Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.2.1.22 (cystathionine beta-synthase)
 965 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

O-Acetylserine (thiol) lyase, the last enzyme in the cysteine biosynthetic pathway, was purified to homogeneity from spinach leaf chloroplasts. The enzyme has a molecular mass of 68,000 and consists of two identical subunits of Mr 35,000. The absorption spectrum obtained at pH 7.5 exhibited a peak at 407 nm due to pyridoxal phosphate, and addition of O-acetylserine induced a considerable modification of the spectrum. The pyridoxal phosphate content was found to be 1.1 per subunit of 35,000, and the chromophore was displaced from the enzyme by O-acetylserine, leading to a progressive inactivation of the holoenzyme. Upon gel filtration chromatography on Superdex 200, part of the chloroplastic O-acetylserine (thiol) lyase eluted in association with serine acetyltransferase at a position corresponding to a molecular mass of 310,000 (such a complex called cysteine synthase has been characterized in bacteria). The activity of O-acetylserine (thiol) lyase was optimum between pH 7.5 and 8.5. The apparent Km for O-acetylserine was 1.3 mM and for sulfide was 0.25 mM. The calculated activation energy was 12.6 kcal/mol at 10 mM O-acetylserine. The overall amino-acid composition of spinach chloroplast O-acetylserine (thiol) lyase was different than that determined for the same enzyme (cytosolic?) obtained from a crude extract of spinach leaves. A polyclonal antibody prepared against the chloroplastic O-acetylserine (thiol) lyase exhibited a very low cross-reactivity with a preparation of mitochondrial matrix and cytosolic proteins suggesting that the chloroplastic isoform was distinct from the mitochondrial and cytosolic counterparts.
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PMID:Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts. 137 15

The last steps of cysteine synthesis in plants involve two consecutive enzymes. The first enzyme, serine acetyltransferase, catalyses the acetylation of L-serine in the presence of acetyl-CoA to form O-acetylserine. The second enzyme, O-acetylserine (thiol) lyase, converts O-acetylserine to L-cysteine in the presence of sulfide. We have, in the present work, over-produced in Escherichia coli harboring various type of plasmids, either a plant serine acetyltransferase or this enzyme with a plant O-acetylserine (thiol) lyase. The free recombinant serine acetyltransferase (subunit mass of 34 kDa) exhibited a high propensity to form high-molecular-mass aggregates and was found to be highly unstable in solution. However, these aggregates were prevented in the presence of O-acetylserine (thiol) lyase (subunit mass of 36 kDa). Under these conditions homotetrameric serine acetyltransferase associated with two molecules of homodimeric O-acetylserine (thiol) lyase to form a bienzyme complex (molecular mass approximately 300 kDa) called cysteine synthase containing 4 mol pyridoxal 5'-phosphate/mol complex. O-Acetylserine triggered the dissociation of the bienzyme complex, whereas sulfide counteracted the action of O-acetylserine. Protein-protein interactions within the bienzyme complex strongly modified the kinetic properties of plant serine acetyltransferase: there was a transition from a typical Michaelis-Menten model to a model displaying positive kinetic co-operativity with respect to serine and acetyl-CoA. On the other hand, the formation of the bienzyme complex resulted in a very dramatic decrease in the catalytic efficiency of bound O-acetylserine (thiol) lyase. The latter enzyme behaved as if it were a structural and/or regulatory subunit of serine acetyltransferase. Our results also indicated that bound serine acetyltransferase produces a build-up of O-acetylserine along the reaction path and that the full capacity for cysteine synthesis can only be achieved in the presence of a large excess of free O-acetylserine (thiol) lyase. These findings contradict the widely held belief that such a bienzyme complex is required to channel the metabolite intermediate O-acetylserine.
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PMID:Interactions between serine acetyltransferase and O-acetylserine (thiol) lyase in higher plants--structural and kinetic properties of the free and bound enzymes. 969 24

O-Acetylserine (thiol)-lyase (cysteine synthase) was purified from Azospirillum brasilense Sp7. After hydrolysis of the purified protein, amino acid sequences of five peptides were obtained, which permitted the cloning and sequencing of the cysK gene. The deduced amino acid sequence of cysteine synthase exhibited homology with several putative proteins from Alpha- and Gammaproteobacteria. Azospirillum brasilense Sp7 cysK exhibited 58% identity (72% similarity) with Escherichia coli K12 and Salmonella enterica serovar Typhimurium cysteine synthase proteins. An E. coli auxotroph lacking cysteine synthase loci could be complemented with A. brasilense Sp7 cysK. The 3.0-kb HindIII-EcoRI fragment bearing cysK contained two additional ORFs encoding a putative transcriptional regulator and dUTPase. Insertional disruption of the cysK gene did not produce a cysteine auxotroph, indicating that gene redundancy in the cysteine biosynthetic or other biosynthetic pathways exists in Azospirillum, as already described in other bacteria. Nitrogen fixation was not altered in the mutant strain as determined by acetylene reduction. However, this strain showed an eight-fold reduction in tellurite resistance as compared to the wild-type strain, which was only observed during growth in minimal medium. These data confirm earlier observations regarding the importance of cysteine metabolism in tellurite resistance.
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PMID:Identification, cloning and characterization of cysK, the gene encoding O-acetylserine (thiol)-lyase from Azospirillum brasilense, which is involved in tellurite resistance. 1690 31