Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.47 (cysteine synthase)
625 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gram-positive human pathogen Staphylococcus aureus is often isolated with media containing potassium tellurite, to which it has a higher level of resistance than Escherichia coli. The S. aureus cysM gene was isolated in a screen for genes that would increase the level of tellurite resistance of E. coli DH5alpha. The protein encoded by S. aureus cysM is sequentially and functionally homologous to the O-acetylserine (thiol)-lyase B family of cysteine synthase proteins. An S. aureus cysM knockout mutant grows poorly in cysteine-limiting conditions, and analysis of the thiol content in cell extracts showed that the cysM mutant produced significantly less cysteine than wild-type S. aureus SH1000. S. aureus SH1000 cannot use sulfate, sulfite, or sulfonates as the source of sulfur in cysteine biosynthesis, which is explained by the absence of genes required for the uptake and reduction of these compounds in the S. aureus genome. S. aureus SH1000, however, can utilize thiosulfate, sulfide, or glutathione as the sole source of sulfur. Mutation of cysM caused increased sensitivity of S. aureus to tellurite, hydrogen peroxide, acid, and diamide and also significantly reduced the ability of S. aureus to recover from starvation in amino acid- or phosphate-limiting conditions, indicating a role for cysteine in the S. aureus stress response and survival mechanisms.
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PMID:Role of a cysteine synthase in Staphylococcus aureus. 1499 87

The O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium catalyzes a beta-replacement reaction in which the beta-acetoxy group of O-acetyl-L-serine (OAS) is replaced by bisulfide to give L-cysteine and acetate. The kinetic mechanism of OASS is ping-pong with a stable alpha-aminoacrylate intermediate. The enzyme is a homodimer with one pyridoxal 5'-phosphate (PLP) bound per subunit deep within the protein in a cleft between the N- and C-terminal domains of each of the monomers. All of the active site residues are contributed by a single subunit. The enzyme cycles through open and closed conformations as it catalyzes its reaction with structural changes largely limited to a subdomain of the N-terminal domain. The elimination of acetic acid from OAS is thought to proceed via an anti-E2 mechanism, and the only catalytic group identified to date is lysine 41, which originally participates in Schiff base linkage to PLP. The transition state for the elimination of acetic acid is thought to be asynchronous and earlier for Cbeta-O bond cleavage than for Calpha-H bond cleavage.
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PMID:Structure and mechanism of O-acetylserine sulfhydrylase. 1507 90

Serine acetyltransferase is a key enzyme in the sulfur assimilation pathway of bacteria and plants, and is known to form a bienzyme complex with O-acetylserine sulfhydrylase, the last enzyme in the cysteine biosynthetic pathway. The biological function of the complex and the mechanism of reciprocal regulation of the constituent enzymes are still poorly understood. In this work the effect of complex formation on the O-acetylserine sulfhydrylase active site has been investigated exploiting the fluorescence properties of pyridoxal 5'-phosphate, which are sensitive to the cofactor microenvironment and to conformational changes within the protein matrix. The results indicate that both serine acetyltransferase and its C-terminal decapeptide bind to the alpha-carboxyl subsite of O-acetylserine sulfhydrylase, triggering a transition from an open to a closed conformation. This finding suggests that serine acetyltransferase can inhibit O-acetylserine sulfhydrylase catalytic activity with a double mechanism, the competition with O-acetylserine for binding to the enzyme active site and the stabilization of a closed conformation that is less accessible to the natural substrate.
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PMID:Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site: evidence from fluorescence spectroscopy. 1598 96

O-Phosphoserine sulfhydrylase is a new enzyme found in a hyperthermophilic archaeon, Aeropyrum pernix K1. This enzyme catalyzes a novel cysteine synthetic reaction from O-phospho-l-serine and sulfide. The crystal structure of the enzyme was determined at 2.0A resolution using the method of multi-wavelength anomalous dispersion. A monomer consists of three domains, including an N-terminal domain with a new alpha/beta fold. The topology folds of the middle and C-terminal domains were similar to those of the O-acetylserine sulfhydrylase-A from Salmonella typhimurium and the cystathionine beta-synthase from human. The cofactor, pyridoxal 5'-phosphate, is bound in a cleft between the middle and C-terminal domains through a covalent linkage to Lys127. Based on the structure determined, O-phospho-l-serine could be rationally modeled into the active site of the enzyme. An enzyme-substrate complex model and a mutation experiment revealed that Arg297, unique to hyperthermophilic archaea, is one of the most crucial residues for O-phosphoserine sulfhydrylation activity. There are more hydrophobic areas and less electric charges at the dimer interface, compared to the S.typhimurium O-acetylserine sulfhydrylase.
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PMID:Three-dimensional structure of a new enzyme, O-phosphoserine sulfhydrylase, involved in l-cysteine biosynthesis by a hyperthermophilic archaeon, Aeropyrum pernix K1, at 2.0A resolution. 1600 86

The metabolically versatile soil bacterium Pseudomonas putida has to cope with numerous abiotic stresses in its habitats. The stress responses of P. putida KT2440 to 4 degrees C, pH 4.5, 0.8 M urea, and 45 mM sodium benzoate were analyzed by determining the global mRNA expression profiles and screening for stress-intolerant nonauxotrophic Tn5 transposon mutants. In 392 regulated genes or operons, 36 gene regions were differentially expressed by more than 2.5-fold, and 32 genes in 23 operons were found to be indispensable for growth during exposure to one of the abiotic stresses. The transcriptomes of the responses to urea, benzoate, and 4 degrees C correlated positively with each other but negatively with the transcriptome of the mineral acid response. The CbrAB sensor kinase, the cysteine synthase CysM, PcnB and VacB, which control mRNA stability, and BipA, which exerts transcript-specific translational control, were essential to cope with cold stress. The cyo operon was required to cope with acid stress. A functional PhoP, PtsP, RelA/SpoT modulon, and adhesion protein LapA were necessary for growth in the presence of urea, and the outer membrane proteins OmlA and FepA and the phosphate transporter PstBACS were indispensable for growth in the presence of benzoate. A lipid A acyltransferase (PP0063) was a mandatory component of the stress responses to cold, mineral acid, and benzoate. Adaptation of the membrane barrier, uptake of phosphate, maintenance of the intracellular pH and redox status, and translational control of metabolism are key mechanisms of the response of P. putida to abiotic stresses.
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PMID:Functional genomics of stress response in Pseudomonas putida KT2440. 1670 99

O-Acetylserine sulfhydrylase is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the beta-acetoxy group of O-acetyl-l-serine by a thiol to give l-cysteine. Two isozymes are found in Salmonella typhimurium, with the A-isozyme expressed under aerobic and the B-isozyme expressed under anaerobic conditions. The structure of O-acetylserine sulfhydrylase B has been solved to 2.3 A and exhibits overall a fold very similar to that of the A-isozyme. The main difference between the two isozymes is the more hydrophilic active site of the B-isozyme with two ionizable residues, C280 and D281, replacing the neutral residues S300 and P299, respectively, in the A-isozyme. D281 is above the re face of the cofactor and is within hydrogen-bonding distance to Y286, while C280 is located about 3.4 A from the pyridine nitrogen (N1) of the internal Schiff base. The B-isozyme has a turnover number (V/Et) 12.5-fold higher than the A-isozyme and an approximately 10-fold lower Km for O-acetyl-l-serine. Studies of the first half-reaction by rapid-scanning stopped-flow indicate a first-order conversion of the internal Schiff base to the alpha-aminoacrylate intermediate at any concentration of O-acetyl-l-serine. The Kd values for formation of the external Schiff base with cysteine and serine, obtained by spectral titration, are pH dependent and exhibit a pKa of 7.0-7.5 (for a group that must be unprotonated for optimum binding) with values, above pH 8.0, of about 3.0 and 30.0 mM, respectively. In both cases the neutral enolimine is favored at high pH. Failure to observe the pKa for the alpha-amines of cysteine and serine in the pKESB vs pH profile suggests a compensatory effect resulting from titration of a group on the enzyme with a pKa in the vicinity of the alpha-amine's pKa. The pH dependence of the first-order rate constant for decay of the alpha-aminoacrylate intermediate to give pyruvate and ammonia gives a pKa of about 9 for the active site lysine (K41), a pH unit higher than that of the A-isozyme. The difference in pH dependence of the pKESB for cysteine and serine, the higher pKa for K41, and the preference for the neutral species at high pH compared to the A-isozyme can be explained by titration of C280 to give the thiolate. Subtle conformational differences between O-acetylserine sulfhydrylase A and O-acetylserine sulfhydrylase B are detected by comparing the absorption and emission spectra of the internal aldimine in the absence and presence of the product acetate and of the external aldimine with l-serine. The two isozymes show a different equilibrium distribution of the enolimine and ketoenamine tautomers, likely as a result of a more polar active site for O-acetylserine sulfhydrylase B. The distribution of cofactor tautomers is dramatically affected by the ligation state of the enzyme. In the presence of acetate, which occupies the alpha-carboxylate subsite, the equilibrium between tautomers is shifted toward the ketoenamine tautomer, as a result of a conformational change affecting the structure of the active site. This finding, in agreement with structural data, suggests for the O-acetylserine sulfhydrylase B-isozyme a higher degree of conformational flexibility linked to catalysis.
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PMID:Structure, mechanism, and conformational dynamics of O-acetylserine sulfhydrylase from Salmonella typhimurium: comparison of A and B isozymes. 1758 14

The biosynthesis of cysteine is a crucial metabolic pathway supplying a building block for de novo protein synthesis but also a reduced thiol as a component of the oxidative defense mechanisms that appear particularly vital in the dormant state of Mycobacterium tuberculosis. We here show that the cysteine synthase CysM is, in contrast to previous annotations, an O-phosphoserine-specific cysteine synthase. CysM belongs to the fold type II pyridoxal 5'-phosphate-dependent enzymes, as revealed by the crystal structure determined at 2.1-angstroms resolution. A model of O-phosphoserine bound to the enzyme suggests a hydrogen bonding interaction of the side chain of Arg220 with the phosphate group as a key feature in substrate selectivity. Replacement of this residue results in a significant loss of specificity for O-phosphoserine. Notably, reactions with sulfur donors are not affected by the amino acid replacement. The specificity of CysM toward O-phosphoserine together with the previously established novel mode of sulfur delivery via thiocarboxylated CysO (Burns, K. E., Baumgart, S., Dorrestein, P. C., Zhai, H., McLafferty, F. W., and Begley, T. P. (2005) J. Am. Chem. Soc. 127, 11602-11603) provide strong evidence for an O-phosphoserine-based cysteine biosynthesis pathway in M. tuberculosis that is independent of both O-acetylserine and the sulfate reduction pathway. The existence of an alternative biosynthetic pathway to cysteine in this pathogen has implications for the design strategy aimed at inhibition of this metabolic route.
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PMID:Cysteine synthase (CysM) of Mycobacterium tuberculosis is an O-phosphoserine sulfhydrylase: evidence for an alternative cysteine biosynthesis pathway in mycobacteria. 1879 56

O-acetylserine sulfhydrylase (OASS) is a key enzyme involved in the pathway of the cysteine biosynthesis. The gene of OASS from Acidithiobacillus ferrooxidans ATCC 23270 was cloned and expressed in E. coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. Colors and UV-vis scanning results of the recombinant protein confirmed that it was a pyridoxal 5'-phosphate (PLP)-containing protein. Sequence alignment and site-directed mutation of the enzyme revealed that the cofactor PLP is covalently bound in Schiff base linkage with K30, as well as the two residues H150 and H168 were the crucial residues for PLP binding and stabilization.
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PMID:K30, H150, and H168 are essential residues for coordinating pyridoxal 5'-phosphate of O-acetylserine sulfhydrylase from Acidithiobacillus ferrooxidans. 2003 72

The regulation of enzyme activity through the transient formation of multiprotein assemblies plays an important role in the control of biosynthetic pathways. One of the first regulatory complexes to be discovered was cysteine synthase (CS), formed by the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). These enzymes are at the branch point of the sulfur, carbon, and nitrogen assimilation pathways. Understanding the mechanism of complex formation helps to clarify the role played by CS in the regulation of sulfur assimilation in bacteria and plants. To this goal, stopped-flow fluorescence spectroscopy was used to characterize the interaction of SAT with OASS, at different temperatures and pH values, and in the presence of the physiological regulators cysteine and bisulfide. Results shed light on the mechanism of complex formation and regulation, so far poorly understood. Cysteine synthase assembly occurs via a two-step mechanism involving rapid formation of an encounter complex between the two enzymes, followed by a slow conformational change. The conformational change likely results from the closure of the active site of OASS upon binding of the SAT C-terminal peptide. Bisulfide, the second substrate and a feedback inhibitor of OASS, stabilizes the CS complex mainly by decreasing the back rate of the isomerization step. Cysteine, the product of the OASS reaction and a SAT inhibitor, slightly affects the kinetics of CS formation leading to destabilization of the complex.
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PMID:A two-step process controls the formation of the bienzyme cysteine synthase complex. 2016 78

The pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) catalyzes the synthesis of cysteine in bacteria and plants. In bacteria two isoenzymes are present, OASS-A and OASS-B, with distinct structural, functional, and regulatory properties. In order to gain a deeper insight into OASS-B dynamic and functional properties, single and double mutants of the three tryptophan residues, Trp28, Trp159, and Trp212, were prepared and their fluorescence emission properties were characterized in the absence and presence of substrate and ligands by steady-state and time-resolved spectrofluorimetry. Residue Trp28 was found to be mainly responsible for Trp fluorescence emission, whereas Trp212, located in a highly flexible region near the active site, is mainly responsible for an energy-transfer to PLP leading to an emission at 500 nm. Not surprisingly, mutation of Trp212 affects OASS-B activity. Trp159 slightly contributes to both direct emission and energy transfer to PLP. Time-resolved fluorescence measurements confirmed these findings, observing a third longer tryptophan lifetime for apo-OASS-B, in addition to the two lifetimes that are present in the holo-enzyme and mutants. A comparison with the emissions previously determined for OASS-A indicates that OASS-B active site is likely to be more polar and flexible, in agreement with a broader substrate specificity and higher catalytic efficiency.
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PMID:Exploring O-acetylserine sulfhydrylase-B isoenzyme from Salmonella typhimurium by fluorescence spectroscopy. 2093 39


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