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)

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grown in solution culture with 2 mM N and either 200 microM S (high-S) or 50 microM S (low-S) and all nutrients were withdrawn at various times from booting until 8 d post-anthesis.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

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


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