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The active site of O-acetylserine sulfhydrylase is the anchor point for bienzyme complex formation with serine acetyltransferase.

ABSTRACT: The biosynthesis of cysteine in bacteria and plants is carried out by a two-step pathway, catalyzed by serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS; O-acetylserine [thiol] lyase). The aerobic form of OASS forms a tight bienzyme complex with SAT in vivo, termed cysteine synthase. We have determined the crystal structure of OASS in complex with a C-terminal peptide of SAT required for bienzyme complex formation. The binding site of the peptide is at the active site of OASS, and its C-terminal carboxyl group occupies the same anion binding pocket as the alpha-carboxylate of the O-acetylserine substrate of OASS. These results explain the partial inhibition of OASS by SAT on complex formation as well as the competitive dissociation of the complex by O-acetylserine.

SUBMITTER: Huang B

PROVIDER: S-EPMC1082839 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

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The active site of O-acetylserine sulfhydrylase is the anchor point for bienzyme complex formation with serine acetyltransferase.

Huang Bin B Vetting Matthew W MW Roderick Steven L SL

Journal of bacteriology 20050501 9

The biosynthesis of cysteine in bacteria and plants is carried out by a two-step pathway, catalyzed by serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS; O-acetylserine [thiol] lyase). The aerobic form of OASS forms a tight bienzyme complex with SAT in vivo, termed cysteine synthase. We have determined the crystal structure of OASS in complex with a C-terminal peptide of SAT required for bienzyme complex formation. The binding site of the peptide is at the active site of OASS ...[more]

PMID: 15838047

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Project description:The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.

Project description:A crystal structure of serine acetyltransferase (SAT) with cysteine bound in the serine subsite of the active site shows that both H154 and H189 are within hydrogen-bonding distance to the cysteine thiol [Olsen, L. R., Huang, B., Vetting, M. W., and Roderick, S. L. (2004) Biochemistry 43, 6013 -6019]. In addition, H154 is in an apparent dyad linkage with D139. The structure suggests that H154 is the most likely catalytic general base and that H189 and D139 may also play important roles during the catalytic reaction. Site-directed mutagenesis was performed to mutate each of these three residues to Asn, one at a time. The V1/Et value of all of the single mutant enzymes decreased, with the largest decrease (approximately 1240-fold) exhibited by the H154N mutant enzyme. Mutation of both histidines, H154N/H189N, gave a V1/Et approximately 23700-fold lower than that of the wild-type enzyme. An increase in K Ser was observed for the H189N, D139N, and H154N/H189N mutant enzymes, while the H154N mutant enzyme gave an 8-fold decrease in K Ser. For all three single mutant enzymes, V1/Et and V1/K Ser Et decrease at low pH and give a pKa of about 7, while the V1/Et of the double mutant enzyme was pH independent. The solvent deuterium kinetic isotope effects on V 1 and V1/K Ser decreased compared to wild type for the H154N mutant enzyme and increased for the H189N mutant enzyme but was about the same as that of wild type for D139N and H154N/H189N. Data suggest that H154, H189, and D139 play different catalytic roles for SAT. H154 likely serves as a general base, accepting a proton from the beta-hydroxyl of serine as the tetrahedral intermediate is formed upon nucleophilic attack on the thioester carbonyl of acetyl-CoA. However, activity is not completely lost upon elimination of H154, and thus, H189 may be able to serve as a backup general base at a lower efficiency compared to H154; it also aids in binding and orienting the serine substrate. Aspartate 139, in dyad linkage with H154, likely facilitates catalysis by increasing the basicity of H154.

Project description:BackgroundThe importance of understanding the detailed mechanism of cysteine biosynthesis in bacteria is underscored by the fact that cysteine is the only sulfur donor for all cellular components containing reduced sulfur. O-acetylserine sulfhydrylase (OASS) catalyzes this crucial last step in the cysteine biosynthesis and has been recognized as an important gene for the survival and virulence of pathogenic bacteria. Structural and kinetic studies have contributed to the understanding of mechanistic aspects of OASS, but details of ligand recognition features of OASS are not available. In the absence of any detailed study on the energetics of ligand binding, we have studied the thermodynamics of OASS from Salmonella typhimurium (StOASS), Haemophilus influenzae (HiOASS), and Mycobacterium tuberculosis (MtOASS) binding to their substrate O-acetylserine (OAS), substrate analogue (methionine), and product (cysteine).ResultsLigand binding properties of three OASS enzymes are studied under defined solution conditions. Both substrate and product binding is an exothermic reaction, but their thermodynamic signatures are very different. Cysteine binding to OASS shows that both enthalpy and entropy contribute significantly to the binding free energy at all temperatures (10-30°C) examined. The analyses of interaction between OASS with OAS (substrate) or methionine (substrate analogue) revealed a completely different mode of binding. Binding of both OAS and methionine to OASS is dominated by a favorable entropy change, with minor contribution from enthalpy change (?H(St-Met) = -1.5 ± 0.1 kJ/mol; T?S(St-Met) = 8.2 kJ/mol) at 20°C. Our salt dependent ligand binding studies indicate that methionine binding affinity is more sensitive to [NaCl] as compared to cysteine affinity.ConclusionsWe show that OASS from three different pathogenic bacteria bind substrate and product through two different mechanisms. Results indicate that predominantly entropy driven methionine binding is not mediated through classical hydrophobic binding, instead, may involve desolvation of the polar active site. We speculate that OASS in general, may exhibit two different binding mechanisms for recognizing substrates and products.

Dataset Information

The active site of O-acetylserine sulfhydrylase is the anchor point for bienzyme complex formation with serine acetyltransferase.

Publications

The active site of O-acetylserine sulfhydrylase is the anchor point for bienzyme complex formation with serine acetyltransferase.

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