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)

O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Only Trp161 is highly conserved in eucaryotes and bacteria. The coenzyme pyridoxal 5'-phosphate is bound in a cleft between the two domains. The enzyme undergoes an open to closed conformational transition upon substrate binding. The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques. The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction. The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface. The W161Y mutation, occurring in the C-terminal domain, produces a reduction of the accessibility of the active site to acrylamide and stabilizes thermodynamically the N-terminal domain, a result consistent with stronger interdomain interactions.
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PMID:Surface-exposed tryptophan residues are essential for O-acetylserine sulfhydrylase structure, function, and stability. 1281 39

We highly purified O-acetylserine sulfhydrylase from the glutamate-producing bacterium Corynebacterium glutamicum. The molecular mass of the purified enzyme was 34,500 as determined by SDS-polyacrylamide gel electrophoresis, and 70,800 as determined by gel filtration chromatography. It had an apparent Km of 7.0 mM for O-acetylserine and a Vmax of 435 micromol min-1 (mg x protein)-1. This is the first report of the cysteine biosynthetic enzyme of C. glutamicum in purified form.
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PMID:Purification and characterization of O-Acetylserine sulfhydrylase of Corynebacterium glutamicum. 1527 66

We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.
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PMID:Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits. 1582 11

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.
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PMID:The active site of O-acetylserine sulfhydrylase is the anchor point for bienzyme complex formation with serine acetyltransferase. 1583 47

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

Compared with traditional two-dimensional (2D) proteome analysis of Streptococcus mutans grown as a biofilm from a planktonic culture at steady state (Rathsam et al., Microbiol. 2005, 151, 1823-1837), the use of 2D fluorescence difference gel electrophoresis (DIGE) led to a 3-fold increase in the number of identified protein spots that were significantly altered in their level of expression (P < 0.050). Of the 73 identified proteins, only nine were up-regulated in biofilm grown cells. The results supported the previously surmised hypothesis that general metabolic functions were down-regulated in response to a reduction in growth rate in mature S. mutans biofilms. Up-regulation of competence proteins without any concomitant increase in stress-responsive proteins was confirmed, while the levels of glucosyltransferase C (GtfC), involved in glucan formation, O-acetylserine sulfhyrylase (cysteine synthetase A; CsyK), implicated in the formation of [Fe-S] clusters, and a hypothetical protein encoded by the open reading frame, SMu0188, were also up-regulated.
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PMID:Two-dimensional fluorescence difference gel electrophoretic analysis of Streptococcus mutans biofilms. 1633 63

Cysteine synthesis in plants represents the final step of assimilatory sulfate reduction and the almost exclusive entry reaction of reduced sulfur into metabolism not only of plants, but also the human food chain in general. It is accomplished by the sequential reaction of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they form the hetero-oligomeric cysteine synthase complex (CSC). Recent evidence is reviewed that identifies the dual function of the CSC as a sensor and as part of a regulatory circuit that controls cellular sulfur homeostasis. Computational modeling of three-dimensional structures of plant SAT and OAS-TL based on the crystal structure of the corresponding bacterial enzymes supports quaternary conformations of SAT as a dimer of trimers and OAS-TL as a homodimer. These findings suggest an overall alpha6beta4 structure of the subunits of the plant CSC. Kinetic measurements of CSC dissociation triggered by the reaction intermediate O-acetylserine as well as CSC stabilization by sulfide indicate quantitative reactions that are suited to fine-tune the equilibrium between free and associated CSC subunits. In addition, in vitro data show that SAT requires binding to OAS-TL for full activity, while at the same time bound OAS-TL becomes inactivated. Since OAS concentrations inside cells increase upon sulfate deficiency, whereas sulfide concentrations most likely decrease, these data suggest the dissociation of the CSC in vivo, accompanied by inactivation of SAT and activation of OAS-TL function in their free homo-oligomer states. Biochemical evidence describes this protein-interaction based mechanism as reversible, thus closing the regulatory circuit. The properties of the CSC and its subunits are therefore consistent with models of positive regulation of sulfate uptake and reduction in plants by OAS as well as a demand-driven repression/de-repression by a sulfur intermediate, such as sulfide.
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PMID:Functional analysis of the cysteine synthase protein complex from plants: structural, biochemical and regulatory properties. 1638 30

O-Acetylserine sulfhydrylase-B (OASS-B, EC 2.5.1.47) is one of the two isozymes produced by Escherichia coli that catalyze the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. The cysM gene encoding OASS-B was cloned and the enzyme was overexpressed in E. coli using pUC19 with a lacUV5 promoter. The enzyme was purified to homogeneity, as evidenced by SDS-PAGE. Approximately 300 mg of purified OASS-B was obtained from 1600 mL of culture broth with a purification yield of 60% or higher. The purified OASS-B was characterized and its properties compared with OASS-A. OASS-B did not form a complex with E. coli serine acetyltransferase (SAT, EC 2.3.1.30) and showed a wide range of substrate specificity in nonproteinaceous amino acid synthesis.
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PMID:Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli. 1654 1

Purification of O-acetylserine sulfhydrylase (OASS) from seedlings of two species of Phaseolus reveals the presence in both species of two forms of this enzyme. The isolation and purification procedure gives purification of 7- to 160-fold for individual isoenzymes with specific activities ranging from 33 IU mg(-1) to 775 IU mg(-1) protein.Detailed study of the basic kinetic parameters of the OASS isoenzymes indicates that both forms from Phaseolus vulgaris (which are of about equal specific activity) display substrate inhibition by S(2-) above 1 mm and positive cooperativity at lower concentrations of S(2-). With respect to O-acetylserine (OAS), the second substrate of the reaction, one P. vulgaris isoenzyme shows substrate inhibition by OAS concentrations above 10 mm, while the second is unaffected by OAS concentrations up to 50 mm. The isoenzymes from Phaseolus polyanthus (one of which has a specific activity 24 times higher than the other) are slightly and approximately equally inhibited by both S(2-) and OAS.
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PMID:Purification and initial kinetic characterization of different forms of o-acetylserine sulfhydrylase from seedlings of two species of phaseolus. 1666 22

The effect of nitrogen and sulfur nutrition on sulfate permease and O-acetylserine sulfhydrylase was studied in tobacco cells.Sulfate transport rates increased 10-fold in cells transferred to sulfur-deficient B-5 medium. The addition of either sulfate or l-cysteine reduced transport 95 and 80%, respectively. The pools of sulfate, cysteine, glutathione, and methionine declined in sulfur-starved cells. The addition of either sulfate or l-cysteine increased the pools of sulfur-containing compounds, but major quantitative differences were measured. Nitrogen-starved cells had low transport rates which were not increased by addition of nitrate/ammonia. The pools of sulfate, cysteine, and methionine were high in nitrogen-starved cells and remained high upon addition of a nitrogen source. The results show that sulfate transport is regulated by the intracellular sulfate pool.O-Acetylserine sulfhydrylase was not affected by sulfur nutrition. The extractable activity was high in B-5-grown cells, sulfur-deficient cells, and cells to which either sulfate or l-cysteine had been added. In contrast, the enzyme declined in cells transferred to nitrogen-deficient medium and the amount of enzyme/g fresh weight increased 10-fold when nitrate/ammonia was added. The addition of nitrate/ammonia had no effect on the cysteine or methionine pools but increased the total amino acid pool. The amount of O-acetylserine was positively correlated with extractable enzyme activity. This enzyme is positively regulated by an effector (possibly O-acetylserine) which is high under conditions of net nitrate assimilation.
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PMID:Regulation of Sulfate Assimilation in Tobacco Cells: EFFECT OF NITROGEN AND SULFUR NUTRITION ON SULFATE PERMEASE AND O-ACETYLSERINE SULFHYDRYLASE. 1666 45


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