Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.47 (cysteine synthase)
 625 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Beta-(pyrazol-1-yl)-L-alanine (beta-PA), a model nonproteinaceous amino acid, was specifically synthesized by two methods using recombinant Escherichia coli cells that express cysteine synthase, comprising serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A (OASS-A) and related enzymes from E. coli. In the first method (method A), recombinant cells that express wild-type SAT, OASS-A, acetate kinase (AK), and phosphotransacetylase (PTA) showed the highest beta-PA production. beta-PA was produced at 140 mM from 200 mM L-serine and 200 mM pyrazole under optimum conditions. Using the cells expressing SATDeltaC20 (truncated SAT), OASS-A, AK, and PTA, beta-PA was produced at a level of only 80 mM, whereas O-acetyl-serine (OAS) was found to be secreted into the broth. Under optimum conditions, OAS accumulated at levels of around 105 mM from 300 mM L-serine. Thus, in the second method (method B), the secreted OAS was used as the substrate for the syntheses of beta-PA and beta-(triazol-1-yl)-L-alanine (beta-TA). The OAS that accumulated in the broth was efficiently converted to beta-PA and beta-TA at levels of around 90 mM from 105 mM OAS using free OASS-A. In both methods A and B, the addition of glucose was essential for the efficient production of beta-PA and OAS, respectively.
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PMID:Production of nonproteinaceous amino acids using recombinant Escherichia coli cells expressing cysteine synthase and related enzymes with or without the secretion of O-acetyl-L-serine. 1623 37

We report here the function of L-serine O-acetyltransferase (SAT) from the glutamic acid-producing bacterium Corynebacterium glutamicum. Based on the genome sequence of C. glutamicum and the NH(2)-terminal amino-acid sequence, the gene encoding SAT (cysE) was cloned and expressed in C. glutamicum. Deletion analysis of the 5'-noncoding region showed a putative -10 region ((-27)TTAAGT(-22) or (-26)TAAGTC(-21)) and a possible ribosome-binding site ((-12)AGA(-10)) just upstream from the start codon. We found that the SAT activity was sensitive to feedback inhibition by L-cysteine, and that SAT synthesis was repressed by L-methionine. Further, cysE-disrupted cells showed L-cysteine auxotrophy, indicating that C. glutamicum synthesizes L-cysteine from L-serine via O-acetyl-L-serine through the pathway involving SAT and O-acetyl-L-serine sulfhydrylase in the same manner as Escherichia coli.
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PMID:Functional analysis of L-serine O-acetyltransferase from Corynebacterium glutamicum. 1643 75

In higher plants the biosynthesis of l-cysteine from l-serine, acetylCoA, and sulfide requires serine transacetylase and O-acetylserine sulfhydrylase. The distribution of these enzymes in kidney bean (Phaseolus vulgaris L. cv. Red Kidney) seedlings was determined. Between one-third and two-thirds of the serine transacetylase activity was associated with mitochondria, whereas all of the O-acetyl-serine sulfhydrylase activity was present in the soluble fraction of cell homogenates. In a 14-day plant approximately two-thirds of the O-acetylserine sulfhydrylase activity and approximately one-half of the serine transacetylase activity was found in the leaves.Sulfur-deficient plants were grown to determine the effect of sulfur status on the levels of cysteine biosynthetic enzymes. Total extractable serine transacetylase activity was not affected by sulfur deficiency; in contrast, there was an increase in O-acetylserine sulfhydrylase activity under these conditions.
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PMID:Studies of l-Cysteine Biosynthetic Enzymes in Phaseolus vulgaris L. 1665 99

During growth on l-cysteine ethylester, Chlorella fusca (211-8b) accumulated a substance which contained bound sulfide, which could be liberated by reduction with dithioerythritol (DTE) as inorganic sulfide. This substance was extracted with hot methanol and purified by thin layer chromatography. This substance liberated free sulfide when incubated with mono- and dithiols, and thiocyanate was formed after heating with KCN. The isolated substance cochromatographed with authentic sulfur flower using different solvent systems for thin layer chromatography, high pressure liquid chromatography, and the identical spectrum with a relative lambda(max) at 263 nm was found. The chemical structure was confirmed by mass spectrometry showing a molecular weight of 256 m/e for the S(8) configuration. No labeled elemental sulfur was detected when the cells were grown on [(35)S]sulfate and l-cysteine ethylester indicating the origin of elemental sulfur from l-cysteine ethylester. C. fusca seems to have enzymes for the metabolism of elemental sulfur, since it disappeared after prolonged growth into the stationary phase. Cysteine was formed from O-acetyl-l-serine and elemental sulfur in the presence of thiol groups and purified cysteine synthase from spinach or Chlorella.
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PMID:Formation of Elemental Sulfur by Chlorella fusca during Growth on l-Cysteine Ethylester. 1666 75

Synechococcus elongatus PCC 7942 was able to grow with several S sources. The sulphur metabolizing enzymes viz. ATP sulphurylase, cysteine synthase, thiosulphate reductase and L- and D-cysteine desulphydrases were regulated by sulphur sources, particularly by sulphur amino acids and organic sulphate esters. Sulphur starvation reduced ATP sulphurylase and cysteine synthase whereas reduced glutathione appreciated Cys degradation activity. With partially purified enzymes apparent Km values for sulphate, ATP, D- and L-Cys, thiosulphate, sulphide and O-acetyl serine were in a range of 12-50 microM. p-Nitrophenyl sulphate inhibited ATP sulphurylase competitively. Met was a feedback inhibitor of several key enzymes.
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PMID:Catalytic and regulatory properties of sulphur metabolizing enzymes in cyanobacterium Synechococcus elongatus PCC 7942. 1699 35

Cysteine biosynthetic genes are up-regulated in the persistent phase of Mycobacterium tuberculosis, and the corresponding enzymes are therefore of interest as potential targets for novel antibacterial agents. cysK1 is one of these genes and has been annotated as coding for an O-acetylserine sulfhydrylase. Recombinant CysK1 is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine to cysteine. The crystal structure of the enzyme was determined to 1.8A resolution. CysK1 belongs to the family of fold type II PLP enzymes and is similar in structure to other O-acetylserine sulfhydrylases. We were able to trap the alpha-aminoacrylate reaction intermediate and determine its structure by cryocrystallography. Formation of the aminoacrylate complex is accompanied by a domain rotation resulting in active site closure. The aminoacrylate moiety is bound in the active site via the covalent linkage to the PLP cofactor and by hydrogen bonds of its carboxyl group to several enzyme residues. The catalytic lysine residue is positioned such that it can protonate the Calpha-carbon atom of the aminoacrylate only from the si-face, resulting in the formation of L-cysteine. CysK1 is competitively inhibited by a four-residue peptide derived from the C-terminal of serine acetyl transferase. The crystallographic analysis reveals that the peptide binds to the enzyme active site, suggesting that CysK1 forms an bi-enzyme complex with serine acetyl transferase, in a similar manner to other bacterial and plant O-acetylserine sulfhydrylases. The structure of the enzyme-peptide complex provides a framework for the design of strong binding inhibitors.
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PMID:Structural insights into catalysis and inhibition of O-acetylserine sulfhydrylase from Mycobacterium tuberculosis. Crystal structures of the enzyme alpha-aminoacrylate intermediate and an enzyme-inhibitor complex. 1756 78

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

Methionine gamma-lyase (MGL) (EC 4.4.1.11), which is present in certain lineages of bacteria, plants, and protozoa but missing in mammals, catalyzes the single-step degradation of sulfur-containing amino acids (SAAs) to alpha-keto acids, ammonia, and thiol compounds. In contrast to other organisms possessing MGL, anaerobic parasitic protists, namely Entamoeba histolytica and Trichomonas vaginalis, harbor a pair of MGL isozymes. The enteric protozoon En. histolytica shows various unique aspects in its metabolism, particularly degradation of SAAs. Trifluoromethionine (TFM), a halogenated analog of Met, has been exploited as a therapeutic agent against cancer as well as against infections by protozoan organisms and periodontal bacteria. However, its mechanism of action remains poorly understood. In addition, the physiological significance of the presence of two MGL isozymes in these protists remains unclear. In this study, we compared kinetic parameters of the wild-type and mutants, engineered by site-directed mutagenesis, of the two MGL isotypes from En. histolytica (EhMGL1 and EhMGL2) for various potential substrates and TFM. Intracellular concentrations of l-Met and l-Cys suggested that these SAAs are predominantly metabolized by EhMGL1, not by EhMGL2. It is unlikely that O-acetyl-l-serine is decomposed by EhMGLs, given the kinetic parameters of cysteine synthase reported previously. Comparison of the wild-type and mutants revealed that the contributions of several amino acids implicated in catalysis differ between the two isozymes, and that the degradation of TFM is less sensitive to alterations of these residues than is the degradation of physiological substrates. These results support the use of TFM to target MGL.
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PMID:Kinetic characterization of methionine gamma-lyases from the enteric protozoan parasite Entamoeba histolytica against physiological substrates and trifluoromethionine, a promising lead compound against amoebiasis. 1819 85

Cysteine plays a major role in the antioxidative defense mechanisms of the human parasite Entameoba histolytica. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine biosynthetic pathway involving two key enzymes O-acetyl-L-serine sulfhydrylase (OASS) and serine acetyl transferase (SAT). The crystal structure of native OASS from Entameoba histolytica (EhOASS) has been determined at 1.86 A resolution and in complex with its product cysteine at 2.4 A resolution. In comparison with other known OASS structures, insertion in the N-terminal region and C-terminal helix reveal critical differences, which may influence the protein-protein interactions. In spite of lacking chloride binding site at the dimeric interface, the N-terminal extension compared with other known cysteine synthases, participates in dimeric interactions in an interesting domain swapping manner, enabling it to form a stronger dimer. Sulfate is bound in the active site of the native structure, which is replaced by cysteine in the cysteine bound form causing reorientation of the small N-terminal domain and thus closure of the active site. Ligand binding constants of OAS, Cys, and Met with EhOASS are comparable with other known OASS indicating similar active site arrangement and dynamics. The cysteine complexed structure represents the snapshot of the enzyme just before releasing the final product with a closed active site. The C-terminal helix positioning in the EhOASS may effect its interactions with EhSAT and thus influencing the formation of the cysteine synthase complex in this organism.
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PMID:Crystal structure of native O-acetyl-serine sulfhydrylase from Entamoeba histolytica and its complex with cysteine: structural evidence for cysteine binding and lack of interactions with serine acetyl transferase. 1835 May 70


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