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:4.2.1.22 (cystathionine beta-synthase)
965 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

Beta-cyanoalanine synthase (CAS, L-3-cyanoalanine synthase; EC 4.4.1.9) is the most important enzyme in cyanide metabolism. In addition to CAS, cysteine synthase (CS, EC 4.2.99.8) possesses CAS activity. To explore the physiological significance of cyanide metabolism, we isolated the cDNA clones corresponding to purified CAS (designated PCAS-1 and PCAS-2) and CS (designated PCS-1 and PCS-2) from potato using the information of these amino acid sequences. The recombinant proteins of PCS-1, PCS-2 and PCAS-1 catalyzed both CAS and CS reactions, although the ratios between CAS and CS activity were remarkably different. PCAS-1 preferred the substrates for the CAS reaction to the substrates for the CS reaction. From the kinetic characters and homology of amino acid sequences with known CS-like proteins, PCS-1, PCS-2 and PCAS-1 were identified as cytosolic CS, plastidic CS and mitochondrial CAS, respectively. The highest level of CAS activity, CAS protein and its mRNA were detected in potato buds. Stimulation of CAS activity and protein accumulation by ethylene without the concomitant increase of its mRNA suggested that ethylene induces CAS protein accumulation at the post-transcriptional level.
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PMID:Beta-cyanoalanine synthase and cysteine synthase from potato: molecular cloning, biochemical characterization, and spatial and hormonal regulation. 1157 29

The present study was undertaken to test whether endurance training in patients with COPD, along with enhancement of muscle bioenergetics, decreases muscle redox capacity as a result of recurrent episodes of cell hypoxia induced by high intensity exercise sessions. Seventeen patients with COPD (FEV(1), 38 +/- 4% pred; PaO2), 69 +/- 2.7 mm Hg; PaCO2, 42 +/- 1.7 mm Hg) and five age-matched control subjects (C) were studied pretraining and post-training. Reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and gamma-glutamyl cysteine synthase heavy subunit chain mRNA expression (gammaGCS-HS mRNA) were measured in the vastus lateralis. Pretraining redox status at rest and after moderate (40% Wpeak) constant-work rate exercise were similar between groups. After training (DeltaWpeak, 27 +/- 7% and 37 +/- 18%, COPD and C, respectively) (p < 0.05 each), GSSG levels increased only in patients with COPD (from 0.7 +/- 0.08 to 1.0 +/- 0.15 nmol/ mg protein, p < 0.05) with maintenance of GSH levels, whereas GSH markedly increased in C (from 4.6 +/- 1.03 to 8.7 +/- 0.41 nmol/ mg protein, p < 0.01). Post-training gammaGCS-HS mRNA levels increased after submaximal exercise in patients with COPD. No evidence of lipid peroxidation was observed. We conclude that although endurance training increased muscle redox potential in healthy subjects, patients with COPD showed a reduced ability to adapt to endurance training reflected in lower capacity to synthesize GSH.
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PMID:Reduced muscle redox capacity after endurance training in patients with chronic obstructive pulmonary disease. 1199 89

The nucleotide sequence of a 4,539 bp fragment of Bacillus stearothermophilus V mediating tellurite resistance in Escherichia coli was determined. Four ORFs of more than 150 amino acids encoding polypeptides of 244, 258, 308, and 421 residues were found in the restriction fragment. E. coli cells harboring a recombinant plasmid containing the Ter determinant express, when challenged with tellurite, a 32 kDa protein with an amino terminal sequence identical to the ten first residues of the 308 ORF. This ORF shows great similarity with the cysteine synthase gene (cysK) of a number of organisms. Recombinant clones carrying the active cysK gene have minimal inhibitory concentrations to K2TeO3 that were tenfold higher than those determined for the host strain or that of clones carrying ORFs 244, 258, and 421. Introduction of the B. stearothermophilus V cysK gene into a cysK strain of Salmonella typhimurium LT2 resulted in complementation of the mutation as well as transfer of tellurite resistance.
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PMID:The product of the cysK gene of Bacillus stearothermophilus V mediates potassium tellurite resistance in Escherichia coli. 1168 9

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

Real time biomolecular interaction analysis based on surface plasmon resonance has been proven useful for studying protein-protein interaction but has not been extended so far to investigate enzyme-enzyme interactions, especially as pertaining to regulation of metabolic activity. We have applied BIAcore technology to study the regulation of enzyme-enzyme interaction during mitochondrial cysteine biosynthesis in Arabidopsis thaliana. The association of the two enzyme subunits in the hetero-oligomeric cysteine synthase complex was investigated with respect to the reaction intermediate and putative effector O-acetylserine. We have determined an equilibrium dissociation constant of the cysteine synthase complex (K(D) = 25 +/- 4 x 10(-9) m), based on a reliable A + B <--> AB model of interaction. Analysis of dissociation kinetics in the presence of O-acetylserine revealed a half-maximal dissociation rate at 77 +/- 4 microm O-acetylserine and strong positive cooperativity for complex dissociation. The equilibrium of interaction was determined using an enzyme activity-based approach and yielded a K(m) value of 58 +/- 7 microm O-acetylserine. Both effector concentrations are in the range of intracellular O-acetylserine fluctuations and support a functional model that integrates effector-driven cysteine synthase complex dissociation as a regulatory switch for the biosynthetic pathway. The results show that BIAcore technology can be applied to obtain quantitative kinetic data of a hetero-oligomeric protein complex with enzymatic and regulatory function.
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PMID:Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana. 1206 44

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

A 0.5 kb fragment of Streptomyces venezuelae ISP5230 genomic DNA was amplified by PCR using primers based on consensus sequences of cysteine synthase isozyme A from bacteria. The deduced amino acid sequence of the PCR product resembled not only cysteine synthase sequences from prokaryotes and eukaryotes but also eukaryotic cystathionine beta-synthase sequences. Probing an Str. venezuelae genomic library with the PCR product located a hybridizing colony from which pJV207 was isolated. Sequencing and analysis of the Str. venezuelae DNA insert in pJV207 detected two ORFs. The deduced amino acid sequence of ORF1 matched both cysteine synthase and cystathionine beta-synthase sequences in GenBank, but its size favoured assignment as a cystathionine beta-synthase. ORF2 in the pJV207 insert was unrelated in function to ORF1; in its sequence the deduced product resembled acetyl-CoA transferases, but disruption of the ORF did not cause a detectable phenotypic change. Disruption of ORF1 failed to elicit cysteine auxotrophy in wild-type Str. venezuelae, but in the cys-28 auxotroph VS263 it prevented restoration of prototrophy with homocysteine or methionine supplements. The change in phenotype implicated loss of the transsulfuration activity that in the wild-type converts these supplements to cysteine. This study concludes that disruption of ORF1 inactivates a cbs gene, the product of which participates in cysteine synthesis by transsulfuration. Enzyme assays of Str. venezuelae mycelial extracts confirmed the formation of cysteine by thiolation of O-acetylserine, providing the first unambiguous detection of this activity in a streptomycete. Enzyme assays also detected cystathionine gamma-synthase, cystathionine beta-lyase and cystathionine gamma-lyase activity in the extracts and showed that the substrate for cystathionine gamma-synthase was O-succinyl-homoserine. Based on assay results, the cys-28 mutation in Str. venezuelae VS263 does not inactivate the cysteine synthase gene but impairs expression in cultures grown in minimal medium.
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PMID:Biosynthesis of sulfur-containing amino acids in Streptomyces venezuelae ISP5230: roles for cystathionine beta-synthase and transsulfuration. 1210 1

Metallothionein (MT) promoter was methylated in rat hepatoma and in mouse lymphosarcoma cells by methylation of cytosine within the CpG dinucleotide region. After demethylation of MT-I promoter in mouse lymphosarcoma cells or in the transplanted rat hepatoma with 5-azacytidine, a potent inhibitor of DNA methyltransferase, the promoter was activated in response to heavy metal treatment. MT-I promoter was also suppressed in human prostate cancer lines PC3 and DU145, probably by promoter methylation, whereas cadmium induced MT-I in the human prostate cancer line LNCaP. In the prostate cancer lines where MT-I was suppressed, glutathione-S-transferase-pi (GST-pi) was expressed. On the contrary, GST-pi gene was repressed in the cell line where MT-I was induced, which suggests an inverse relationship between MT-I induction and GST-pi expression in some prostate cancer lines. The expressions of GST-pi and gamma-glutamyl cysteine synthase were also significantly higher (5- to 12-fold) in the lymphosarcoma cells and the hepatoma relative to the parental tissues. The higher expressions of these two genes suggest a compensatory mechanism in the cells where the gene for the antioxidant MT-I/II is not induced. MT-I/II may function as a growth suppressor either alone or in concert with other factor(s), and consequently their lack of expression could facilitate the tumor growth. In addition to suppression of MT-I/II expression by promoter methylation, the lack of MT induction could also be brought about by nuclear factor I (NFI), probably by interaction with the metal transcription factor MTF-1. An inverse relationship was observed between the level of NFI and MT-I expression in some cells, which suggests a role for NFI in the relatively low constitutive levels of MT-I expression in these cells.
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PMID:Suppression of metallothionein-I/II expression and its probable molecular mechanisms. 1242 40

Illiminated intact chromatophore of chromatium vinosum in the presence of O-acetylserine(OAS) catalysed incorporation of SeO3(2-) into selenocysteine at rate of 359 nmol.mgBchl-1.h-1. Sonicated chromatophore catalysed SeO3(2-) incorporation at 1.1% of the rate of intact chromatophore. Addition of GSH and NADPH increased the rate to 88.3% of intact rate, but SeO3(2-) incorporation under these conditions was essentially light dependent. The purified GSH reductase from Chromatium vinosum in the presence of cysteine synthase OAs and NADPH catalysed incorporation of SeO3(2-) into selenocysteine. It is proposed that SeO3(2-) is reduced by light-coupled GSH reductase and that Se2- produced is incorporated into selenocysteine by cysteine synthase.
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PMID:[Light-dependent incorporation of selenite into selenocysteine by isolated chromatophore of Chromatium vinosum]. 1255 43


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