirmed by the spectroscopic analysis ( Fig. S1 C and Fig. 2 C, D). Finally, the C-terminal domain is organized into a five-stranded antiparallel β-sheet (exhibiting the following topology: ↑β8↓β9↑β12↓β11↓β10), decorated with five α-helices (α10–14) on one side which protect it from the solvent. Similarly to all previously characterized CGLs, Pa CGL self-assembles into a homotetramer that can be described as a dimer of dimers that include subunits A-C and B-D, respectively, in which the two subunits of each dimer are related by a 2-fold axis, and the dimers themselves maintain a 2-fold symmetry in relation to each other (Fig. 4 D). The four subunits of the tetramer exhibit high similarity (average rmsd = 0.140 Å), differing only in the conformation of two long loops (L23-60 and L347-370), that appear partially disordered in some of the monomers or present a slightly different conformation. Interestingly, in the crystals both segments adopt a conformation that differs from what was observed in the predicted AF2 model, or in the topological equivalent regions of CGLs from other species. Notably, the loop, L347-370 adopts an extended conformation in PaCGL, diverging from the typical two-turn helix (α13) observed in other homologs ( Fig. S3A ). This structural feature directly impacts the accessibility to the large cleft (chamber-2) located behind the catalytic site (chamber-1) that accommodates the PLP cofactor (Fig. 5 ). Of note, the loop L347-370 region does not display significant sequence conservation among the different Pa CGL homologs. Within helix a13, merely three amino acid residues remain consistent across all sequences-specifically, a proline, an arginine, and a glycine (P357, R361, G365 in PaCGL)-found within the loop´s second segment ( Fig. S3 ). Strikingly, the AF2-predicted Pa CGL model maintains the two-turns helicity fold like other CGL enzymes ( Fig. S3 ) suggesting the probability of two different (and stable) conformations for this segment. Differential conformation of this loop may modulate the accessibility of chamber-2, known to host inhibitors of Staphylococcus aureus CGL ( Sa CGL) [ 4 ] and dictate the specificity of such inhibitors to Pa CGL versus other species. The active site of Pa CGL (Fig. 5 ) is characterized by a deep cavity situated at the dimerization interface between subunits A-B (or equivalently C-D). The PLP-interacting residues, which are well-conserved across CGLs, in Pa CGL are represented by Y110, R372, K208, S337, and S205. K208 is covalently bound to PLP through its ε-amino group and forms a Schiff- base linkage at the C4A position of PLP. The orientation of PLP is fixed by H-bond interactions between its phosphate group and the main chain nitrogen of residues G86 and L87. The hydroxyl group of S205 and T207 also stabilizes the PLP phosphate moiety. The complementary subunit interacts with O2P and O3P of PLP via the guanidine group of R58. Most CGLs also show interactions between the PLP and the conserved tyrosine of the neighboring subunit. In Pa CGL, residue Y56 does not interact with the phosphate moiety of PLP, but rather establishes an H-bond with the N-terminal residue Q45. This contact is possible thanks to the flexibility provided by residue G57 to the polypeptide main chain. Interestingly, residue G57 of Pa CGL is usually substituted by a conserved serine in other CGLs. Finally, the pyridoxal ring of PLP is stacked with the phenol ring of Y110. Chamber-2 of Pa CGL as potential drug binding site The crystal structure of Pa CGL provided valuable insights for comparing critical regions with both bacterial and human counterparts. Special focus was given to the chamber-2, which has been found in Sa CGL to host pharmacological inhibitors [ 4 ]. Chamber-2 can be reached only via a channel with limited accessibility determined by residues from the long loop connecting strands b11-b12 containing helix a13 (loop L347-370 in Pa CGL), and by a tyrosine residue (Y103 in Sa CGL; Y102 in Bc CGL) located in helix a4. Some CGL enzymes lack this tyrosine, which is substituted by either another bulky hydrophobic residue (i.e., F114 in Pa CGL; F101 in Lp CGL) (Fig. 6 ), or alternatively an asparagine (N137 in Tg CGL; N118 in Hs CGL). The crystal structures of Sa CGL, obtained in complex with novel pharmacological inhibitors (named NL1, NL2, and NL3) revealed the significance of the residue Y103 in Sa CGL catalysis and its role in stabilizing the inhibitors within the cavity through a π-stacking interaction [ 4 ]. The Y103A mutation of Sa CGL, or even the "humanized" Y103N variant, eliminated the H 2 S production activity of Sa CGL and disrupted the interaction with the NL1 and NL2 inhibitors [ 4 ]. To assess the role of the equivalent chamber-2 residue F114 in Pa CGL, we replaced it with alanine or asparagine and compared the resulting variants with the wild type protein. The two mutations did not impair the overall structural properties as well as the tetrameric oligomerization of the purified mutant enzymes ( Fig. S4A-C ). Interestingly, both the F114A and the "humanized" F114N mutations resulted in only a slight reduction in enzyme’s catalytic efficiency for both the canonical and H 2 S-producing activities of Pa CGL (Table 2 and Fig. S4D ). This reduction is mainly attributed to decreases in k cat values, while K m remained similar to the wild type. Table 2 Kinetic parameters for reactions catalyzed by Pa CGL F114A and F114N variants. Values correspond to the means ± SEMs of at least three independent experiments. Reaction number (Fig. 1 B) k cat (s − 1 ) K m (mM) Ki (mM) k cat / K m (mM − 1 s − 1 ) Hydrolysis of L-Cth a 1 + 2 Wild type 5.2 ± 0.2 0.30 ± 0.04 17 ± 3 F114A 3.2 ± 0.2 0.6 ± 0.1 5.3 ± 1.2 F114N 3.2 ± 0.1 0.5 ± 0.1 6.4 ± 1.5 H 2 S generation from L-Cys b Wild type 3 0.31 ± 0.02 0.43 ± 0.03 16 ± 4 0.7 ± 0.1 4 0.6 ± 0.1 33 ± 6 0.02 ± 0.01 F114A 3 0.13 ± 0.01 0.49 ± 0.01 20 ± 6 0.27 ± 0.03 4 0.3 ± 0.1 33 ± 8 0.0091 ± 0.005 F114N 3 0.38 ± 0.06 1.6 ± 0.3 17 ± 6 0.24 ± 0.08 4 0.5 ± 0.1 23 ± 3 0.02 ± 0.01 a Activity was determined using the DTNB assay. b Activity was determined using the lead acetate assay. Two additional amino acids situated at the base of the chamber-2 have been suggested to define the connecting gate between chamber-2 and the catalytic site (chamber-1). The first is a conserved histidine (H339 in Sa CGL; H356 in Hs CGL; H338 in Bc CGL; H337 in Lp CGL; and H376 in Tg CGL) (Fig. 6 ). Pa CGL also contains an equivalent histidine in its amino acid chain (H353 in Pa CGL), but its spatial location differs from that found in other CGLs due to the more extended conformation of the loop L347-370, and the topological position of H339 in Sa CGL is occupied by the M351 in Pa CGL structure (Fig. 6 A). The second residue connecting chamber-1 and chamber-2 is a conserved tyrosine that packs against the pyridine ring of PLP and helps to orient the cofactor (Y99 in Sa CGL; Y114 in Hs CGL; Y98 in Bc CGL; Y97 in Lp CGL; Y133 in Tg CGL; and Y110 in Pa CGL, respectively). The volume and access of chamber-2 differ significantly in Sa CGL and Pa CGL (Fig. 6 B). In apo- Hs CGL (no PLP cofactor present, PDB ID 3ELP), this tyrosine (Y114) appears displaced towards the bottom of the chamber-2 cavity, due to a partial unwinding of the last turn of helix α4. Upon binding of PLP (holo- Hs CGL), the last turn of this same helix recovers its helicity and reorients the tyrosine towards the interior of the catalytic chamber-1. This conformational change is thought to function as an access gate from chamber-2 to the catalytic site. DISCUSSION We conducted a biochemical and structural characterization of the H 2 S-producing enzyme CGL from P. aeruginosa . Our results clearly show an enzymatic competence of Pa CGL to generate H 2 S using alternative substrates in addition to the canonical hydrolysis of L-Cth. Detailed structural comparison of Pa CGL with CGL enzymes from other species has revealed distinctive structural features within the primary cavities of the enzyme, which may modulate the access of substrates and/or inhibitors. The reaction catalyzed by CGL in the transsulfuration pathway involves elimination at the γ-carbon of L-Cth. However, CGLs are prone to β-elimination of L-Cth as a side reaction. Our kinetic analysis demonstrated that Pa CGL can catalyze both the α, γ- and α, β-cleavage of L-Cth to yield L-Cys and L-Hcys, respectively. Absorbance in the region of 440–480 nm in the UV-Vis and CD spectra in the presence of L-Cth provided support for the formation of α-aminocrotonate and α-aminoacrylate, the reaction intermediates of the γ- and β-elimination mechanism, respectively. Our spectra of the enzyme-substrate complex in the long-wavelength region are similar to spectra of the Citrobacter freundii methionine γ-lyase complex with methionine [ 27 , 28 ]. Two overlapping absorption bands with maxima at ~ 460 and ~ 485 nm were also observed in the yeast CGL [ 29 ]. The absorption at 480 nm likely corresponds to the formation of α-aminocrotonate, an essential intermediate of the γ-elimination reaction [ 29 ], while absorption at around 460 nm is probably due to α-aminoacrylate formation, which has been observed under steady-state conditions for β-elimination reactions of different PLP-dependent enzymes [ 29 – 31 ]. In addition, Pa CGL exhibited a notably high catalytic efficiency for the hydrolysis of L-Cth (17 mM − 1 s − 1 ), surpassing the activities of CGLs from other organisms ( Lb CGL, 1.1 mM − 1 s − 1 ; Tg CGL, 2.2 mM − 1 s − 1 ; Bc CGL, 3.2 mM − 1 s − 1 ; Sc CGL, 2.1 mM − 1 s − 1 ; Hs CGL, 8.2 mM − 1 s − 1 ) [ 17 , 19 , 22 , 24 , 32 , 33 ]. Recombinant purified Pa CGL also produces H 2 S using L-Cys or/and L-Hcys as alternative substrates. This aligns with the previous reports showing that inactivation of the Cse gene, encoding for the Pa CGL, leads to a significant reduction of H 2 S production in clinical isolates of P. aeruginosa [ 4 ]. We investigated various reactions catalyzed by Pa CGL that lead to H 2 S biogenesis and, as side products, the uncommon thioether-bond containing amino acids L-Lanthionine and L-Homolanthionine. We found that Pa CGL efficiently produced H 2 S via different mechanisms such as (i) a β -elimination reaction where L-Cys was degraded to form H 2 S and L-Ser, (ii) a β -replacement reaction where two molecules of L-Cys were condensed to generate H 2 S and L-Lanthionine, (iii) a γ -elimination reaction where L-Hcys was degraded to form H 2 S and L-Homoserine, (iv) a γ -replacement reaction where two molecules of L-Hcys were condensed to generate H 2 S and L-Homolanthionine, and (v) the replacement of L-Hcys and L-Cys producing H 2 S and L-Cth. Under substrate saturating conditions, the catalytic efficiency ( k cat / K m ) for the H 2 S elimination from L-Cys and L-Hcys is approximately 25- and − 68-fold lower, respectively, than for the canonical hydrolysis of L-Cth (Table 1 ). However, the occurrence and regulation of these alternative reactions in the cell remain unknown. The bacterial intracellular concentrations of L-Cys are tightly regulated during biosynthesis. Notably, P. aeruginosa displays a high redundancy in L-Cys production. In addition to the CGL and CBS enzymes in the RTP, this pathogen possesses genes encoding the enzymes for the de novo L-Cys synthesis pathway, i.e., serine acetyltransferase (SAT) catalyzing the condensation of L-Ser and the acetyl group of acetyl-CoA to form O-acetylserine (OAS), and the cysteine synthase (CS), which catalyzes the nucleophilic attack of sulfide (H 2 S) on OAS to form L-Cys and releasing acetate. L-Cys is also known to participate in the allosteric inhibition of SAT, leading to the production of OAS. OAS, in turn, is converted into N-acetylserine, an auto-inducer of the transcription regulator (the CysB protein), which acts as a sensor and regulator of the intracellular content of L-Cys and sulfur [ 34 ]. Further studies are required to determine whether Pa CGL serves as the primary checkpoint in the RTP in P. aeruginosa and its involvement in L-Cys production. Shatalin et al [ 4 ] identified three potential CGL enzyme inhibitors, NL1, NL2, and NL3, which demonstrated strong specificity against bacterial CGL, with no impact on mammalian CGL. Experiments on the co-crystallization of these inhibitors with S. aureus CGL made it possible to determine the binding sites of all three inhibitors to the enzyme. Our crystal structure of Pa CGL has enabled us to compare essential regions of this enzyme (chamber-1 and chamber-2) with the corresponding regions in other bacterial counterparts (including S. aureus ) and the human enzyme. This analysis revealed distinct structural characteristics that set Pa CGL apart, potentially paving the way for future drug development targeting this important metabolic enzyme. While chamber-1, corresponding to the catalytic site, is highly conserved among PLP-dependent enzymes, chamber-2 has unique physical-chemical properties and distinct conformation compared to other CGLs (Fig. 6 ). An intriguing difference in the chamber-2 of Pa CGL is the presence of the residue F114 (Fig. 6 A) which occupies equivalent position of the conserved residue Y103 in Sa CGL (Fig. 6 B). Strikingly, the F114A mutation in Pa CGL did not result in loss of enzyme activity, as it occurred for the equivalent Y103A mutation in Sa CGL, supporting the notion that this residue not only determines the general characteristics of the entrance channel to chamber-2 (volume, size, steric hindrance), but modulates the overall volume of the chamber and, consequently, the type of molecule that can be accommodated within the chamber-2. In addition, the conformation of a long loop containing helix α13 (L347-370 in Pa CGL) in important for accessibility of the chamber-2 (Fig. 5 ). This loop determines the width of the chamber-2 entrance and affects the void volume of the entire cleft ( Fig. S5 ). Interestingly, our crystal structure of Pa CGL revealed that the helicity of this loop is partially lost, resulting in a more extended peptide segment that allows for a wider access into chamber-2 (a comparison of the internal cavity volume for chamber-2 and the fold of the equivalent regions to loop L347-370 in CGLs from different organisms is shown in Fig. S5 ). However, despite the cavity opening being bigger, the reorientation and shape of the loop L347-370 in Pa CGL reconfigured the internal contour and consequently, made the volume of the cavity smaller compared to what was observed, for example, in Sa CGL (loop L332-356) or the human enzyme (loop L349-373) (Fig. 6 and Fig. S5 ). The comparison of the crystal structures of Pa CGL and Sa CGL complexed with NL1, NL2, and NL3 inhibitors suggests that the distinct arrangement of Pa CGL observed in this region would not hinder the binding of these molecules to Pa CGL, requiring only minor shifts of side chain to accommodate them inside. This is consistent with the inhibitory effectiveness of these molecules in P. aeruginosa [ 4 ]. The Pa CGL model predicted with AF2 ( Fig. S5 ) exhibited a helical conformation of the loop L347-370 like that of other CGLs but different from the conformation found in our Pa CGL crystals. This suggests that the opening and closing of the chamber-2 cavity may differ from what was proposed based on the apo- and holo-states of the human enzyme. As demonstrated in Fig. 6 , Fig S5, and Movie S1 , the increased helicity of this loop in the Pa CGL model predicted by AF2 corresponds to a closed conformation of the chamber-2. In this state, the cavity significantly restricts its accessibility and internal volume. In the AF2-predicted closed conformation of Pa CGL, H353 residue would occupy a position like that found in other CGLs. On the other hand, the extended conformation observed in the crystals made the chamber-2 larger, thus likely more accessible for small molecules, such as NL1, NL2, and NL3 inhibitors. Interestingly, in this open state observed in the crystals, residue M351 occupies the equivalent position to the conserved histidine in other CGLs, suggesting a role in modulating the type of molecule that can be hosted inside the cavity. Overall, our findings revealed the fundamental structural traits of the P. aeruginosa CGL enzyme, which has gained significant attention as a potential pharmacological target due to its role in H 2 S biogenesis in this emerging and concerning pathogen. EXPERIMENTAL SECTION Protein production Gene sequence encoding for Pa CGL (PAO1_PA0400) with a N-terminal 6xHis-Tag was synthesized by Genscript, PCR amplified and cloned into a modified pET28a expression vector (Novagen). The F114A and F114N point mutations were introduced by site-directed mutagenesis using QuikChange II Kit (Agilent), using the primers in Table S1 . All constructs were verified by DNA sequencing performed by Eurofins Genomics. The Pa CGL constructs were transformed into E. coli Rosetta (DE3) expression host cells (Novagen). Cells were grown in Luria-Bertani medium at 37°C to a turbidity of 0.6–0.8 at 600 nm. Expression was induced with 0.5 mM IPTG for 16 h at 24°C. Cells were harvested and resuspended in 20 mM sodium phosphate pH 8.0, 150 mM NaCl, 0.1 mM DTT containing a protease inhibitor cocktail EDTA free. After sonication, the suspension was centrifuged at 30,000x g for 20 min at 4°C. The supernatant was recovered and loaded on an Ni-NTA Sepharose column (GE-Healthcare) equilibrated with 20 mM sodium phosphate pH 8.0, 150 mM NaCl, 0.1 mM DTT and 10 mM imidazole. A linear gradient from 10 to 500 mM imidazole was then applied. Fractions enriched in Pa CGL were pooled together, concentrated and buffer exchanged into 20 mM sodium phosphate pH 8.0, 150 mM NaCl, 0.1 mM DTT buffer using Vivaspin concentrators (Sartorius). Each purification yielded about 100 mg of pure protein per liter of bacterial culture. To facilitate crystallization, an additional construct for Pa CGL wild-type with the C-terminal 6x-His tag was prepared using similar strategy as described above. The theoretical extinction coefficient of monomeric Pa CGL at 280 nm was 28,545 M − 1 cm − 1 ( http://www.expasy.ch/tools/protparam.html ). The PLP content of the enzyme was determined by releasing the coenzyme in 0.1 M NaOH and by using ε M = 6,600 M − 1 cm − 1 at 388 nm. The oligomeric state of Pa CGL variants was determined by gel filtration using a Sephacryl S-200 16/60 HR column in 20 mM sodium phosphate pH 8.0, 150 mM NaCl and 0.1 mM DTT. The calibration curve was generated following the protocols in [ 35 , 36 ]. The apo-form of Pa CGL was obtained by incubating the enzyme with phenylhydrazine hydrochloride following the protocol in [ 37 ]. The dissociation constant for PLP ( K d) was obtained by monitoring the change of intrinsic fluorescence (excitation was set at 295 nm) of 1 µM apo-protein at different concentrations of PLP (0.01–4 µM) in 20 mM sodium phosphate pH 8.0 at 25° C on a FP8200 Jasco spectrofluorimeter [ 24 , 38 ]. Spectroscopic measurements Absorption spectra of 15 µM Pa CGL were collected on a Jasco V-750 UV-visible spectrophotometer in 20 mM sodium phosphate pH 8.0 at 25°C [ 30 ]. CD spectra were recorded on CD spectropolarimeter Jasco J-1500 equipped with a Peltier type thermostated cell holder, as previously described [ 30 , 39 ]. Briefly, far-UV (190–250 nm) spectra of 0.2 mg mL − 1 Pa CGL variants were collected in using a 0.1-cm path length quartz cuvette. Near UV-Vis (250–600 nm) spectra of 1 mg mL − 1 Pa CGL variants were recorded in 1-cm path length quartz cuvette at 25°C. A minimum of three accumulations were made for each scan, averaged, and corrected for the blank solution of corresponding buffer. Thermal unfolding profiles were collected by recording ellipticity at 222 nm in a temperature range between 15 to 90°C (scan rate 90°C/h) using 0.1-cm path length quartz cuvettes and protein concentration of 0.2 mg mL − 1 . All CD measurements were recorded in 20 mM sodium phosphate pH 8.0 [ 40 ]. Enzyme activity assays The CGL activity in the L-Cth γ-elimination reaction was determined by a previously described 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) assay [ 24 ]. Briefly, the purified enzyme (1 µM) was assayed in a 200 µL reaction (50 mM MOPS, 50 mM bicine, 50 mM proline pH 8.0, 20 µM PLP, 0.2 mM DTNB) at 37°C in the presence of different concentration of the L-Cth substrate. The activities in the H 2 S-generating alternative reactions were measured using the lead acetate assay as described elsewhere [ 30 , 41 – 43 ]. The enzyme (1–4 µM) was added to 0.4 mL of reaction mixture containing 50 mM Hepes pH 7.4, 20 µM PLP, 0.4 mM lead (II) acetate, and 0–50 mM L-Cys or 0–50 mM L-Hcys. Pyruvate formation was measured by monitoring NADH oxidation (ε 340 = 6,200 M − 1 cm − 1 ) via LDH assay [ 35 ]. Data for H 2 S production from L-Cys by Pa CGL were fitted following the kinetic models described prevoiusly [ 19 , 43 ]. Briefly, H 2 S production from L-Cys is the sum of two possible reactions, the b-elimination of L-Cys to generate L-Ser (reaction 3) or the condensation of two molecules of L-Cys to generate L-Lanthionine (reaction 4 ). Data for overall H 2 S production from L-Cys was fitted using Eq. ( 1 ) where v L−ser and v Lanthionine are defined by Eq. ( 2 ) and Eq. ( 3 ), respectively [ 19 , 30 , 43 ]. $${v}_{H2S}={v}_{L-Ser}+ {v}_{Lanthionine}$$ 1 $${v}_{L-Ser}=\frac{{v}_{max1 }[L-Cys]}{{K}_{m1(L-Cys)}+[L-Cys](1+\frac{\left[L-Cys\right]}{{K}_{i}})}$$ 2 $${v}_{Lanthionine}=\frac{{v}_{max2 [L-Cys]{[L-Cys]}^{n}}}{\left[L-Cys\right]{\left[L-Cys\right]}^{n}+{K}_{m1 }{[L-Cys]}^{n}+\left[L-Cys\right]{K}_{m2}^{n}}$$ 3 where K m1 and V max1 are associated to the unimolecular reaction, K m2 and V max2 to substrate binding at the second site and the reaction velocity of the bimolecular reaction and n represents Hill coefficient. Liquid chromatography mass spectrometry (LC-MS/MS) A TSQ Fortis Triple Quadrupole mass spectrometer (Thermo Scientific) coupled to Ultimate 3000 HPLC system (Thermo Scientific) was used for this analysis. The products separation was performed on a Luna C18(2) column (150 x 4.6 mm, 3 µm particle size, Phenomenex) with gradient elution. The mobile phase was composed of formic acid (A, 0.1% formic acid in water) and acetonitrile (B, 0.1% formic acid in ACN). Chromatographic gradient elution was the following: constant flow of 0.4 mL min − 1 ; 98% phase A at time 0, then decreased up to 5% A in 10 min and maintained at 5% A for 2 min and re-equilibrated for 5 min. The ESI source settings were ion spray voltage, + 3,500 V; ion transfer tube, 300°C; sheath gas and aux gas, 50 and 10, respectively, vaporizer temperature 350°C. Multiple reaction monitoring was optimized using nitrogen as collision gas (with pressure set at 1.5 mTorr). Two transitions for each substance were chosen for identification. Data acquisition and elaboration were performed by the Chromeleon (version 7.2, Thermo Fisher). Protein crystallization For crystallization, the enzymes were buffer exchanged into 50 mM HEPES, 150 mM NaCl, 0.1 mM DTT pH 8.0. Preliminary crystallization trials were carried out by the vapor-diffusion technique in a sitting drop format with 96-well MRC crystallization plates, following a previously described protocol [ 22 ]. Drops consisted of 200 nL protein solution (20 mg mL − 1 ) were mixed with 200 nL precipitant solution and incubated at 293K. The successful condition was scaled-up in a hanging-drop format using 24-well VDX plates (Hampton Research) in a reservoir with drops consisting of 0.5 µL protein (protein concentration of 20 mg mL − 1 ) with 0.5 µL precipitant solution. This reservoir was composed of 9% v/v polyethylene glycol 4000 and 0.1 M sodium acetate pH 4.6 with a volume of 0.5 mL. The crystals were transferred to a crystallization buffer containing 9% (w/v) PEG 4000, 0.1 M sodium acetate pH 4.6, and 20% glycerol for a few seconds before being flash frozen in liquid nitrogen. Structural determination by X-ray crystallography All X-rays datasets were collected at Synchrotron beamlines XALOC (ALBA), I03/I24 (DIAMOND, UK) and ID29 of ESRF (Grenoble). Datasets were collected over a range of 0.1–0.25° and the distance to the detector was set to reach resolution data between 1.6–3.8 Å depending on the crystal, and according to the diffraction parameters previously determined by several test images. Several data set were collected but only one allowed the structural determination of Pa CGL ( Table S2 ). Diffraction data were processed using HKL2000 511 or XDS 483 programs. The three-dimensional structure of Pa CGL was determined by MR method 488 with the Phaser-MR program489 from Phenix Suite 500 using the coordinates of Hs CGL holoenzyme (PDB ID 2NMP) as initial search model. The geometric quality of the models was assessed with MolProbity 490 integrated in Phenix suite. Figures were done with Pymol (The PyMOL Molecular Graphics System, Version 2.2.3, Schrödinger, LLC) and UCSF Chimera [ 44 ]. Deep Learning Structural Comparison Protein structure predictions were performed with AlphaFold 2.3.0 [ 25 ] using an adapted version of the AF2 code ( https://github.com/deepmind/alphafold ). Declarations Supporting Information List of mutagenic primers used to generate Pa CGL variants ( Table S1 ). Statistics for data collection and refinement ( Table S2 ). Properties of recombinant Pa CGL ( Fig. S1 ). Effect of pH and temperature on Pa CGL γ-elimination of L-Cth ( Fig. S2 ). Conformation of the loop 347–370 in Pa CGL ( Fig. S3 ). Structural and kinetic properties of Pa CGL variants ( Fig. S4 ). Main cavities found in CGLs ( Fig. S5 ). COMPETING INTERESTS The authors declare no competing interests. Author Contribution M.P., C.F.-R, C.C and I.O conceived the study, conducted the experiments, analysed the data, and edited the manuscript. F.F., P.D., M.L.M-C and M. PET. contributed to the discussion of results and edited the manuscript. A.DM., M.L.M.-C, T.M. supported funding acquisition and edited the manuscript. A.A. and L.A.M.-C. conceived the study, provided financial support, analysed the data, and wrote the manuscript. All authors read and approved the final manuscript. ACKNOWLEDGMENTS This research was supported by the MUR-PRIN 2022 grant No. 20224BYR59 to AA, by the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4 - Call for tender No. 341 of 03.15.2022 of Italian Ministry of University and Research funded by the European Union – NextGenerationEU to ADM, by Spanish Ministry of Economy and Competitiveness Grant BFU2016-77408-R and by Spanish Ministerio de Ciencia e Innovación (MICINN), Grants No PID2019-109055RB-I00 and PID2022-141748OB-I00, to LAM-C. We also thank MINECO for the Severo Ochoa Excellence Accreditation (CEX2021-001136-S). TM acknowledges the support from University of Fribourg Research Pool grant (22 − 15). We thank the Centro Piattaforme Tecnologiche of the University of Verona for providing access to the spectroscopic and mass spectrometry platforms. References Tacconelli, E., E. Carrara, A. Savoldi, S. Harbarth, M. Mendelson, D.L. Monnet, C. Pulcini, G. Kahlmeter, J. Kluytmans, Y. Carmeli, M. 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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-3869461","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":268696437,"identity":"077c808f-b976-4069-9a0e-b327b35c2643","order_by":0,"name":"Marco Pedretti","email":"","orcid":"","institution":"University of Verona","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Pedretti","suffix":""},{"id":268696438,"identity":"8845d54f-b860-41d5-bbdb-abbedd3f218b","order_by":1,"name":"Carmen Fernández-Rodríguez","email":"","orcid":"","institution":"Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA)","correspondingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Fernández-Rodríguez","suffix":""},{"id":268696439,"identity":"34fed39e-b974-44c9-965a-fb7be8a0a283","order_by":2,"name":"Carolina Conter","email":"","orcid":"","institution":"University of Verona","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Conter","suffix":""},{"id":268696440,"identity":"94e2bcc2-4805-471a-9ba8-a3b091b2a3eb","order_by":3,"name":"Iker Oyenarte","email":"","orcid":"","institution":"Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA)","correspondingAuthor":false,"prefix":"","firstName":"Iker","middleName":"","lastName":"Oyenarte","suffix":""},{"id":268696441,"identity":"5c1fb8c1-1cdb-4af0-af15-fd960c3c42dc","order_by":4,"name":"Filippo Favretto","email":"","orcid":"","institution":"University of Verona","correspondingAuthor":false,"prefix":"","firstName":"Filippo","middleName":"","lastName":"Favretto","suffix":""},{"id":268696442,"identity":"063b001e-134b-4a75-bb8f-4b87a19ed769","order_by":5,"name":"Adele di Matteo","email":"","orcid":"","institution":"CNR Institute of Molecular Biology and Pathology","correspondingAuthor":false,"prefix":"","firstName":"Adele","middleName":"di","lastName":"Matteo","suffix":""},{"id":268696443,"identity":"09c7b3ac-adad-4721-bf0c-3f4c990f5a83","order_by":6,"name":"Paola Dominici","email":"","orcid":"","institution":"University of Verona","correspondingAuthor":false,"prefix":"","firstName":"Paola","middleName":"","lastName":"Dominici","suffix":""},{"id":268696444,"identity":"425d6dbc-0048-4132-9064-01dc4aca3230","order_by":7,"name":"Maria Petrosino","email":"","orcid":"","institution":"University of Fribourg","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Petrosino","suffix":""},{"id":268696445,"identity":"8b12e5d9-2530-46bb-97e1-298fa114cc27","order_by":8,"name":"Maria Luz Martinez-Chantar","email":"","orcid":"","institution":"Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA)","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Luz","lastName":"Martinez-Chantar","suffix":""},{"id":268696446,"identity":"c14b3af4-86be-4f49-9424-8392db71e45a","order_by":9,"name":"Tomas Majtan","email":"","orcid":"","institution":"University of Fribourg","correspondingAuthor":false,"prefix":"","firstName":"Tomas","middleName":"","lastName":"Majtan","suffix":""},{"id":268696447,"identity":"563bceec-b743-4cc6-98fb-bdbcb906bcde","order_by":10,"name":"Alessandra Astegno","email":"","orcid":"","institution":"University of Verona","correspondingAuthor":false,"prefix":"","firstName":"Alessandra","middleName":"","lastName":"Astegno","suffix":""},{"id":268696448,"identity":"ed91eedb-0cd9-4e66-b47e-b179ee79fa18","order_by":11,"name":"Luis Alfonso Martinez-Cruz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYNACAyBmb2A4wNggQaSOAyAtPAdI0gIiJBIYGBgbiFDNP+3ws88fCu7Iyc98/PDAzx0WcvIRuQcfMFTU4dQicTvNeMYBg2fGjLPTDA72npEwNryRl2zAcOYwbmtuJxgD/XI4sVk6h+EwY5tE4sYZOWYSjG0HcOqQv53+GaSlvk3yDFyL+Q/Gf7gdZnA7B2xLAo8ED0TLfIkcM2A4MOPUYng7p5jhjMFhwxk8UL8Y8Lwxlkg4htsvcrfTNzNU/DksL99++PGHnzvq5OTbcww/fKjB7TAsTj0AJBJI0AAMkAaSlI+CUTAKRsEIAAChOlikmDbaLwAAAABJRU5ErkJggg==","orcid":"","institution":"Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA)","correspondingAuthor":true,"prefix":"","firstName":"Luis","middleName":"Alfonso","lastName":"Martinez-Cruz","suffix":""}],"badges":[],"createdAt":"2024-01-16 10:17:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3869461/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3869461/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-57625-7","type":"published","date":"2024-04-23T22:51:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50079538,"identity":"fe6f29f9-75e0-4cc4-acc9-804f1ec63967","added_by":"auto","created_at":"2024-01-24 07:03:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1239702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBacterial H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS synthesis and reactions catalyzed by CGLs. (A) \u003c/strong\u003eScheme of the known H\u003csub\u003e2\u003c/sub\u003eS production pathways in bacteria: SRP (grey arrows), 3-MST pathway (dashed arrows), and RTP (black arrows). The first step in the SRP is the activation of sulfate by the bifunctional sulfate adenylyltransferase subunit1/adenylylsulfate kinase (CysND), which converts ATP and sulfate to AMP and adenosine 5'-phosphosulfate (APS). The APS is transformed into 3′-phosphoadenosine 5′-phosphosulfate (PAPS) by adenylyl-sulfate kinase (CysC), and then reduced to sulfite (SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e) by phosphoadenosine phosphosulfate reductase (CysH). Sulfite is subsequently reduced to hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) by sulfite reductase (SR, CysI). Abbreviations: SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e(sulfate); SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e (sulfite); 3-mercaptopyruvate sulfurtransferase (3-MST); persulfidated 3-MST (3-MST-SS); Cystathionine β-synthase (CBS); Cystathionine g-lyase (CGL); \u003cstrong\u003e(B) \u003c/strong\u003eH\u003csub\u003e2\u003c/sub\u003eS-producing reactions catalyzed by CGL.\u003cstrong\u003e \u003c/strong\u003eThe first α,g-elimination of L-Cth represents the canonical CGL reaction, which does not lead to production of H\u003csub\u003e2\u003c/sub\u003eS. However, CGL catalyzes several alternative reactions using L-Hcys and/or L-Cys as substrates generating H\u003csub\u003e2\u003c/sub\u003eS.\u003c/p\u003e","description":"","filename":"image1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3869461/v1/928a366cef1ba13379d8471d.jpg"},{"id":50079539,"identity":"6bc3abb3-2c27-4c1c-b078-2408218c4c91","added_by":"auto","created_at":"2024-01-24 07:03:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1578802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHydrolysis of L-Cth by recombinant \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eCGL.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) LC-MS/MS chromatogram of the amino acid products of L-Cth hydrolysis by \u003cem\u003ePa\u003c/em\u003eCGL. Parent ions with\u0026nbsp;\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e\u0026nbsp;values of 122 (L-Cys), 136 (L-Hcys), and 223 (L-Cth) are seen. (\u003cstrong\u003eB\u003c/strong\u003e) Steady-state initial velocity kinetic for \u003cem\u003ePa\u003c/em\u003eCGL in the hydrolysis of L-Cth measured using the DTNB assay. (\u003cstrong\u003eC\u003c/strong\u003e) Kinetics of the L-Cth hydrolysis by \u003cem\u003ePa\u003c/em\u003eCGL followed by measuring the pyruvate production via the LDH assay. Each data point in (\u003cstrong\u003eB\u003c/strong\u003e) and (\u003cstrong\u003eC\u003c/strong\u003e) represents the mean ± SEM of at least three independent experiments. (\u003cstrong\u003eD\u003c/strong\u003e) The UV-Vis absorbance spectra of 15 µM \u003cem\u003ePa\u003c/em\u003eCGL in the absence (black line) and presence of 2.5 mM L-Cth (red line). \u003cstrong\u003e(E)\u003c/strong\u003e CD spectra of 1 mgmL\u003csup\u003e-1\u003c/sup\u003e \u003cem\u003ePa\u003c/em\u003eCGL in the absence (black line) and presence of 2.5 mM L-Cth (red line).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-3869461/v1/20735ad2d3b7a05d135d6516.png"},{"id":50079681,"identity":"f6447dcf-694d-4673-83d3-d4220e70b6c8","added_by":"auto","created_at":"2024-01-24 07:11:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1018853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS alternative reactions.\u003c/strong\u003e (\u003cstrong\u003eA-C)\u003c/strong\u003e Product analysis by LC-MS/MS of the \u003cem\u003ePa\u003c/em\u003eCGL-catalyzed reactions in the presence of L-Cys alone (\u003cstrong\u003eA\u003c/strong\u003e), L-Hcys alone (\u003cstrong\u003eB\u003c/strong\u003e) or L-Cys and L-Hcys (\u003cstrong\u003eC\u003c/strong\u003e). Parent ions with\u0026nbsp;\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e\u0026nbsp;values of 122 (L-Cys), 106 (L-Ser), 209 (L-Lanthionine), 136 (L-Hcys), 120 (L-Homoserine), 237 (L-Homolanthionine) and 223 (L-Cth) are seen. (\u003cstrong\u003eD\u003c/strong\u003e) Kinetics of\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eS generation by \u003cem\u003ePa\u003c/em\u003eCGL in the presence of L-Cys.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e Shown are the kinetics of pyruvate (red) (reaction\u0026nbsp;\u003cstrong\u003e3\u003c/strong\u003e) and H\u003csub\u003e2\u003c/sub\u003eS (black) (reactions\u0026nbsp;\u003cstrong\u003e3\u003c/strong\u003e\u0026nbsp;+\u0026nbsp;\u003cstrong\u003e4\u003c/strong\u003e) generation from L-Cys. Each data point represents the mean ± SEM of at least three independent experiments. (\u003cstrong\u003eE\u003c/strong\u003e) Kinetics of\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eS generation (reactions\u0026nbsp;\u003cstrong\u003e5\u003c/strong\u003e\u0026nbsp;+\u0026nbsp;\u003cstrong\u003e6\u003c/strong\u003e) by \u003cem\u003ePa\u003c/em\u003eCGL in the presence of L-Hcys. (\u003cstrong\u003eF\u003c/strong\u003e) LC-MS/MS analysis of L-Cth formation at 10 mM\u0026nbsp;L-Cys and varying concentrations of L-Hcys.\u0026nbsp;\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-3869461/v1/93d06cb3abc61fc1a3f9f5ac.png"},{"id":50079541,"identity":"91096994-89ea-47b4-882a-dd0888ee8705","added_by":"auto","created_at":"2024-01-24 07:03:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5037175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructure of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eCGL. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Domain architecture of the \u003cem\u003ePa\u003c/em\u003eCGL monomer. (\u003cstrong\u003eB\u003c/strong\u003e) Crystal structure of \u003cem\u003ePa\u003c/em\u003eCGL. The N-terminal domain (residues 1-62), the PLP-binding domain (residues 63-260), and the C-terminal module (green, residues 261-394) are colored in blue, orange, and green, respectively. PLP is in pink sticks. (\u003cstrong\u003eC\u003c/strong\u003e) Structure of \u003cem\u003ePa\u003c/em\u003eCGL predicted with AF2. Modelled amino acid residues 1-8, and 46-57 not visible in the crystals, are colored in red. The inset shows the loop L347-370 (in red, and pointed out with an asterisk), which partially defines the entrance to the catalytic cavity, and adopts a markedly different conformation in the crystals than in the predicted \u003cem\u003ePa\u003c/em\u003eCGL model. The latter is consistent with the overall structure found in other CGLs (\u003cstrong\u003eFig. S5\u003c/strong\u003e). (\u003cstrong\u003eD\u003c/strong\u003e) \u003cem\u003ePa\u003c/em\u003eCGL tetrameric organization. The four subunits, A to D, are represented in different surface colors. The secondary elements from each subunit are colored according to the domain architecture colors shown in panel A.\u003c/p\u003e","description":"","filename":"image4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3869461/v1/ab5349de83df4c1764af45c2.jpg"},{"id":50079543,"identity":"ac7ab6d2-a94b-4b90-b79d-165e8dec1d01","added_by":"auto","created_at":"2024-01-24 07:03:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1603307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMain cavities and active site of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eCGL.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Main structural elements configuring the active site. Asterisks indicate elements from a complementary subunit. PLP is in sticks. The two main cavities, chamber-1 (active site) and chamber-2 (known to host inhibitors of \u003cem\u003eSa\u003c/em\u003eCGL), are shown as red surface areas (Cavity detection cutoff= 4 solvent radii; Cavity detection radii= 7 Angstrom). (\u003cstrong\u003eB\u003c/strong\u003e) Stick representation of main amino acid residues within the PLP-binding cavity. Residues are colored according to the protein domain representation shown in Fig. 4. Polar interactions are represented in dashed lines. PLP is depicted in pink and red spheres correspond to water molecules.\u003c/p\u003e","description":"","filename":"image5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3869461/v1/16a67ba106106619efc1db6b.jpg"},{"id":50079544,"identity":"c02f747b-9b94-40d7-9b96-c7f34eea16e3","added_by":"auto","created_at":"2024-01-24 07:03:45","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6664045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChamber-1 and chamber-2 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eCGL and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eCGL\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) (Top) Surface representation of the \u003cem\u003ePa\u003c/em\u003eCGL monomer. Chamber-1 and chamber-2 are colored in blue and magenta, respectively. (Bottom) Main amino acid residues within the cavities. PLP is in sticks. \u003cstrong\u003e(B) \u003c/strong\u003e(Top) Surface representation of the \u003cem\u003eSa\u003c/em\u003eCGL monomer. Chamber-1- and chamber-2 are colored in blue and magenta, respectively. (Bottom) Main amino acid residues within the cavities. PLP is in sticks. NL1 inhibitor is in blue. The atom coordinates used to make the figure were obtained from PDB IDs 7BA4 (\u003cem\u003ePa\u003c/em\u003eCGL) and 7MCT (\u003cem\u003eSa\u003c/em\u003eCGL+NL1).\u003c/p\u003e","description":"","filename":"image6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3869461/v1/4de59bba3c86831b2fb76e3a.jpg"},{"id":55690781,"identity":"b03f62e5-91b1-451f-a88b-dea8488f19e6","added_by":"auto","created_at":"2024-05-01 22:51:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2035081,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3869461/v1/445541eb-c0ca-4081-94e1-52ead474bd16.pdf"},{"id":50079542,"identity":"83cc2d1e-4d67-4a0d-8668-8a2f94fb44b4","added_by":"auto","created_at":"2024-01-24 07:03:45","extension":"mov","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1122143,"visible":true,"origin":"","legend":"","description":"","filename":"SuppMovieS1PaCGLSciRepDec29.mov","url":"https://assets-eu.researchsquare.com/files/rs-3869461/v1/3e9e8d00ee1dda7e142f7a96.mov"},{"id":50079545,"identity":"395176a6-9243-455d-b7f3-fd0cbb7da2ed","added_by":"auto","created_at":"2024-01-24 07:03:45","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4889927,"visible":true,"origin":"","legend":"","description":"","filename":"SupplinfoJan182024SciRep.docx","url":"https://assets-eu.researchsquare.com/files/rs-3869461/v1/2c0aa2a21352c0d3fe4093b3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe diseases caused by bacteria, fungi, or parasites pose a growing problem for today's healthcare system. Infectious and parasitic diseases are among the top ten global causes of mortality, as identified by the World Health Organization [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Of particular concern is the opportunistic Gram-negative bacterium \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, which has been designated as a critical priority for study, discovery, and the development of new antibiotics. Infections caused by \u003cem\u003eP. aeruginosa\u003c/em\u003e can progress to extensive colonization and be more severe with a higher mortality rate, especially in cystic fibrosis patients and immunocompromised hospitalized individuals [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. At present, treating \u003cem\u003eP. aeruginosa\u003c/em\u003e infections effectively poses a substantial challenge owing to the bacterium's increasing resistance to numerous clinically available antibiotics.\u003c/p\u003e \u003cp\u003eRecent studies have shown that a defense mechanism of bacteria against reactive oxygen species (ROS) and antibiotic-induced oxidative damage relies on the endogenous production of hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Based on these findings, inhibiting the endogenous generation of this gasotransmitter has been proposed as a strategy to combat these pathogens [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], although it is still a matter of debate whether this defensive role applies equally to all bacterial species [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, the detailed study and characterization of the enzymes involved in the potential production of H\u003csub\u003e2\u003c/sub\u003eS in these organisms becomes particularly relevant for selecting suitable targets upon which to design new antibiotics.\u003c/p\u003e \u003cp\u003eThe synthesis of H\u003csub\u003e2\u003c/sub\u003eS in bacteria varies across species and depends on substrate availability and presence of the specific enzymes. Under anaerobic conditions, the primary route for H\u003csub\u003e2\u003c/sub\u003eS production is the sulfate reduction pathway (SRP) involving the reduction of inorganic sulfate (SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e) to H\u003csub\u003e2\u003c/sub\u003eS [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). However, under aerobic conditions, the production of H\u003csub\u003e2\u003c/sub\u003eS by the SRP is quite inefficient compared to specialized sulfate-reducing bacteria. Therefore, some bacteria can synthetize H\u003csub\u003e2\u003c/sub\u003eS through 3-mercaptopyruvate sulfurtransferase (3-MST) under conditions of sulfur limitation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The 3-MST enzyme is part of a larger pathway involved in the biosynthesis of cysteine, which also includes other enzymes such as cysteine synthase and O-acetylserine sulfhydrylase. Finally, the third known bacterial mechanism to produce H\u003csub\u003e2\u003c/sub\u003eS is the reverse transsulfuration pathway (RTP), which involves the conversion of L-homocysteine (L-Hcys) to L-cysteine (L-Cys) through two consecutive steps catalyzed by two distinct pyridoxal-5\u0026rsquo;-phosphate (PLP)-dependent enzymes, the cystathionine b-synthase (CBS) and the cystathionine g-lyase (CGL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). CBS catalyzes a β-replacement reaction in which the hydroxyl group of L-serine (L-Ser) is replaced by L-Hcys, yielding L-cystathionine (L-Cth) and H\u003csub\u003e2\u003c/sub\u003eO (reviewed in [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]). Next, CGL catalyzes the α,γ-elimination of L-Cth into L-Cys, α-ketobutyrate, and ammonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, reaction \u003cb\u003e1\u003c/b\u003e). In addition, many CGL can catalyze β-elimination of L-Cth as a side reaction, producing L-Hcys, pyruvate, and ammonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, reaction \u003cb\u003e2\u003c/b\u003e). This reaction is referred to as the β-lyase activity of CGL. Besides these canonical reactions, both CBS and CGL can catalyze the synthesis of H\u003csub\u003e2\u003c/sub\u003eS using L-Cys and L-Hcys as substrates exploiting alternative reactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, reactions \u003cb\u003e3 to 7\u003c/b\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIt has recently been demonstrated that inhibitors of human CGL (\u003cem\u003eHs\u003c/em\u003eCGL) require much higher concentration to achieve inhibition or may entirely lack any efficacy on homolog enzymes encoded by different bacteria [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This is likely due to structural and physico-chemical differences within each CGL enzyme, which could be exploited to develop new antibiotics and/or adjuvants to treat recurrent infections caused by the specific pathogen. Recent studies have revealed the presence of the \u003cem\u003eCse\u003c/em\u003e gene within \u003cem\u003eP. aeruginosa\u003c/em\u003e genome encoding for the CGL (\u003cem\u003ePa\u003c/em\u003eCGL) and that inactivation of the \u003cem\u003eCse\u003c/em\u003e gene leads to a significant reduction of H\u003csub\u003e2\u003c/sub\u003eS production in clinical isolates of \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study we expressed, purified, and biochemically characterized \u003cem\u003ePa\u003c/em\u003eCGL as well as solved its crystal structure. We found that \u003cem\u003ePa\u003c/em\u003eCGL can hydrolyze L-Cth via g- as well as β-elimination mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, reactions \u003cb\u003e1,2\u003c/b\u003e) and, in addition, can catalyze the generation of H\u003csub\u003e2\u003c/sub\u003eS from L-Cys or/and L-Hcys (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, reactions \u003cb\u003e3\u003c/b\u003e\u0026ndash;\u003cb\u003e6\u003c/b\u003e). Structural comparisons to other CGL enzymes and a complementary analysis of \u003cem\u003ePa\u003c/em\u003eCGL using deep learning predictions by AlphaFold2 (AF2) revealed significant structural diversity within the main cavities of the enzymes as well as a differently organized entrance loop that may direct the access of substrates and/or inhibitors into the PLP-containing catalytic center. These findings open new avenues for the design of more selective inhibitors of \u003cem\u003ePa\u003c/em\u003eCGL and provide significant insights on the structural evolution of CGL enzymes across different pathogenic organisms.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eProduction and biochemical properties of\u003c/b\u003e \u003cb\u003ePa\u003c/b\u003e\u003cb\u003eCGL\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003ePa\u003c/em\u003eCGL was overexpressed in \u003cem\u003eE. coli\u003c/em\u003e and purified to homogeneity using Ni-NTA chromatography (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). Gel filtration showed a molecular mass of 173 kDa for \u003cem\u003ePa\u003c/em\u003eCGL, suggesting that it adopts a tetrameric structure in solution, in accordance with a monomer molecular mass of ~\u0026thinsp;44 kDa (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB)\u003c/b\u003e. The UV-Vis absorption spectrum of \u003cem\u003ePa\u003c/em\u003eCGL at pH 8.0 exhibited, in addition to the protein band centered at 278 nm, a peak at 425 nm that is typical of the ketoenamine tautomer of the internal aldimine (protein-bound PLP) (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC)\u003c/b\u003e. \u003cem\u003ePa\u003c/em\u003eCGL binds\u0026thinsp;~\u0026thinsp;1 mol of PLP/mol of monomer with a \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e value for PLP of 0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u0026micro;M, as calculated by fluorescence titrations of apo-\u003cem\u003ePa\u003c/em\u003eCGL with PLP (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD)\u003c/b\u003e. The apo-\u003cem\u003ePa\u003c/em\u003eCGL displayed substantially decreased thermal stability with a melting temperature (T\u003csub\u003em\u003c/sub\u003e) of 57\u0026deg;C compared to the PLP-loaded holo-enzyme yielding T\u003csub\u003em\u003c/sub\u003e of 68\u0026deg;C (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEnzymatic properties of\u003c/b\u003e \u003cb\u003ePa\u003c/b\u003e\u003cb\u003eCGL enzyme\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCanonical reactions\u003c/h2\u003e \u003cp\u003eThe reaction catalyzed by CGL in the transsulfuration pathway involves elimination at the γ-carbon of L-Cth. However, it is well established [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] that CGLs, owing to the chemistry of the catalyzed reaction, exhibit an appreciable cystathionine β-lyase (CBL)-like activity, \u003cem\u003ei.e.\u003c/em\u003e, they can cleave both the C\u0026ndash;γ\u0026ndash;S and C\u0026ndash;β\u0026ndash;S bonds of L-Cth producing L-Cys or L-Hcys, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, reaction \u003cb\u003e1\u0026ndash;2\u003c/b\u003e). In our first approach to distinguish between these types of elimination reactions catalyzed by \u003cem\u003ePa\u003c/em\u003eCGL (γ- or β-elimination), the reaction products were analyzed by applying LC-MS/MS. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, when L-Cth (\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;223) was used as a substrate, both L-Cys (\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;122) and L-Hcys (\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;136) products were observed, consistent with the ability of the enzyme to catalyse both γ-elimination (reaction \u003cb\u003e1\u003c/b\u003e) and β-elimination (reaction \u003cb\u003e2\u003c/b\u003e) of L-Cth.\u003c/p\u003e \u003cp\u003eThe kinetics of L-Cth hydrolysis by \u003cem\u003ePa\u003c/em\u003eCGL were then characterized using the DTNB assay. Firstly, we determined the optimal pH and temperature for \u003cem\u003ePa\u003c/em\u003eCGL by measuring the CGL activity at various conditions. \u003cem\u003ePa\u003c/em\u003eCGL displayed its highest enzymatic activity at pH 8.0 and around 42\u0026deg;C (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e)\u003c/b\u003e. However, considering the physiological temperature of 37\u0026deg;C, we opted to conduct enzymatic characterization of \u003cem\u003ePa\u003c/em\u003eCGL at pH 8.0 and 37\u0026deg;C. The obtained enzymatic kinetics for the hydrolysis of L-Cth catalyzed by \u003cem\u003ePa\u003c/em\u003eCGL followed the Michaelis\u0026ndash;Menten profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) with \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e and K\u003csub\u003em\u003c/sub\u003e values of 5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 mM, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The kinetics of pyruvate formation (reaction \u003cb\u003e2\u003c/b\u003e) from L-Cth was also characterized using the LDH assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC \u003cb\u003eand\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe also monitored the behavior of the enzyme-bound PLP in the presence of L-Cth by absorption and CD spectroscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E\u003cb\u003e)\u003c/b\u003e. Addition of L-Cth to \u003cem\u003eP\u003c/em\u003eaCGL resulted in significant changes in the spectrum of the enzyme. The most pronounced difference is the presence of two chromophores with absorption in the region of 440\u0026ndash;480 nm with positive CD of their absorption bands. For PLP-dependent enzymes involved in γ- and β-elimination reactions, absorbance bands in the region of 440\u0026ndash;480 nm usually are assigned to the α-aminocrotonate or α-aminoacrylate intermediates [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The absorption region of 320\u0026ndash;340 nm is not considered because pyruvate produced during the turnover also contributes to this region.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAlternative reactions\u003c/h3\u003e\n\u003cp\u003eThe ability of \u003cem\u003ePa\u003c/em\u003eCGL to produce H\u003csub\u003e2\u003c/sub\u003eS was assessed by examining the various reactions outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB (reactions \u003cb\u003e3\u0026ndash;7\u003c/b\u003e). The use of LC-MS/MS for product analysis provided direct evidence for all the five potential H\u003csub\u003e2\u003c/sub\u003eS-generating reactions attributed to CGL. When L-Cys served as substrate, we observed the formation of both the L-Ser (\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;106) and L-Lanthionine (\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;209) products, confirming the enzyme's proficiency in catalyzing both L-Cys β-lyase (reaction \u003cb\u003e3\u003c/b\u003e) and L-Cys β-replacement (reaction \u003cb\u003e4\u003c/b\u003e) activities, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similarly, in the presence of L-Hcys alone, L-Homoserine (\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;120) and L-Homolanthionine (\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;237) were detected, consistent with γ-elimination (reaction \u003cb\u003e4\u003c/b\u003e) and γ-replacement (reaction \u003cb\u003e5\u003c/b\u003e) reactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In the presence of L-Hcys and L-Cys, L-Cth (\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;223) was detected, consistent with a replacement reaction (reaction \u003cb\u003e7\u003c/b\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eNext, we determined the kinetics of H\u003csub\u003e2\u003c/sub\u003eS generation from L-Cys or/and L-Hcys. The active site pocket of CGL has binding requirements for two amino acids, since the main substrate of the enzyme, which is L-Cth, is a condensation product of two amino acids, L-Ser and L-Hcys. In the H\u003csub\u003e2\u003c/sub\u003eS-generating reactions catalyzed by PaCGL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, reactions \u003cb\u003e3\u0026ndash;7)\u003c/b\u003e, either one (reaction \u003cb\u003e3\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e) or both (reactions \u003cb\u003e4\u003c/b\u003e, \u003cb\u003e6\u003c/b\u003e, and \u003cb\u003e7\u003c/b\u003e) amino acid binding pockets are occupied. We analyzed the kinetic parameters associated with the single substrate reaction (\u003cem\u003ei.e.\u003c/em\u003e ignoring H\u003csub\u003e2\u003c/sub\u003eO) or bimolecular reactions involving two amino acids, as outlined in the Methods section.\u003c/p\u003e \u003cp\u003eThe dependence of the rate of H\u003csub\u003e2\u003c/sub\u003eS formation on L-Cys concentration is markedly biphasic, simplifying the deconvolution of kinetic parameters related to two distinct phases corresponding to reactions \u003cb\u003e3\u003c/b\u003e (β-elimination of L-Cys) and \u003cb\u003e4\u003c/b\u003e (β- replacement of L-Cys). The enzyme kinetics of pyruvate (reaction \u003cb\u003e3\u003c/b\u003e) and of H\u003csub\u003e2\u003c/sub\u003eS (reactions \u003cb\u003e3\u003c/b\u003e\u0026thinsp;+\u0026thinsp;\u003cb\u003e4\u003c/b\u003e) formation from L-Cys are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and the resulting kinetic parameters are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Notably, \u003cem\u003ePa\u003c/em\u003eCGL exhibits a significant higher affinity for L-Cys binding at site 1 (0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mM) compared to site 2 (33\u0026thinsp;\u0026plusmn;\u0026thinsp;6 mM), with observed cooperativity in the binding of the second L-Cys molecule (n\u0026thinsp;=\u0026thinsp;3\u0026thinsp;\u0026plusmn;\u0026thinsp;1). Interestingly, like many other CGLs [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], \u003cem\u003ePa\u003c/em\u003eCGL also displayed L-Cys inhibition (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe dependence of the rate of H\u003csub\u003e2\u003c/sub\u003eS formation on L-Hcys concentration is not as markedly biphasic as observed with L-Cys, even though mass spectrometry data clearly indicate that both L-Homoserine (reaction \u003cb\u003e5\u003c/b\u003e) and L-Homolanthionine (reaction \u003cb\u003e6\u003c/b\u003e) are produced when L-Hcys serves as a substrate. We reported the kinetic parameters for the overall rate of H\u003csub\u003e2\u003c/sub\u003eS formation (reaction \u003cb\u003e5\u0026thinsp;+\u0026thinsp;6\u003c/b\u003e) without applying deconvolution analysis of the two phases associated with the single substrate reaction (reaction \u003cb\u003e5\u003c/b\u003e) or double substrate reaction (reaction \u003cb\u003e6\u003c/b\u003e). The kinetic data for H\u003csub\u003e2\u003c/sub\u003eS production are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Analysis of reaction \u003cb\u003e7\u003c/b\u003e (i.e., the condensation of L-Hcys and L-Cys) was done by monitoring the L-Cth formation through LC-MS/MS, showing that L-Cth increases with increasing L-Hcys concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eKinetic parameters determined for reactions catalyzed by\u003c/b\u003e \u003cb\u003ePa\u003c/b\u003e\u003cb\u003eCGL.\u003c/b\u003e Values correspond to the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs of at least three independent experiments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReaction number (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ek\u003csub\u003ecat\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eK\u003csub\u003em\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKi\u003c/p\u003e \u003cp\u003e(mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ek\u003csub\u003ecat\u003c/sub\u003e/ K\u003csub\u003em\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHydrolysis of L-Cth\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e17\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyruvate generation from L-Cth\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS generation from L-Cys\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e33\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyruvate generation from L-Cys\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS generation from L-Hcys\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e5\u0026thinsp;+\u0026thinsp;6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e Activity was determined using the DTNB assay.\u003c/p\u003e \u003cp\u003e \u003csup\u003eb\u003c/sup\u003e Activity was determined using the LDH assay.\u003c/p\u003e \u003cp\u003e \u003csup\u003ec\u003c/sup\u003e Activity was determined using the lead acetate assay.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOverall structure\u003c/b\u003e \u003cb\u003eof Pa\u003c/b\u003e\u003cb\u003eCGL\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe crystal structure of \u003cem\u003ePa\u003c/em\u003eCGL complexed with PLP was solved at 2.0 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The few missing or disordered segments not visible in the crystals were predicted by AF2 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. As expected, the overall fold of \u003cem\u003ePa\u003c/em\u003eCGL is consistent with the type-I PLP-dependent enzymes, resembling human, yeast, and bacteria CGLs, as well as enzymes like cystathionine γ-synthase (CGS), or cystathionine β-lyase (CBL) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Each \u003cem\u003ePa\u003c/em\u003eCGL subunit consists of 394 amino acids distributed in three domains: (i) an N-terminal domain (residues 1\u0026ndash;62), (ii) a central PLP-binding domain (residues 63\u0026ndash;260), and (iii) a C-terminal domain (residues 261\u0026ndash;394). The N-terminal domain begins with an unstructured segment (residues 1\u0026ndash;13), followed by a short α-helix (α0, residues 14\u0026ndash;22) and a long-disordered loop (L23-60). However, residues 46\u0026ndash;57 are not visible in three out of the four molecules in the asymmetric unit. The PLP-binding domain is built up of a seven-stranded, mostly parallel, β-sheet (\u0026uarr;β1\u0026darr;β7\u0026uarr;β6\u0026uarr;β5\u0026uarr;β4\u0026uarr;β2\u0026uarr;β3), with strand β7 (residues 219\u0026ndash;223) antiparallel to the rest. This domain additionally contains eight α-helices, split in two sets (α1, α2, α5, α6, α7 and α3, α4, α8, respectively) that flank the central β-sheet core at both sides. In our crystals, the short helix α1 (residues 55\u0026ndash;60), inserted in the loop L23-60, is only visible in one protein subunit.\u003c/p\u003e \u003cp\u003eThe PLP-binding domain houses the enzyme\u0026rsquo;s catalytic center containing PLP covalently anchored to the enzyme via a conserved K208 residue and forming an internal aldimine as confirmed by the spectroscopic analysis (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC and\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Finally, the C-terminal domain is organized into a five-stranded antiparallel β-sheet (exhibiting the following topology: \u0026uarr;β8\u0026darr;β9\u0026uarr;β12\u0026darr;β11\u0026darr;β10), decorated with five α-helices (α10\u0026ndash;14) on one side which protect it from the solvent.\u003c/p\u003e \u003cp\u003eSimilarly to all previously characterized CGLs, \u003cem\u003ePa\u003c/em\u003eCGL self-assembles into a homotetramer that can be described as a dimer of dimers that include subunits A-C and B-D, respectively, in which the two subunits of each dimer are related by a 2-fold axis, and the dimers themselves maintain a 2-fold symmetry in relation to each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The four subunits of the tetramer exhibit high similarity (average rmsd\u0026thinsp;=\u0026thinsp;0.140 \u0026Aring;), differing only in the conformation of two long loops (L23-60 and L347-370), that appear partially disordered in some of the monomers or present a slightly different conformation. Interestingly, in the crystals both segments adopt a conformation that differs from what was observed in the predicted AF2 model, or in the topological equivalent regions of CGLs from other species. Notably, the loop, L347-370 adopts an extended conformation in PaCGL, diverging from the typical two-turn helix (α13) observed in other homologs (\u003cb\u003eFig. S3A\u003c/b\u003e). This structural feature directly impacts the accessibility to the large cleft (chamber-2) located behind the catalytic site (chamber-1) that accommodates the PLP cofactor (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Of note, the loop L347-370 region does not display significant sequence conservation among the different \u003cem\u003ePa\u003c/em\u003eCGL homologs. Within helix a13, merely three amino acid residues remain consistent across all sequences-specifically, a proline, an arginine, and a glycine (P357, R361, G365 in PaCGL)-found within the loop\u0026acute;s second segment (\u003cb\u003eFig. S3\u003c/b\u003e). Strikingly, the AF2-predicted \u003cem\u003ePa\u003c/em\u003eCGL model maintains the two-turns helicity fold like other CGL enzymes (\u003cb\u003eFig. S3\u003c/b\u003e) suggesting the probability of two different (and stable) conformations for this segment. Differential conformation of this loop may modulate the accessibility of chamber-2, known to host inhibitors of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e CGL (\u003cem\u003eSa\u003c/em\u003eCGL) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and dictate the specificity of such inhibitors to \u003cem\u003ePa\u003c/em\u003eCGL versus other species.\u003c/p\u003e \u003cp\u003eThe active site of \u003cem\u003ePa\u003c/em\u003eCGL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) is characterized by a deep cavity situated at the dimerization interface between subunits A-B (or equivalently C-D). The PLP-interacting residues, which are well-conserved across CGLs, in \u003cem\u003ePa\u003c/em\u003eCGL are represented by Y110, R372, K208, S337, and S205. K208 is covalently bound to PLP through its ε-amino group and forms a Schiff- base linkage at the C4A position of PLP. The orientation of PLP is fixed by H-bond interactions between its phosphate group and the main chain nitrogen of residues G86 and L87. The hydroxyl group of S205 and T207 also stabilizes the PLP phosphate moiety. The complementary subunit interacts with O2P and O3P of PLP via the guanidine group of R58. Most CGLs also show interactions between the PLP and the conserved tyrosine of the neighboring subunit. In \u003cem\u003ePa\u003c/em\u003eCGL, residue Y56 does not interact with the phosphate moiety of PLP, but rather establishes an H-bond with the N-terminal residue Q45. This contact is possible thanks to the flexibility provided by residue G57 to the polypeptide main chain. Interestingly, residue G57 of \u003cem\u003ePa\u003c/em\u003eCGL is usually substituted by a conserved serine in other CGLs. Finally, the pyridoxal ring of PLP is stacked with the phenol ring of Y110.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChamber-2 of\u003c/b\u003e \u003cb\u003ePa\u003c/b\u003e\u003cb\u003eCGL as potential drug binding site\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe crystal structure of \u003cem\u003ePa\u003c/em\u003eCGL provided valuable insights for comparing critical regions with both bacterial and human counterparts. Special focus was given to the chamber-2, which has been found in \u003cem\u003eSa\u003c/em\u003eCGL to host pharmacological inhibitors [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Chamber-2 can be reached only via a channel with limited accessibility determined by residues from the long loop connecting strands b11-b12 containing helix a13 (loop L347-370 in \u003cem\u003ePa\u003c/em\u003eCGL), and by a tyrosine residue (Y103 in \u003cem\u003eSa\u003c/em\u003eCGL; Y102 in \u003cem\u003eBc\u003c/em\u003eCGL) located in helix a4. Some CGL enzymes lack this tyrosine, which is substituted by either another bulky hydrophobic residue (i.e., F114 in \u003cem\u003ePa\u003c/em\u003eCGL; F101 in \u003cem\u003eLp\u003c/em\u003eCGL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), or alternatively an asparagine (N137 in \u003cem\u003eTg\u003c/em\u003eCGL; N118 in \u003cem\u003eHs\u003c/em\u003eCGL). The crystal structures of \u003cem\u003eSa\u003c/em\u003eCGL, obtained in complex with novel pharmacological inhibitors (named NL1, NL2, and NL3) revealed the significance of the residue Y103 in \u003cem\u003eSa\u003c/em\u003eCGL catalysis and its role in stabilizing the inhibitors within the cavity through a π-stacking interaction [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The Y103A mutation of \u003cem\u003eSa\u003c/em\u003eCGL, or even the \"humanized\" Y103N variant, eliminated the H\u003csub\u003e2\u003c/sub\u003eS production activity of \u003cem\u003eSa\u003c/em\u003eCGL and disrupted the interaction with the NL1 and NL2 inhibitors [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo assess the role of the equivalent chamber-2 residue F114 in \u003cem\u003ePa\u003c/em\u003eCGL, we replaced it with alanine or asparagine and compared the resulting variants with the wild type protein. The two mutations did not impair the overall structural properties as well as the tetrameric oligomerization of the purified mutant enzymes (\u003cb\u003eFig. S4A-C\u003c/b\u003e). Interestingly, both the F114A and the \"humanized\" F114N mutations resulted in only a slight reduction in enzyme\u0026rsquo;s catalytic efficiency for both the canonical and H\u003csub\u003e2\u003c/sub\u003eS-producing activities of \u003cem\u003ePa\u003c/em\u003eCGL (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eand Fig. S4D\u003c/b\u003e). This reduction is mainly attributed to decreases in k\u003csub\u003ecat\u003c/sub\u003e values, while K\u003csub\u003em\u003c/sub\u003e remained similar to the wild type.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eKinetic parameters for reactions catalyzed by\u003c/b\u003e \u003cb\u003ePa\u003c/b\u003e\u003cb\u003eCGL F114A and F114N variants.\u003c/b\u003e Values correspond to the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs of at least three independent experiments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReaction number (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ek\u003csub\u003ecat\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eK\u003csub\u003em\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKi\u003c/p\u003e \u003cp\u003e(mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ek\u003csub\u003ecat\u003c/sub\u003e/ K\u003csub\u003em\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHydrolysis of L-Cth\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e1\u0026thinsp;+\u0026thinsp;2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWild type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e17\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF114A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF114N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS generation from L-Cys\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWild type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e33\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF114A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e33\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.0091\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF114N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e17\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e Activity was determined using the DTNB assay.\u003c/p\u003e \u003cp\u003e \u003csup\u003eb\u003c/sup\u003e Activity was determined using the lead acetate assay.\u003c/p\u003e \u003cp\u003eTwo additional amino acids situated at the base of the chamber-2 have been suggested to define the connecting gate between chamber-2 and the catalytic site (chamber-1). The first is a conserved histidine (H339 in \u003cem\u003eSa\u003c/em\u003eCGL; H356 in \u003cem\u003eHs\u003c/em\u003eCGL; H338 in \u003cem\u003eBc\u003c/em\u003eCGL; H337 in \u003cem\u003eLp\u003c/em\u003eCGL; and H376 in \u003cem\u003eTg\u003c/em\u003eCGL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). \u003cem\u003ePa\u003c/em\u003eCGL also contains an equivalent histidine in its amino acid chain (H353 in \u003cem\u003ePa\u003c/em\u003eCGL), but its spatial location differs from that found in other CGLs due to the more extended conformation of the loop L347-370, and the topological position of H339 in \u003cem\u003eSa\u003c/em\u003eCGL is occupied by the M351 in \u003cem\u003ePa\u003c/em\u003eCGL structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The second residue connecting chamber-1 and chamber-2 is a conserved tyrosine that packs against the pyridine ring of PLP and helps to orient the cofactor (Y99 in \u003cem\u003eSa\u003c/em\u003eCGL; Y114 in \u003cem\u003eHs\u003c/em\u003eCGL; Y98 in \u003cem\u003eBc\u003c/em\u003eCGL; Y97 in \u003cem\u003eLp\u003c/em\u003eCGL; Y133 in \u003cem\u003eTg\u003c/em\u003eCGL; and Y110 in \u003cem\u003ePa\u003c/em\u003eCGL, respectively). The volume and access of chamber-2 differ significantly in \u003cem\u003eSa\u003c/em\u003eCGL and \u003cem\u003ePa\u003c/em\u003eCGL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In apo-\u003cem\u003eHs\u003c/em\u003eCGL (no PLP cofactor present, PDB ID 3ELP), this tyrosine (Y114) appears displaced towards the bottom of the chamber-2 cavity, due to a partial unwinding of the last turn of helix α4. Upon binding of PLP (holo-\u003cem\u003eHs\u003c/em\u003eCGL), the last turn of this same helix recovers its helicity and reorients the tyrosine towards the interior of the catalytic chamber-1. This conformational change is thought to function as an access gate from chamber-2 to the catalytic site.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe conducted a biochemical and structural characterization of the H\u003csub\u003e2\u003c/sub\u003eS-producing enzyme CGL from \u003cem\u003eP. aeruginosa\u003c/em\u003e. Our results clearly show an enzymatic competence of \u003cem\u003ePa\u003c/em\u003eCGL to generate H\u003csub\u003e2\u003c/sub\u003eS using alternative substrates in addition to the canonical hydrolysis of L-Cth. Detailed structural comparison of \u003cem\u003ePa\u003c/em\u003eCGL with CGL enzymes from other species has revealed distinctive structural features within the primary cavities of the enzyme, which may modulate the access of substrates and/or inhibitors.\u003c/p\u003e \u003cp\u003eThe reaction catalyzed by CGL in the transsulfuration pathway involves elimination at the γ-carbon of L-Cth. However, CGLs are prone to β-elimination of L-Cth as a side reaction. Our kinetic analysis demonstrated that \u003cem\u003ePa\u003c/em\u003eCGL can catalyze both the α, γ- and α, β-cleavage of L-Cth to yield L-Cys and L-Hcys, respectively. Absorbance in the region of 440\u0026ndash;480 nm in the UV-Vis and CD spectra in the presence of L-Cth provided support for the formation of α-aminocrotonate and α-aminoacrylate, the reaction intermediates of the γ- and β-elimination mechanism, respectively. Our spectra of the enzyme-substrate complex in the long-wavelength region are similar to spectra of the \u003cem\u003eCitrobacter freundii\u003c/em\u003e methionine γ-lyase complex with methionine [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Two overlapping absorption bands with maxima at ~\u0026thinsp;460 and ~\u0026thinsp;485 nm were also observed in the yeast CGL [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The absorption at 480 nm likely corresponds to the formation of α-aminocrotonate, an essential intermediate of the γ-elimination reaction [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], while absorption at around 460 nm is probably due to α-aminoacrylate formation, which has been observed under steady-state conditions for β-elimination reactions of different PLP-dependent enzymes [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, \u003cem\u003ePa\u003c/em\u003eCGL exhibited a notably high catalytic efficiency for the hydrolysis of L-Cth (17 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), surpassing the activities of CGLs from other organisms (\u003cem\u003eLb\u003c/em\u003eCGL, 1.1 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003cem\u003eTg\u003c/em\u003eCGL, 2.2 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003cem\u003eBc\u003c/em\u003eCGL, 3.2 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003cem\u003eSc\u003c/em\u003eCGL, 2.1 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003cem\u003eHs\u003c/em\u003eCGL, 8.2 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecombinant purified \u003cem\u003ePa\u003c/em\u003eCGL also produces H\u003csub\u003e2\u003c/sub\u003eS using L-Cys or/and L-Hcys as alternative substrates. This aligns with the previous reports showing that inactivation of the \u003cem\u003eCse\u003c/em\u003e gene, encoding for the \u003cem\u003ePa\u003c/em\u003eCGL, leads to a significant reduction of H\u003csub\u003e2\u003c/sub\u003eS production in clinical isolates of \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. We investigated various reactions catalyzed by \u003cem\u003ePa\u003c/em\u003eCGL that lead to H\u003csub\u003e2\u003c/sub\u003eS biogenesis and, as side products, the uncommon thioether-bond containing amino acids L-Lanthionine and L-Homolanthionine. We found that \u003cem\u003ePa\u003c/em\u003eCGL efficiently produced H\u003csub\u003e2\u003c/sub\u003eS via different mechanisms such as (i) a \u003cem\u003eβ\u003c/em\u003e-elimination reaction where L-Cys was degraded to form H\u003csub\u003e2\u003c/sub\u003eS and L-Ser, (ii) a \u003cem\u003eβ\u003c/em\u003e-replacement reaction where two molecules of L-Cys were condensed to generate H\u003csub\u003e2\u003c/sub\u003eS and L-Lanthionine, (iii) a \u003cem\u003eγ\u003c/em\u003e -elimination reaction where L-Hcys was degraded to form H\u003csub\u003e2\u003c/sub\u003eS and L-Homoserine, (iv) a \u003cem\u003eγ\u003c/em\u003e-replacement reaction where two molecules of L-Hcys were condensed to generate H\u003csub\u003e2\u003c/sub\u003eS and L-Homolanthionine, and (v) the replacement of L-Hcys and L-Cys producing H\u003csub\u003e2\u003c/sub\u003eS and L-Cth. Under substrate saturating conditions, the catalytic efficiency (\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e) for the H\u003csub\u003e2\u003c/sub\u003eS elimination from L-Cys and L-Hcys is approximately 25- and \u0026minus;\u0026thinsp;68-fold lower, respectively, than for the canonical hydrolysis of L-Cth (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the occurrence and regulation of these alternative reactions in the cell remain unknown. The bacterial intracellular concentrations of L-Cys are tightly regulated during biosynthesis. Notably, \u003cem\u003eP. aeruginosa\u003c/em\u003e displays a high redundancy in L-Cys production. In addition to the CGL and CBS enzymes in the RTP, this pathogen possesses genes encoding the enzymes for the \u003cem\u003ede novo\u003c/em\u003e L-Cys synthesis pathway, i.e., serine acetyltransferase (SAT) catalyzing the condensation of L-Ser and the acetyl group of acetyl-CoA to form O-acetylserine (OAS), and the cysteine synthase (CS), which catalyzes the nucleophilic attack of sulfide (H\u003csub\u003e2\u003c/sub\u003eS) on OAS to form L-Cys and releasing acetate. L-Cys is also known to participate in the allosteric inhibition of SAT, leading to the production of OAS. OAS, in turn, is converted into N-acetylserine, an auto-inducer of the transcription regulator (the CysB protein), which acts as a sensor and regulator of the intracellular content of L-Cys and sulfur [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Further studies are required to determine whether \u003cem\u003ePa\u003c/em\u003eCGL serves as the primary checkpoint in the RTP in \u003cem\u003eP. aeruginosa\u003c/em\u003e and its involvement in L-Cys production.\u003c/p\u003e \u003cp\u003eShatalin et al [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] identified three potential CGL enzyme inhibitors, NL1, NL2, and NL3, which demonstrated strong specificity against bacterial CGL, with no impact on mammalian CGL. Experiments on the co-crystallization of these inhibitors with \u003cem\u003eS. aureus\u003c/em\u003e CGL made it possible to determine the binding sites of all three inhibitors to the enzyme. Our crystal structure of \u003cem\u003ePa\u003c/em\u003eCGL has enabled us to compare essential regions of this enzyme (chamber-1 and chamber-2) with the corresponding regions in other bacterial counterparts (including \u003cem\u003eS. aureus\u003c/em\u003e) and the human enzyme. This analysis revealed distinct structural characteristics that set \u003cem\u003ePa\u003c/em\u003eCGL apart, potentially paving the way for future drug development targeting this important metabolic enzyme. While chamber-1, corresponding to the catalytic site, is highly conserved among PLP-dependent enzymes, chamber-2 has unique physical-chemical properties and distinct conformation compared to other CGLs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). An intriguing difference in the chamber-2 of \u003cem\u003ePa\u003c/em\u003eCGL is the presence of the residue F114 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) which occupies equivalent position of the conserved residue Y103 in \u003cem\u003eSa\u003c/em\u003eCGL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Strikingly, the F114A mutation in \u003cem\u003ePa\u003c/em\u003eCGL did not result in loss of enzyme activity, as it occurred for the equivalent Y103A mutation in \u003cem\u003eSa\u003c/em\u003eCGL, supporting the notion that this residue not only determines the general characteristics of the entrance channel to chamber-2 (volume, size, steric hindrance), but modulates the overall volume of the chamber and, consequently, the type of molecule that can be accommodated within the chamber-2. In addition, the conformation of a long loop containing helix α13 (L347-370 in \u003cem\u003ePa\u003c/em\u003eCGL) in important for accessibility of the chamber-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This loop determines the width of the chamber-2 entrance and affects the void volume of the entire cleft (\u003cb\u003eFig. S5\u003c/b\u003e). Interestingly, our crystal structure of \u003cem\u003ePa\u003c/em\u003eCGL revealed that the helicity of this loop is partially lost, resulting in a more extended peptide segment that allows for a wider access into chamber-2 (a comparison of the internal cavity volume for chamber-2 and the fold of the equivalent regions to loop L347-370 in CGLs from different organisms is shown in \u003cb\u003eFig. S5\u003c/b\u003e). However, despite the cavity opening being bigger, the reorientation and shape of the loop L347-370 in \u003cem\u003ePa\u003c/em\u003eCGL reconfigured the internal contour and consequently, made the volume of the cavity smaller compared to what was observed, for example, in \u003cem\u003eSa\u003c/em\u003eCGL (loop L332-356) or the human enzyme (loop L349-373) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cb\u003eFig. S5\u003c/b\u003e). The comparison of the crystal structures of \u003cem\u003ePa\u003c/em\u003eCGL and \u003cem\u003eSa\u003c/em\u003eCGL complexed with NL1, NL2, and NL3 inhibitors suggests that the distinct arrangement of \u003cem\u003ePa\u003c/em\u003eCGL observed in this region would not hinder the binding of these molecules to \u003cem\u003ePa\u003c/em\u003eCGL, requiring only minor shifts of side chain to accommodate them inside. This is consistent with the inhibitory effectiveness of these molecules in P. aeruginosa [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePa\u003c/em\u003eCGL model predicted with AF2 (\u003cb\u003eFig. S5\u003c/b\u003e) exhibited a helical conformation of the loop L347-370 like that of other CGLs but different from the conformation found in our \u003cem\u003ePa\u003c/em\u003eCGL crystals. This suggests that the opening and closing of the chamber-2 cavity may differ from what was proposed based on the apo- and holo-states of the human enzyme. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cb\u003eFig S5, and Movie S1\u003c/b\u003e, the increased helicity of this loop in the \u003cem\u003ePa\u003c/em\u003eCGL model predicted by AF2 corresponds to a closed conformation of the chamber-2. In this state, the cavity significantly restricts its accessibility and internal volume. In the AF2-predicted closed conformation of \u003cem\u003ePa\u003c/em\u003eCGL, H353 residue would occupy a position like that found in other CGLs. On the other hand, the extended conformation observed in the crystals made the chamber-2 larger, thus likely more accessible for small molecules, such as NL1, NL2, and NL3 inhibitors. Interestingly, in this open state observed in the crystals, residue M351 occupies the equivalent position to the conserved histidine in other CGLs, suggesting a role in modulating the type of molecule that can be hosted inside the cavity.\u003c/p\u003e \u003cp\u003eOverall, our findings revealed the fundamental structural traits of the \u003cem\u003eP. aeruginosa\u003c/em\u003e CGL enzyme, which has gained significant attention as a potential pharmacological target due to its role in H\u003csub\u003e2\u003c/sub\u003eS biogenesis in this emerging and concerning pathogen.\u003c/p\u003e "},{"header":"EXPERIMENTAL SECTION","content":"\n\u003ch3\u003eProtein production\u003c/h3\u003e\n\u003cp\u003eGene sequence encoding for \u003cem\u003ePa\u003c/em\u003eCGL (PAO1_PA0400) with a N-terminal 6xHis-Tag was synthesized by Genscript, PCR amplified and cloned into a modified pET28a expression vector (Novagen). The F114A and F114N point mutations were introduced by site-directed mutagenesis using QuikChange II Kit (Agilent), using the primers in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. All constructs were verified by DNA sequencing performed by Eurofins Genomics. The \u003cem\u003ePa\u003c/em\u003eCGL constructs were transformed into \u003cem\u003eE. coli\u003c/em\u003e Rosetta (DE3) expression host cells (Novagen). Cells were grown in Luria-Bertani medium at 37\u0026deg;C to a turbidity of 0.6\u0026ndash;0.8 at 600 nm. Expression was induced with 0.5 mM IPTG for 16 h at 24\u0026deg;C. Cells were harvested and resuspended in 20 mM sodium phosphate pH 8.0, 150 mM NaCl, 0.1 mM DTT containing a protease inhibitor cocktail EDTA free. After sonication, the suspension was centrifuged at 30,000x\u003cem\u003eg\u003c/em\u003e for 20 min at 4\u0026deg;C. The supernatant was recovered and loaded on an Ni-NTA Sepharose column (GE-Healthcare) equilibrated with 20 mM sodium phosphate pH 8.0, 150 mM NaCl, 0.1 mM DTT and 10 mM imidazole. A linear gradient from 10 to 500 mM imidazole was then applied. Fractions enriched in \u003cem\u003ePa\u003c/em\u003eCGL were pooled together, concentrated and buffer exchanged into 20 mM sodium phosphate pH 8.0, 150 mM NaCl, 0.1 mM DTT buffer using Vivaspin concentrators (Sartorius). Each purification yielded about 100 mg of pure protein per liter of bacterial culture. To facilitate crystallization, an additional construct for \u003cem\u003ePa\u003c/em\u003eCGL wild-type with the C-terminal 6x-His tag was prepared using similar strategy as described above.\u003c/p\u003e \u003cp\u003eThe theoretical extinction coefficient of monomeric \u003cem\u003ePa\u003c/em\u003eCGL at 280 nm was 28,545 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.expasy.ch/tools/protparam.html\u003c/span\u003e\u003cspan address=\"http://www.expasy.ch/tools/protparam.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The PLP content of the enzyme was determined by releasing the coenzyme in 0.1 M NaOH and by using ε\u003csub\u003eM\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6,600 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 388 nm.\u003c/p\u003e \u003cp\u003eThe oligomeric state of \u003cem\u003ePa\u003c/em\u003eCGL variants was determined by gel filtration using a Sephacryl S-200 16/60 HR column in 20 mM sodium phosphate pH 8.0, 150 mM NaCl and 0.1 mM DTT. The calibration curve was generated following the protocols in [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe apo-form of \u003cem\u003ePa\u003c/em\u003eCGL was obtained by incubating the enzyme with phenylhydrazine hydrochloride following the protocol in [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The dissociation constant for PLP (\u003cem\u003eK\u003c/em\u003ed) was obtained by monitoring the change of intrinsic fluorescence (excitation was set at 295 nm) of 1 \u0026micro;M apo-protein at different concentrations of PLP (0.01\u0026ndash;4 \u0026micro;M) in 20 mM sodium phosphate pH 8.0 at 25\u0026deg; C on a FP8200 Jasco spectrofluorimeter [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eSpectroscopic measurements\u003c/h3\u003e\n\u003cp\u003eAbsorption spectra of 15 \u0026micro;M \u003cem\u003ePa\u003c/em\u003eCGL were collected on a Jasco V-750 UV-visible spectrophotometer in 20 mM sodium phosphate pH 8.0 at 25\u0026deg;C [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. CD spectra were recorded on CD spectropolarimeter Jasco J-1500 equipped with a Peltier type thermostated cell holder, as previously described [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Briefly, far-UV (190\u0026ndash;250 nm) spectra of 0.2 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cem\u003ePa\u003c/em\u003eCGL variants were collected in using a 0.1-cm path length quartz cuvette. Near UV-Vis (250\u0026ndash;600 nm) spectra of 1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cem\u003ePa\u003c/em\u003eCGL variants were recorded in 1-cm path length quartz cuvette at 25\u0026deg;C. A minimum of three accumulations were made for each scan, averaged, and corrected for the blank solution of corresponding buffer. Thermal unfolding profiles were collected by recording ellipticity at 222 nm in a temperature range between 15 to 90\u0026deg;C (scan rate 90\u0026deg;C/h) using 0.1-cm path length quartz cuvettes and protein concentration of 0.2 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. All CD measurements were recorded in 20 mM sodium phosphate pH 8.0 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eEnzyme activity assays\u003c/h3\u003e\n\u003cp\u003eThe CGL activity in the L-Cth γ-elimination reaction was determined by a previously described 5,5\u0026prime;-dithiobis-2-nitrobenzoic acid (DTNB) assay [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, the purified enzyme (1 \u0026micro;M) was assayed in a 200 \u0026micro;L reaction (50 mM MOPS, 50 mM bicine, 50 mM proline pH 8.0, 20 \u0026micro;M PLP, 0.2 mM DTNB) at 37\u0026deg;C in the presence of different concentration of the L-Cth substrate.\u003c/p\u003e \u003cp\u003eThe activities in the H\u003csub\u003e2\u003c/sub\u003eS-generating alternative reactions were measured using the lead acetate assay as described elsewhere [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The enzyme (1\u0026ndash;4 \u0026micro;M) was added to 0.4 mL of reaction mixture containing 50 mM Hepes pH 7.4, 20 \u0026micro;M PLP, 0.4 mM lead (II) acetate, and 0\u0026ndash;50 mM L-Cys or 0\u0026ndash;50 mM L-Hcys.\u003c/p\u003e \u003cp\u003ePyruvate formation was measured by monitoring NADH oxidation (ε\u003csub\u003e340\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6,200 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) via LDH assay [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Data for H\u003csub\u003e2\u003c/sub\u003eS production from L-Cys by \u003cem\u003ePa\u003c/em\u003eCGL were fitted following the kinetic models described prevoiusly [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Briefly, H\u003csub\u003e2\u003c/sub\u003eS production from L-Cys is the sum of two possible reactions, the b-elimination of L-Cys to generate L-Ser (reaction \u003cb\u003e3)\u003c/b\u003e or the condensation of two molecules of L-Cys to generate L-Lanthionine (reaction \u003cb\u003e4\u003c/b\u003e). Data for overall H\u003csub\u003e2\u003c/sub\u003eS production from L-Cys was fitted using Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) where v\u003csub\u003eL\u0026minus;ser\u003c/sub\u003e and v\u003csub\u003eLanthionine\u003c/sub\u003e are defined by Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and Eq.\u0026nbsp;(\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), respectively [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$${v}_{H2S}={v}_{L-Ser}+ {v}_{Lanthionine}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$${v}_{L-Ser}=\\frac{{v}_{max1 }[L-Cys]}{{K}_{m1(L-Cys)}+[L-Cys](1+\\frac{\\left[L-Cys\\right]}{{K}_{i}})}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$${v}_{Lanthionine}=\\frac{{v}_{max2 [L-Cys]{[L-Cys]}^{n}}}{\\left[L-Cys\\right]{\\left[L-Cys\\right]}^{n}+{K}_{m1 }{[L-Cys]}^{n}+\\left[L-Cys\\right]{K}_{m2}^{n}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere K\u003csub\u003em1\u003c/sub\u003e and V\u003csub\u003emax1\u003c/sub\u003e are associated to the unimolecular reaction, K\u003csub\u003em2\u003c/sub\u003e and V\u003csub\u003emax2\u003c/sub\u003e to substrate binding at the second site and the reaction velocity of the bimolecular reaction and n represents Hill coefficient.\u003c/p\u003e\n\u003ch3\u003eLiquid chromatography mass spectrometry (LC-MS/MS)\u003c/h3\u003e\n\u003cp\u003eA TSQ Fortis Triple Quadrupole mass spectrometer (Thermo Scientific) coupled to Ultimate 3000 HPLC system (Thermo Scientific) was used for this analysis. The products separation was performed on a Luna C18(2) column (150 x 4.6 mm, 3 \u0026micro;m particle size, Phenomenex) with gradient elution. The mobile phase was composed of formic acid (A, 0.1% formic acid in water) and acetonitrile (B, 0.1% formic acid in ACN). Chromatographic gradient elution was the following: constant flow of 0.4 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; 98% phase A at time 0, then decreased up to 5% A in 10 min and maintained at 5% A for 2 min and re-equilibrated for 5 min. The ESI source settings were ion spray voltage, +\u0026thinsp;3,500 V; ion transfer tube, 300\u0026deg;C; sheath gas and aux gas, 50 and 10, respectively, vaporizer temperature 350\u0026deg;C. Multiple reaction monitoring was optimized using nitrogen as collision gas (with pressure set at 1.5 mTorr). Two transitions for each substance were chosen for identification. Data acquisition and elaboration were performed by the Chromeleon (version 7.2, Thermo Fisher).\u003c/p\u003e\n\u003ch3\u003eProtein crystallization\u003c/h3\u003e\n\u003cp\u003eFor crystallization, the enzymes were buffer exchanged into 50 mM HEPES, 150 mM NaCl, 0.1 mM DTT pH 8.0. Preliminary crystallization trials were carried out by the vapor-diffusion technique in a sitting drop format with 96-well MRC crystallization plates, following a previously described protocol [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDrops consisted of 200 nL protein solution (20 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were mixed with 200 nL precipitant solution and incubated at 293K. The successful condition was scaled-up in a hanging-drop format using 24-well VDX plates (Hampton Research) in a reservoir with drops consisting of 0.5 \u0026micro;L protein (protein concentration of 20 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with 0.5 \u0026micro;L precipitant solution. This reservoir was composed of 9% v/v polyethylene glycol 4000 and 0.1 M sodium acetate pH 4.6 with a volume of 0.5 mL. The crystals were transferred to a crystallization buffer containing 9% (w/v) PEG 4000, 0.1 M sodium acetate pH 4.6, and 20% glycerol for a few seconds before being flash frozen in liquid nitrogen.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStructural determination by X-ray crystallography\u003c/h2\u003e \u003cp\u003eAll X-rays datasets were collected at Synchrotron beamlines XALOC (ALBA), I03/I24 (DIAMOND, UK) and ID29 of ESRF (Grenoble). Datasets were collected over a range of 0.1\u0026ndash;0.25\u0026deg; and the distance to the detector was set to reach resolution data between 1.6\u0026ndash;3.8 \u0026Aring; depending on the crystal, and according to the diffraction parameters previously determined by several test images. Several data set were collected but only one allowed the structural determination of \u003cem\u003ePa\u003c/em\u003eCGL (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Diffraction data were processed using HKL2000 511 or XDS 483 programs. The three-dimensional structure of \u003cem\u003ePa\u003c/em\u003eCGL was determined by MR method 488 with the Phaser-MR program489 from Phenix Suite 500 using the coordinates of \u003cem\u003eHs\u003c/em\u003eCGL holoenzyme (PDB ID 2NMP) as initial search model. The geometric quality of the models was assessed with MolProbity 490 integrated in Phenix suite. Figures were done with Pymol (The PyMOL Molecular Graphics System, Version 2.2.3, Schr\u0026ouml;dinger, LLC) and UCSF Chimera [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDeep Learning Structural Comparison\u003c/h2\u003e \u003cp\u003eProtein structure predictions were performed with AlphaFold 2.3.0 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] using an adapted version of the AF2 code (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/deepmind/alphafold\u003c/span\u003e\u003cspan address=\"https://github.com/deepmind/alphafold\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eSupporting Information\u003c/h2\u003e \u003cp\u003eList of mutagenic primers used to generate \u003cem\u003ePa\u003c/em\u003eCGL variants (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Statistics for data collection and refinement (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Properties of recombinant \u003cem\u003ePa\u003c/em\u003eCGL (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Effect of pH and temperature on \u003cem\u003ePa\u003c/em\u003eCGL γ-elimination of L-Cth (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Conformation of the loop 347\u0026ndash;370 in \u003cem\u003ePa\u003c/em\u003eCGL (\u003cb\u003eFig. S3\u003c/b\u003e). Structural and kinetic properties of \u003cem\u003ePa\u003c/em\u003eCGL variants (\u003cb\u003eFig. S4\u003c/b\u003e). Main cavities found in CGLs (\u003cb\u003eFig. S5\u003c/b\u003e).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.P., C.F.-R, C.C and I.O conceived the study, conducted the experiments, analysed the data, and edited the manuscript. F.F., P.D., M.L.M-C and M. PET. contributed to the discussion of results and edited the manuscript. A.DM., M.L.M.-C, T.M. supported funding acquisition and edited the manuscript. A.A. and L.A.M.-C. conceived the study, provided financial support, analysed the data, and wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e \u003cp\u003eThis research was supported by the MUR-PRIN 2022 grant No. 20224BYR59 to AA, by the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4 - Call for tender No. 341 of 03.15.2022 of Italian Ministry of University and Research funded by the European Union \u0026ndash; NextGenerationEU to ADM, by Spanish Ministry of Economy and Competitiveness Grant BFU2016-77408-R and by Spanish Ministerio de Ciencia e Innovaci\u0026oacute;n (MICINN), Grants No PID2019-109055RB-I00 and PID2022-141748OB-I00, to LAM-C. We also thank MINECO for the Severo Ochoa Excellence Accreditation (CEX2021-001136-S). TM acknowledges the support from University of Fribourg Research Pool grant (22\u0026thinsp;\u0026minus;\u0026thinsp;15). We thank the Centro Piattaforme Tecnologiche of the University of Verona for providing access to the spectroscopic and mass spectrometry platforms.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTacconelli, E., E. Carrara, A. Savoldi, S. Harbarth, M. Mendelson, D.L. Monnet, C. Pulcini, G. Kahlmeter, J. Kluytmans, Y. Carmeli, M. Ouellette, K. Outterson, J. Patel, M. Cavaleri, E.M. Cox, C.R. Houchens, M.L. Grayson, P. Hansen, N. Singh, U. Theuretzbacher, and N. Magrini, \u003cem\u003eDiscovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis\u003c/em\u003e. Lancet Infect Dis, 2018. 18(3): p. 318\u0026ndash;327.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSadikot, R.T., T.S. Blackwell, J.W. Christman, and A.S. Prince, \u003cem\u003ePathogen-host interactions in Pseudomonas aeruginosa pneumonia\u003c/em\u003e. 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Ding, and J. Li, \u003cem\u003eCurrent Advances on Structure-Function Relationships of Pyridoxal 5\u0026prime;-Phosphate-Dependent Enzymes\u003c/em\u003e. Frontiers in Molecular Biosciences, 2019. 6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorozova, E.A., N.P. Bazhulina, N.V. Anufrieva, D.V. Mamaeva, Y.V. Tkachev, S.A. Streltsov, V.P. Timofeev, N.G. Faleev, and T.V. Demidkina, \u003cem\u003eKinetic and spectral parameters of interaction of Citrobacter freundii methionine γ-lyase with amino acids\u003c/em\u003e. Biochemistry (Moscow), 2010. 75(10): p. 1272\u0026ndash;1280.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnufrieva, N.V., N.G. Faleev, E.A. Morozova, N.P. Bazhulina, S.V. Revtovich, V.P. Timofeev, Y.V. Tkachev, A.D. Nikulin, and T.V. Demidkina, \u003cem\u003eThe role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase.\u003c/em\u003e Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2015. 1854(9): p. 1220\u0026ndash;1228.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cem\u003eConversion of the Aminocrotonate Intermediate Limits the Rate of γ-Elimination Reaction Catalyzed by L-Cystathionine γ-lyase of the Yeast\u0026thinsp;\u0026lt;\u0026thinsp;i\u0026thinsp;\u0026gt;\u0026thinsp;Saccharomyces cerevisiae\u003c/em\u003e. The Journal of Biochemistry, 2003. 134(4): p. 607\u0026ndash;613.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConter, C., S. Fruncillo, C. Fern\u0026aacute;ndez-Rodr\u0026iacute;guez, L.A. Mart\u0026iacute;nez-Cruz, P. Dominici, and A. Astegno, \u003cem\u003eCystathionine β-synthase is involved in cysteine biosynthesis and H(2)S generation in Toxoplasma gondii\u003c/em\u003e. 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Kim, \u003cem\u003eStructural and Functional Characterization of Cystathionine γ-lyase from Bacillus cereus ATCC 14579.\u003c/em\u003e Journal of Agricultural and Food Chemistry, 2020. 68(51): p. 15267\u0026ndash;15274.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKredich, N.M., \u003cem\u003eThe molecular basis for positive regulation of cys promoters in Salmonella typhimurium and Escherichia coli\u003c/em\u003e. Mol Microbiol, 1992. 6(19): p. 2747\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAstegno, A., A. Giorgetti, A. Allegrini, B. Cellini, and P. Dominici, \u003cem\u003eCharacterization of C-S Lyase from C. diphtheriae: a possible target for new antimicrobial drugs.\u003c/em\u003e Biomed Res Int, 2013. 2013: p. 701536.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAstegno, A., G. Capitani, and P. Dominici, \u003cem\u003eFunctional roles of the hexamer organization of plant glutamate decarboxylase\u003c/em\u003e. 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Astegno, \u003cem\u003eInsights into Domain Organization and Regulatory Mechanism of Cystathionine Beta-Synthase from Toxoplasma gondii\u003c/em\u003e. Int J Mol Sci, 2022. 23(15).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh, S., D. Padovani, R.A. Leslie, T. Chiku, and R. Banerjee, \u003cem\u003eRelative contributions of cystathionine beta-synthase and gamma-cystathionase to H2S biogenesis via alternative trans-sulfuration reactions\u003c/em\u003e. J Biol Chem, 2009. 284(33): p. 22457\u0026ndash;22466.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePettersen, E.F., T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, and T.E. Ferrin, \u003cem\u003eUCSF Chimera\u0026ndash;a visualization system for exploratory research and analysis\u003c/em\u003e. J Comput Chem, 2004. 25(13): p. 1605\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pseudomonas aeruginosa, Cystathionine γ-lyase, Hydrogen sulfide, Multidrug resistant bacteria, Catalytic specificity, Crystal structure.","lastPublishedDoi":"10.21203/rs.3.rs-3869461/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3869461/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS), via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (\u003cem\u003ePa\u003c/em\u003eCGL). In addition to a canonical L-cystathionine hydrolysis, purified \u003cem\u003ePa\u003c/em\u003eCGL can catalyze the production of H\u003csub\u003e2\u003c/sub\u003eS using L-cysteine and/or L-homocysteine as alternative substrates. Comparative analysis with counterparts in other pathogens and humans revealed distinct structural features within the primary enzyme cavities, including a differently folded entrance loop to the catalytic site, potentially influencing substrate and/or inhibitor access. These findings offer opportunities for developing specific inhibitors to limit or eliminate bacterial H\u003csub\u003e2\u003c/sub\u003eS synthesis, weakening a defense barrier against the host immune system.\u003c/p\u003e","manuscriptTitle":"Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-24 07:03:40","doi":"10.21203/rs.3.rs-3869461/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-19T13:27:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-08T14:42:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32205c4f-6eef-4b40-ba2d-ca7cca357b9f","date":"2024-01-26T15:56:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-26T15:13:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-26T07:41:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-01-22T04:18:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-22T04:13:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-01-16T10:16:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"adf27095-4b56-46ee-ab96-dae6299b5f4e","owner":[],"postedDate":"January 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28304040,"name":"Health sciences/Diseases/Infectious diseases"},{"id":28304041,"name":"Health sciences/Diseases/Infectious diseases/Bacterial infection"},{"id":28304042,"name":"Biological sciences/Biochemistry"},{"id":28304043,"name":"Biological sciences/Biophysics"},{"id":28304044,"name":"Biological sciences/Structural biology"},{"id":28304045,"name":"Biological sciences/Structural biology/X ray crystallography"}],"tags":[],"updatedAt":"2024-05-01T22:51:04+00:00","versionOfRecord":{"articleIdentity":"rs-3869461","link":"https://doi.org/10.1038/s41598-024-57625-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-04-23 22:51:04","publishedOnDateReadable":"April 23rd, 2024"},"versionCreatedAt":"2024-01-24 07:03:40","video":"","vorDoi":"10.1038/s41598-024-57625-7","vorDoiUrl":"https://doi.org/10.1038/s41598-024-57625-7","workflowStages":[]},"version":"v1","identity":"rs-3869461","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3869461","identity":"rs-3869461","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}