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)

Our studies of the reaction mechanism of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme that forms a series of intermediates in the reaction of L-serine and L-homocysteine to form L-cystathionine. To characterize these reaction intermediates, we have carried out rapid-scanning stopped-flow and single-wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluorescence spectroscopy under steady-state conditions. We find that the gem-diamine and external aldimine of aminoacrylate are the primary intermediates in the forward half-reaction with L-serine and that the external aldimine of aminoacrylate or its complex with L-homocysteine is the primary intermediate in the reverse half-reaction with L-cystathionine. The second forward half-reaction of aminoacrylate with L-homocysteine is rapid. No primary kinetic isotope effect was obtained in the forward half-reaction with L-serine. The results provide evidence (1) that the formation of the external aldimine of L-serine is faster than the formation of the aminoacrylate intermediate, (2) that aminoacrylate is formed by the concerted removal of the alpha-proton and the hydroxyl group of L-serine, and (3) that the rate of the overall reaction is rate-limited by the conversion of aminoacrylate to L-cystathionine. We compare our results with cystathionine beta-synthase with those of related investigations of tryptophan synthase and O-acetylserine sulfhydrylase.
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PMID:The reaction of yeast cystathionine beta-synthase is rate-limited by the conversion of aminoacrylate to cystathionine. 1153 64

Sulfur metabolism in gram-positive bacteria is poorly characterized. Information on the molecular mechanisms of regulation of genes involved in sulfur metabolism is limited, and no regulator genes have been identified. Here we describe the regulation of the lactococcal metC-cysK operon, encoding a cystathionine beta-lyase (metC) and cysteine synthase (cysK). Its expression was shown to be negatively affected by high concentrations of cysteine, methionine, and glutathione in the culture medium, while sulfur limitation resulted in a high level of expression. Other sulfur sources tested showed no significant effect on metC-cysK gene expression. In addition we found that O-acetyl-l-serine, the substrate of cysteine synthase, was an inducer of the metC-cysK operon. Using a random mutagenesis approach, we identified two genes, cmbR and cmbT, involved in regulation of metC-cysK expression. The cmbT gene is predicted to encode a transport protein, but its precise role in regulation remains unclear. Disruption of cmbT resulted in a two- to threefold reduction of metC-cysK transcription. A 5.7-kb region containing the cmbR gene was cloned and sequenced. The encoded CmbR protein is homologous to the LysR family of regulator proteins and is an activator of the metC-cysK operon. In analogy to CysB from Escherichia coli, we propose that CmbR requires acetylserine to be able to bind the activation sites and subsequently activate transcription of the metC-cysK operon.
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PMID:Regulation of the metC-cysK operon, involved in sulfur metabolism in Lactococcus lactis. 1174 47

Serine acetyltransferase (SATase) and cysteine synthase (O-acetylserine (thiol)-lyase) (CSase) are committed in the final step of cysteine biosynthesis. Six cDNA clones encoding SATase have been isolated from several plants, e.g. watermelon, spinach, Chinese chive and Arabidopsis thaliana. Feedback-inhibition pattern and subcellular localization of plant SATases were evaluated. Two types of SATase that differ in their sensitivity to the feedback inhibition by L-cysteine were found in plants. In Arabidopsis, cytosolic SATase was inhibited by L-cysteine at a physiological concentration in an allosteric manner, but the plastidic and mitochondrial forms were not subjected to this feedback regulation. These results suggest that the regulation of cysteine biosynthesis through feedback inhibition may differ depending on the subcellular compartment. The allosteric domain responsible for L-cysteine inhibition was characterized, using several SATase mutants. The single change of amino acid residue, glycine-277 to cysteine, in the C-terminal region of watermelon SATase caused a significant decrease of the feedback-inhibition sensitivity of watermelon SATase. We made the transgenic Arabidopsis overexpressing point-mutated watermelon SATase gene whose product was not inhibited by L-cysteine. The contents of OAS, cysteine, and glutathione in transgenic Arabidopsis were significantly increased as compared to the wild-type Arabidopsis. Transgenic tobacco (Nicotiana tabacum) (F1) plants with enhanced CSase activities both in the cytosol and in the chloroplasts were generated by cross-fertilization of two transgenic tobacco expressing either cytosolic CSase or chloroplastic CSase. Upon fumigation with 0.1 microLL(-1) sulfur dioxide, both the cysteine and glutathione contents in leaves of F1 plants were increased significantly, but not in leaves of non-transformed control plants. These results indicated that both SATase and CSase play important roles in cysteine biosynthesis and its regulation in plants.
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PMID:Molecular and biochemical analysis of serine acetyltransferase and cysteine synthase towards sulfur metabolic engineering in plants. 1208 67

There is an increasing demand for peptide-mimicking molecules to modulate the interactions between proteins of pharmaceutical and agrochemical interest and their target polypeptides. Unnatural L-alpha-amino acids differing from the 20 naturally proteinogenic amino acids only in their side chain are ideal for this purpose, but their chemical synthesis is complex. Here we describe a fermentation-based approach for biosynthesis of unnatural amino acids after re-engineering the cysteine-biosynthetic pathway in Escherichia coli. O-acetylation of serine, the committed step of the pathway, was released from feedback inhibition by mutating the serine acetyltransferase gene. Next, the naturally broad substrate specificity of O-acetylserine sulfhydrylase was exploited for the direct in vivo incorporation of an unnatural side chain in a semisynthetic fermentation process comparable to the production of beta-lactams. O-acetyl-L-serine extruded from the cells by way of the O-acetylserine efflux protein was amenable to further biotransformations.
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PMID:Semisynthetic production of unnatural L-alpha-amino acids by metabolic engineering of the cysteine-biosynthetic pathway. 1264 Apr 65

An O-acetylserine sulfhydrylase (OASS) from the hyperthermophilic archaeon Aeropyrum pernix K1, which shares the pyridoxal 5'-phosphate binding motif with both OASS and cystathionine beta-synthase (CBS), was cloned and expressed by using Escherichia coli Rosetta(DE3). The purified protein was a dimer and contained pyridoxal 5'-phosphate. It was shown to be an enzyme with CBS activity as well as OASS activity in vitro. The enzyme retained 90% of its activity after a 6-h incubation at 100 degrees C. In the O-acetyl-L-serine sulfhydrylation reaction, it had a pH optimum of 6.7, apparent K(m) values for O-acetyl-L-serine and sulfide of 28 and below 0.2 mM, respectively, and a rate constant of 202 s(-1). In the L-cystathionine synthetic reaction, it showed a broad pH optimum in the range of 8.1 to 8.8, apparent K(m) values for L-serine and L-homocysteine of 8 and 0.51 mM, respectively, and a rate constant of 0.7 s(-1). A. pernix OASS has a high activity in the L-cysteine desulfurization reaction, which produces sulfide and S-(2,3-hydroxy-4-thiobutyl)-L-cysteine from L-cysteine and dithiothreitol.
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PMID:Characterization of a novel thermostable O-acetylserine sulfhydrylase from Aeropyrum pernix K1. 1264 99

Variations in proteome profiles of Escherichia coli in response to the overproduction of human leptin, a serine-rich (11.6% of total amino acids) protein, were examined by two-dimensional gel electrophoresis. The levels of heat shock proteins increased, while those of protein elongation factors, 30S ribosomal protein, and some enzymes involved in amino acid biosynthesis decreased, after leptin overproduction. Most notably, the levels of enzymes involved in the biosynthesis of serine family amino acids significantly decreased. Based on this information, we designed a strategy to enhance the leptin productivity by manipulating the cysK gene, encoding cysteine synthase A. By coexpression of the cysK gene, we were able to increase the cell growth rate by approximately twofold. Also, the specific leptin productivity could be increased by fourfold. In addition, we found that cysK coexpression can improve the production of another serine-rich protein, interleukin-12 beta chain, suggesting that this strategy may be useful for the production of other serine-rich proteins as well. The approach taken in this study should be useful in designing a strategy for improving recombinant protein production.
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PMID:Engineering Escherichia coli for increased productivity of serine-rich proteins based on proteome profiling. 1453 24

The last steps of cysteine biosynthesis are catalysed by a bi-enzyme complex composed of serine acetyltransferase (SAT) and cysteine synthase, also called O-acetyl-serine (thiol) lyase (OASTL). SAT is responsible for the production of O-acetyl-serine (OAS) from serine and acetyl-coenzyme A, while OASTL catalyses the formation of cysteine from OAS and hydrogen sulphide. Several distinct nuclear genes for SAT and OASTL enzymes exist in plants. Products of these genes are targeted into at least three cellular compartments: cytosol, chloroplasts, and mitochondria. The SAT and OASTL enzymes are strongly evolutionary conserved, both structurally and functionally. Therefore, isoenzymes from various cellular compartments can be substituted, not only by their plant counterparts from the other cellular compartments but also by their bacterial homologues. During the last decade transgenic plants overproducing SAT, OASTL or both enzymes simultaneously were obtained independently by several research groups. These manipulations led not only to the elevated levels of the respective products, namely OAS and cysteine, but also to increased amounts of glutathione and changes in the levels of other metabolites and enzymatic activities. In several cases, the transgenic plants were also shown to be less susceptible to applied abiotic stresses. In this review, all published and some unpublished results from this laboratory related to heterologous overproduction of SAT and OASTL in transgenic plants are discussed and summarized.
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PMID:Overproduction of SAT and/or OASTL in transgenic plants: a survey of effects. 1520 50

The cysK gene encoding a cysteine synthase of Geobacillus stearothermophilus V was overexpressed in E. coli and the recombinant protein was purified and characterized. The enzyme is a thermostable homodimer (32 kDa/monomer) belonging to the beta family of pyridoxal phosphate (PLP)-dependent enzymes. UV-visible spectra showed absorption bands at 279 and 410 nm. The band at 279 nm is due to tyrosine residues as the enzyme lacks tryptophan. The 410 nm band represents absorption of the coenzyme bound as a Schiff base to a lysine residue of the protein. Fluorescence characteristics of CysK's Schiff base were influenced by temperature changes suggesting different local structures at the cofactor binding site. The emission of the Schiff base allowed the determination of binding constants for products at both 20 degrees C and 50 degrees C. At 50 degrees C and in the absence of sulphide the enzyme catalyzes the decomposition of O-acetyl-l-serine to pyruvate and ammonia. At 20 degrees C, however, a stable alpha-aminoacrylate intermediate is formed.
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PMID:Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V. 1530 37

Serine acetyltransferase is a member of the left-handed parallel beta-helix family of enzymes that catalyzes the committed step in the de novo synthesis of l-cysteine in bacteria and plants. The enzyme has an ordered kinetic mechanism with acetyl CoA bound prior to l-serine and O-acetyl-l-serine released prior to CoA. The rate-limiting step along the reaction pathway is the nucleophilic attack of the serine hydroxyl on the thioester of acetyl CoA. Product release contributes to rate-limitation at saturating concentrations of reactants. The reaction is catalyzed by an active site general base with a pK of 7, which accepts a proton from the serine hydroxyl as a tetrahedral intermediate is formed between the reactants, and donates it to the thiol of CoA as the intermediate collapses to give products. This mechanism is likely the same for all O-acyltransferases that catalyze their reaction by direct attack of the alcohol on the acyl donor, using an active-site histidine as the general base. Serine acetyltransferase is regulated by feedback inhibition by the end product l-cysteine, which acts by binding to the serine site in the active site and inducing a conformational change that prevents reactant binding. The enzyme also associates with O-acetylserine sulfhydrylase, the final enzyme in the biosynthetic pathway, which contributes to stabilizing the acetyltransferase.
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PMID:The serine acetyltransferase reaction: acetyl transfer from an acylpantothenyl donor to an alcohol. 1558 68

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


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