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)

Three cDNA clones encoding putative cysteine synthases (O-acetylserine (thiol) lyase, EC 4.2.99.8) were isolated from Arabidopsis thaliana and designated AtcysC1, AtcysD1 and AtcysD2, respectively. Southern blot analyses suggested that the corresponding genes were present as a single copy, or at most two copies, in the A. thaliana genome. Escherichia coli complementation analyses confirmed that the cDNAs encode cysteine synthase and the corresponding proteins produced in E. coli clearly showed cysteine synthase activity. In addition, AtcysC1 protein showed beta-cyanoalanine synthase (EC 4.4.1.9) activity, but the other two did not. Kinetic analysis suggests that AtcysC1 actually functions as beta-cyanoalanine synthase rather than cysteine synthase in vivo. The mRNA accumulation of AtcysC1, AtcysD1 and AtcysD2 differed in various organs, but did not change markedly when A. thaliana seedlings were subjected to various stresses, including nutrient deprivation. In vivo targeting experiments indicated that AtcysD1 and AtcysD2 are cytoplasmic isozymes, and AtcysC1 is a mitochondrial isozyme.
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PMID:Three Arabidopsis genes encoding proteins with differential activities for cysteine synthase and beta-cyanoalanine synthase. 1084 60

The final step of cysteine biosynthesis in plants is catalyzed by O-acetylserine (thiol) lyase (OAS-TL), which occurs as several isoforms found in the cytosol, the plastids and the mitochondria. Genomic DNA blot hybridization and isolation of genomic clones indicate single copy genes (oasA1, oasA2, oasB and oasC) that encode the activities of OAS-TL A, B and C found in separate subcellular compartments in the model plant Arabidopsis thaliana. Sequence analysis reveals that the newly discovered oasA2 gene represents a pseudogene that is still transcribed, but is not functionally translated. The comparison of gene structures suggests that oasA1/oasA2 and oasB/oasC are closely related and may be derived from a common ancestor by subsequent duplications. OAS-TL A, B and C were overexpressed in an Escherichia coli mutant lacking cysteine synthesis and exhibited bifunctional OAS-TL and beta-cyanoalanine synthase (CAS) activities. However, all three proteins represent true OAS-TLs according to kinetic analysis and are unlikely to function in cyanide detoxification or secondary metabolism. In addition, it was demonstrated that the mitochondrial OAS-TL C exhibits in vivo protein-protein interaction capabilities with respect to cysteine synthase complex formation similar to cytosolic OAS-TL A and plastid OAS-TL B. Multiple database accessions for each of the A. thaliana OAS-TL isoforms can thus be attributed to a specified number of oas genes to which functionally defined gene products are assigned, and which are responsible for compartment-specific cysteine synthesis.
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PMID:Genomic and functional characterization of the oas gene family encoding O-acetylserine (thiol) lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana. 1094 May 62

Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyltransferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left-handed parallel beta-helix consisting of beta-sheets alternating with turns, resulting in a prism-like structure. This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization. The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.
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PMID:The cysteine synthase complex from plants. Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction. 1116 7

A cell extract of an extremely thermophilic bacterium, Thermus thermophilus HB8, cultured in a synthetic medium catalyzed cystathionine gamma-synthesis with O-acetyl-L-homoserine and L-cysteine as substrates but not beta-synthesis with DL-homocysteine and L-serine (or O-acetyl-L-serine). The amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts. The syntheses of cystathionine beta-lyase (EC 4.4.1.8) and O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) were markedly repressed by L-methionine supplemented to the medium. L-Cysteine and glutathione, both at 0.5 mM, added to the medium as the sole sulfur source repressed the synthesis of O-acetylserine sulfhydrylase by 55 and 73%, respectively, confirming that this enzyme functions as a cysteine synthase. Methionine employed at 1 to 5 mM in the same way derepressed the synthesis of O-acetylserine sulfhydrylase 2.1- to 2.5-fold. A method for assaying a low concentration of sulfide (0.01 to 0.05 mM) liberated from homocysteine by determining cysteine synthesized with it in the presence of excess amounts of O-acetylserine and a purified preparation of the sulfhydrylase was established. The extract of cells catalyzed the homocysteine gamma-lyase reaction, with a specific activity of 5 to 7 nmol/min/mg of protein, but not the methionine gamma-lyase reaction. These results suggested that cysteine was also synthesized under the conditions employed by the catalysis of O-acetylserine sulfhydrylase using sulfur of homocysteine derived from methionine. Methionine inhibited O-acetylserine sulfhydrylase markedly. The effects of sulfur sources added to the medium on the synthesis of O-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organism has two proteins having O-acetylhomoserine sulfhydrylase activity, one of which is cystathionine gamma-synthase. Although it has been reported that homocysteine is directly synthesized in T. thermophilus HB27 by the catalysis of O-acetylhomoserine sulfhydrylase on the basis of genetic studies (T. Kosuge, D. Gao, and T. Hoshino, J. Biosci. Bioeng. 90:271-279, 2000), the results obtained in this study for the behaviors of related enzymes indicate that sulfur is first incorporated into cysteine and then transferred to homocysteine via cystathionine in T. thermophilus HB8.
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PMID:Occurrence of transsulfuration in synthesis of L-homocysteine in an extremely thermophilic bacterium, Thermus thermophilus HB8. 1122 9

Experiments were conducted to investigate the effect of S nutrition and availability on the forms of S and N in the endosperm cavity and endosperm of wheat, and on the capacity of the endosperm to utilize those compounds for the synthesis of proteins. Plants were grown in solution culture with 2 mM N and either 200 microM S (high-S) or 50 microM S (low-S) and all nutrients were withdrawn at various times from booting until 8 d post-anthesis. Sulphate was the major form of soluble S in the endosperm cavity and endosperm of high-S plants during the time of rapid grain development. By contrast, glutathione (GSH) was the major form of soluble S in the endosperm cavity and in the endosperm in low-S plants. Crude extracts of endosperm tissue from both high-S and low-S plants supported (i) the hydrolysis of GSH to gamma-glutamyl cysteine and glycine, and of gamma-glutamyl cysteine to glutamate and cysteine, and (ii) sulphate-dependent PPi-ATP exchange and the sulphydration of O-acetylserine catalysed by ATP sulphurylase and cysteine synthase, respectively. High-S nutrition enhanced the in vitro rates of ATP sulphurylase and cysteine synthase.
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PMID:Sulphur nutrition affects delivery and metabolism of S in developing endosperms of wheat. 1145 12

Proteins utilizing pyridoxal 5'-phosphate as a coenzyme constitute a large superfamily and are currently classified into three functional groups and five structural fold types. Despite the variability of sequences and catalyzed reactions, they share relevant structural, dynamic and functional properties. Therefore, they constitute an optimal system to investigate the relative influence of primary sequence and coenzyme interactions on folding pathways, structural stability and enzymatic function. O-Acetylserine sulfhydrylase is a dimeric pyridoxal 5'-phosphate dependent enzyme that catalyzes the synthesis of L-cysteine from O-acetylserine and sulfide. The time-resolved fluorescence study of O-acetylserine sulfhydrylase unfolding, here reported, indicates that the coenzyme stabilizes the protein structure. The dependence on denaturant concentration of tryptophan lifetimes in the holo- and apo-enzyme demonstrates that the interactions with the coenzyme stabilize the C-terminal domain to a higher extent with respect to the N-terminal domain. This result is discussed in terms of a linkage between the differential stabilization brought about by the coenzyme and the different degrees of conformational flexibility required by the specialized functional role of distinct protein regions.
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PMID:Unfolding of pyridoxal 5'-phosphate-dependent O-acetylserine sulfhydrylase probed by time-resolved tryptophan fluorescence. 1198 20

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

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

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

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


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