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)

Entamoeba histolytica, the causative agent of human amoebiasis, is essentially anaerobic, requiring a small amount of oxygen for growth. It cannot tolerate the higher concentration of oxygen present in human tissues or blood. However, during tissue invasion it is exposed to a higher level of oxygen, leading to oxygen stress. Cysteine, which is a vital thiol in E. histolytica, plays an essential role in its oxygen-defence mechanisms. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine-biosynthetic pathway, which involves cysteine synthase (EhCS) as a key enzyme. In this study, EhCS was cloned, expressed in Escherichia coli and purified by affinity and size-exclusion chromatography. The purified protein was crystallized in space group P4(1) with two molecules per asymmetric unit and a complete data set was collected to a resolution of 1.86 A. A molecular-replacement solution was obtained using the Salmonella typhimurium O-acetylserine sulfhydrylase structure as a probe and had a correlation coefficient of 37.7% and an R factor of 48.8%.
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PMID:Crystallization and preliminary crystallographic analysis of cysteine synthase from Entamoeba histolytica. 1755 75

Cysteine biosynthetic genes are up-regulated in the persistent phase of Mycobacterium tuberculosis, and the corresponding enzymes are therefore of interest as potential targets for novel antibacterial agents. cysK1 is one of these genes and has been annotated as coding for an O-acetylserine sulfhydrylase. Recombinant CysK1 is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine to cysteine. The crystal structure of the enzyme was determined to 1.8A resolution. CysK1 belongs to the family of fold type II PLP enzymes and is similar in structure to other O-acetylserine sulfhydrylases. We were able to trap the alpha-aminoacrylate reaction intermediate and determine its structure by cryocrystallography. Formation of the aminoacrylate complex is accompanied by a domain rotation resulting in active site closure. The aminoacrylate moiety is bound in the active site via the covalent linkage to the PLP cofactor and by hydrogen bonds of its carboxyl group to several enzyme residues. The catalytic lysine residue is positioned such that it can protonate the Calpha-carbon atom of the aminoacrylate only from the si-face, resulting in the formation of L-cysteine. CysK1 is competitively inhibited by a four-residue peptide derived from the C-terminal of serine acetyl transferase. The crystallographic analysis reveals that the peptide binds to the enzyme active site, suggesting that CysK1 forms an bi-enzyme complex with serine acetyl transferase, in a similar manner to other bacterial and plant O-acetylserine sulfhydrylases. The structure of the enzyme-peptide complex provides a framework for the design of strong binding inhibitors.
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PMID:Structural insights into catalysis and inhibition of O-acetylserine sulfhydrylase from Mycobacterium tuberculosis. Crystal structures of the enzyme alpha-aminoacrylate intermediate and an enzyme-inhibitor complex. 1756 78

The terminal step of soybean cysteine synthesis is catalyzed by O-acetylserine(thiol)lyase (OAS-TL, EC 2.5.1.47). In this study, we isolated and characterized an OAS-TL gene from a wild soybean material (designated as GsOAS-TL1). GsOAS-TL1 cDNA sequence showed strict conservation at both nucleotide and amino acid levels compared with that from cultivated soybean. Genomic structure analysis of GsOAS-TL1 indicated that it contained 10 exons and 9 introns in the coding region with conserved exon sizes and intron locations compared with Arabidopsis thaliana OAS-TL-like genes. Among the complete GsOAS-TL1 cDNA and three part-deletion fragments, only expression of the full-length cDNA could rescue the NK3 cys(-) Escherichia coli auxotroph, which was coherent with the assayed enzyme activity of purified fusion proteins. For RT-PCR analysis in different wild soybean tissues, GsOAS-TL1 showed lower expression in roots and developing seeds, whereas total OAS-TL activity of corresponding tissues showed significantly higher level in seeds than other tissues. To our knowledge, this is the first report on cloning and characterization of an OAS-TL gene from wild soybean. Our results are informative to further elucidate the function and evolution of OAS-TL in soybean.
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PMID:Cloning and functional characterization of an O-acetylserine(thiol)lyase-encoding gene in wild soybean (Glycine soja). 1761 17

The crystal structure of the dimeric O-acetylserine sulfhydrylase isozyme B from Escherichia coli (CysM), complexed with the substrate analog citrate, has been determined at 1.33 A resolution by X-ray diffraction analysis. The C1-carboxylate of citrate was bound at the carboxylate position of O-acetylserine, whereas the C6-carboxylate adopted two conformations. The activity of the enzyme and of several active center mutants was determined using an assay based on O-acetylserine and thio-nitrobenzoate (TNB). The unnatural substrate TNB was modeled into the reported structure. The substrate model and the observed mutant activities may facilitate future protein engineering attempts designed to broaden the substrate spectrum of the enzyme. A comparison of the reported structure with previously published CysM structures revealed large conformational changes. One of the crystal forms contained two dimers, each of which comprised one subunit in a closed and one in an open conformation. Although the homodimer asymmetry was most probably caused by crystal packing, it indicates that the enzyme can adopt such a state in solution, which may be relevant for the catalytic reaction.
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PMID:High resolution structure and catalysis of O-acetylserine sulfhydrylase isozyme B from Escherichia coli. 1789 25

Cysteine plays a major role in the antioxidative defense mechanisms of the human parasite Entameoba histolytica. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine biosynthetic pathway involving two key enzymes O-acetyl-L-serine sulfhydrylase (OASS) and serine acetyl transferase (SAT). The crystal structure of native OASS from Entameoba histolytica (EhOASS) has been determined at 1.86 A resolution and in complex with its product cysteine at 2.4 A resolution. In comparison with other known OASS structures, insertion in the N-terminal region and C-terminal helix reveal critical differences, which may influence the protein-protein interactions. In spite of lacking chloride binding site at the dimeric interface, the N-terminal extension compared with other known cysteine synthases, participates in dimeric interactions in an interesting domain swapping manner, enabling it to form a stronger dimer. Sulfate is bound in the active site of the native structure, which is replaced by cysteine in the cysteine bound form causing reorientation of the small N-terminal domain and thus closure of the active site. Ligand binding constants of OAS, Cys, and Met with EhOASS are comparable with other known OASS indicating similar active site arrangement and dynamics. The cysteine complexed structure represents the snapshot of the enzyme just before releasing the final product with a closed active site. The C-terminal helix positioning in the EhOASS may effect its interactions with EhSAT and thus influencing the formation of the cysteine synthase complex in this organism.
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PMID:Crystal structure of native O-acetyl-serine sulfhydrylase from Entamoeba histolytica and its complex with cysteine: structural evidence for cysteine binding and lack of interactions with serine acetyl transferase. 1835 May 70

The structure of the protein complex CysM-CysO from a new cysteine biosynthetic pathway found in the H37Rv strain of Mycobacterium tuberculosis has been determined at 1.53 A resolution. CysM (Rv1336) is a PLP-containing beta-replacement enzyme and CysO (Rv1335) is a sulfur carrier protein with a ubiquitin-like fold. CysM catalyzes the replacement of the acetyl group of O-acetylserine by CysO thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reaction. The protein complex in the crystal structure is asymmetric with one CysO protomer binding to one end of a CysM dimer. Additionally, the structures of CysM and CysO were determined individually at 2.8 and 2.7 A resolution, respectively. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, revealed high conservation of active site residues; however, residues in CysM responsible for CysO binding are not conserved. Comparison of the CysM-CysO binding interface with other sulfur carrier protein complexes revealed a similarity in secondary structural elements that contribute to complex formation in the ThiF-ThiS and MoeB-MoaD systems, despite major differences in overall folds. Comparison of CysM with and without bound CysO revealed conformational changes associated with CysO binding.
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PMID:Crystal structure of a sulfur carrier protein complex found in the cysteine biosynthetic pathway of Mycobacterium tuberculosis. 1877 Dec 96

The biosynthesis of cysteine is a crucial metabolic pathway supplying a building block for de novo protein synthesis but also a reduced thiol as a component of the oxidative defense mechanisms that appear particularly vital in the dormant state of Mycobacterium tuberculosis. We here show that the cysteine synthase CysM is, in contrast to previous annotations, an O-phosphoserine-specific cysteine synthase. CysM belongs to the fold type II pyridoxal 5'-phosphate-dependent enzymes, as revealed by the crystal structure determined at 2.1-angstroms resolution. A model of O-phosphoserine bound to the enzyme suggests a hydrogen bonding interaction of the side chain of Arg220 with the phosphate group as a key feature in substrate selectivity. Replacement of this residue results in a significant loss of specificity for O-phosphoserine. Notably, reactions with sulfur donors are not affected by the amino acid replacement. The specificity of CysM toward O-phosphoserine together with the previously established novel mode of sulfur delivery via thiocarboxylated CysO (Burns, K. E., Baumgart, S., Dorrestein, P. C., Zhai, H., McLafferty, F. W., and Begley, T. P. (2005) J. Am. Chem. Soc. 127, 11602-11603) provide strong evidence for an O-phosphoserine-based cysteine biosynthesis pathway in M. tuberculosis that is independent of both O-acetylserine and the sulfate reduction pathway. The existence of an alternative biosynthetic pathway to cysteine in this pathogen has implications for the design strategy aimed at inhibition of this metabolic route.
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PMID:Cysteine synthase (CysM) of Mycobacterium tuberculosis is an O-phosphoserine sulfhydrylase: evidence for an alternative cysteine biosynthesis pathway in mycobacteria. 1879 56

Several enzymes have evolved as sensors in signal transduction pathways to control gene expression, thereby allowing bacteria to adapt efficiently to environmental changes. We recently identified the master regulator of cysteine metabolism in Bacillus subtilis, CymR, which belongs to the poorly characterized Rrf2 family of regulators. We now report that the signal transduction mechanism controlling CymR activity in response to cysteine availability involves the formation of a stable complex with CysK, a key enzyme for cysteine biosynthesis. We carried out a comprehensive quantitative characterization of this regulator-enzyme interaction by surface plasmon resonance and analytical ultracentrifugation. We also showed that O-acetylserine plays a dual role as a substrate of CysK and as an effector modulating the CymR-CysK complex formation. The ability of B. subtilis CysK to bind to CymR appears to be correlated to the loss of its capacity to form a cysteine synthase complex with CysE. We propose an original model, supported by the determination of the intracellular concentrations of the different partners, by which CysK positively regulates CymR in sensing the bacterial cysteine pool.
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PMID:The CymR regulator in complex with the enzyme CysK controls cysteine metabolism in Bacillus subtilis. 1897 48

Genome mining and biochemical analyses have shown that Leishmania major possesses two pathways for cysteine synthesis--the de novo biosynthesis pathway comprising SAT (serine acetyltransferase) and CS (cysteine synthase) and the RTS (reverse trans-sulfuration) pathway comprising CBS (cystathionine beta-synthase) and CGL (cystathionine gamma-lyase). The LmjCS (L. major CS) is similar to the type A CSs of bacteria and catalyses the synthesis of cysteine using O-acetylserine and sulfide with Kms of 17.5 and 0.13 mM respectively. LmjCS can use sulfide provided by the action of MST (mercaptopyruvate sulfurtransferase) on 3-MP (3-mercaptopyruvate). LmjCS forms a bi-enzyme complex with Leishmania SAT (and Arabidopsis SAT), with residues Lys222, His226 and Lys227 of LmjCS being involved in the complex formation. LmjCBS (L. major CBS) catalyses the synthesis of cystathionine from homocysteine, but, unlike mammalian CBS, also has high cysteine synthase activity (but with the Km for sulfide being 10.7 mM). In contrast, LmjCS does not have CBS activity. CS was up-regulated when promastigotes were grown in medium with limited availability of sulfur amino acids. Exogenous methionine stimulated growth under these conditions and also the levels of intracellular cysteine, glutathione and trypanothione, whereas cysteine had no effect on growth or the intracellular cysteine levels, correlating with the low rate of transport of cysteine into the cell. These results suggest that cysteine is generated endogenously by promastigotes of Leishmania. The absence of CS from mammals and the clear differences between CBS of mammals and Leishmania suggest that each of the parasite enzymes could be a viable drug target.
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PMID:Two pathways for cysteine biosynthesis in Leishmania major. 1929 28

In bacteria, the biosynthesis of Cys is accomplished by two enzymes that are encoded by the cysK and cysM genes. CysM is also able to use thiosulfate as a substrate to produce S-sulfocysteine. In plant cells, the biosynthesis of Cys occurs in the cytosol, mitochondria, and chloroplasts. Chloroplasts contain two O-acetylserine(thiol)lyase homologs, which are encoded by the OAS-B and CS26 genes in Arabidopsis thaliana. An in vitro enzymatic analysis of the recombinant CS26 protein demonstrated that this isoform possesses S-sulfocysteine synthase activity and lacks O-acetylserine(thiol)lyase activity. In vivo functional analysis of this enzyme in knockout mutants demonstrated that mutation of CS26 suppressed the S-sulfocysteine synthase activity that was detected in the wild type; furthermore, the cs26 mutants exhibited a reduction in size and showed paleness, but penetrance of the growth phenotype depended on the light regime. The cs26 mutant plants also had reductions in chlorophyll content and photosynthetic activity (neither of which were observed in oas-b mutants) as well as elevated glutathione levels. However, cs26 leaves were not able to properly detoxify reactive oxygen species, which accumulated to high levels under long-day growth conditions. The transcriptional profile of the cs26 mutant revealed that the mutation had a pleiotropic effect on many cellular and metabolic processes. Our findings reveal that S-sulfocysteine and the activity of S-sulfocysteine synthase play important roles in chloroplast function and are essential for light-dependent redox regulation within the chloroplast.
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PMID:Arabidopsis S-sulfocysteine synthase activity is essential for chloroplast function and long-day light-dependent redox control. 2017 39


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