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)

Saturation curves for O-acetylserine sulfhydrylase using the substrate analogues O-propionyl-L-serine, O-butyryl-L-serine, and beta-chloro-L-alanine all exhibit substrate inhibition and yield Km values comparable to O-acetyl-L-serine, except the O-butyryl derivative which has a Km 5-fold higher. Since all analogues are used as substrates and yield similar kinetic parameters in most cases, it is possible that they share a common intermediate. This evidence also suggests that specificity of O-acetylserine sulfhydrylase resides in the fact that the beta-substituted moiety on L-serine is a good leaving group. The overall rate equation for O-acetylserine sulfhydrylase was derived. A comparison of the numerical integration of the rate equation and an experimental time course is given.
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PMID:Overall mechanism and rate equation for O-acetylserine sulfhydrylase. 86 90

Serine acetyltransferase (SAT) from Escherichia coli is subject to feedback inhibition by L-cysteine. A mutant was isolated which excretes L-cysteine because of a lesion in cysE, the structural gene for SAT, rendering the enzyme less feedback sensitive. To analyse the structural basis for this mutation the cysE genes both from wild-type E. coli and the mutant strain were cloned and their nucleotide sequences determined. The cysE gene contained an open reading frame consisting of 819 bp, equivalent to a protein of 273 amino acids. The mutant gene showed a single base change in position 767 resulting in a methionine to isoleucine substitution. A causal connection between this SAT sequence alteration, feedback insensitivity and L-cysteine excretion was demonstrated. The SAT from the wild-type strain was purified. It was composed of a single polypeptide chain migrating in SDS gels according to an Mr of 34,000. As in Salmonella typhimurium, the enzyme was associated in a bifunctional complex with O-acetylserine (thiol)-lyase.
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PMID:L-cysteine biosynthesis in Escherichia coli: nucleotide sequence and expression of the serine acetyltransferase (cysE) gene from the wild-type and a cysteine-excreting mutant. 330 58

The incorporation of deuterated serine into cysteine during the metabolism of cystine by Escherichia coli was studied in order to determine the extent to which the carbon-sulfur bond(s) of the cystine is cleaved. The results indicate that the major route (approximately 80%) for cystine metabolism consists of a reductive cleavage of the cystine disulfide bond to form cysteine. Evidence is presented which shows that a portion of the remaining cystine is broken down by a pathway(s) which results in cleavage of the carbon-sulfur bond of the cystine. This pathway would be the same as that expected for the beta-elimination of pyruvate from cystine catalysed by the enzyme beta-cystathionase. In addition, a small portion of the resulting cysteine is shown to undergo a reversible dissociation to serine and hydrogen sulfide. Evidence is presented which shows that this dissociation is caused by the enzyme cysteine synthetase [O-acetyl-L-serine acetate-lyase (adding H2S)].
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PMID:An evaluation of the in vivo metabolism of cystine in Escherichia coli using stable isotopes. 642 9

Extracts of Desulfovibrio vulgaris were found to contain serine transacetylase and cysteine synthase activities. When extracts were incubated with bisulfite and o-acetylserine, or acetyl coenzyme A plus L-serine, under a hydrogen atmosphere, cysteine was formed. Pyruvate served as a reductant for bisulfite reduction to sulfide and concomitantly provided the acetyl moiety for acetyl coenzyme A formation. Consequently, when extracts were incubated with pyruvate, bisulfite, and L-serine, cysteine synthesis resulted.
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PMID:Cysteine synthesis by Desulfovibrio vulgaris extracts. 736 31

The O-acetylserine sulfhydrylase (OASS) reaction has been studied using a number of spectral probes including UV--visible, fluorescence, circular dichroism, and 31P NMR spectroscopy. The addition of L-cysteine, L-alanine, and glycine to OASS results in a shift in lambda max of 412 nm for the internal Schiff base to 418 nm resulting from the formation of the external Schiff base. The addition of L-serine or O-methyl-D,L-serine gives decreases of the absorbance of unliganded enzyme at 412 nm of about 50% and 20%, respectively, concomitant with an increase in the absorbance at 320 nm and a shift in the lambda max of the remaining visible absorbance to 418 nm. The spectral shifts observed in the presence of L-serine are suggestive of establishing an equilibrium between different forms of external Schiff base. The concentration dependence of the changes at 440 (L-cysteine) and 320 nm (L-serine) provides an estimate of the dissociation constant for the external aldimine. The pH dependence of the dissociation constant suggests the alpha-amine of the amino acid must be unprotonated for nucleophilic attack at C4' of PLP, and an enzyme side chain must be unprotonated to hydrogen-bond the thiol or hydroxyl side chain of the amino acid. When L-cysteine is the amino acid, the thiol side chain must be protonated to hydrogen-bond to the unprotonated enzyme side chain. The 31P NMR chemical shift is increased from 5.2 ppm for unliganded enzyme to 5.3 ppm in the presence of L-cysteine, signaling a tighter interaction at the 5'-phosphate upon formation of the external Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification and spectral characterization of the external aldimine of the O-acetylserine sulfhydrylase reaction. 754 55

Serine acetyltransferase (SATase; EC 2.3.1.30), which catalyzes the reaction connecting serine and cysteine/methionine metabolism, plays a regulatory role in cysteine biosynthesis in plants. We have isolated a cDNA clone encoding SATase by direct genetic complementation of a Cys- mutation in Escherichia coli using an expression library of Citrullus vulgaris (watermelon) cDNA. The cDNA encodes a polypeptide of 294 amino acids (31,536 Da) exhibiting 51% homology with that of E. coli SATase. DNA-blot analysis indicated the presence of a single copy of the SATase gene (sat) in watermelon. RNA hybridization analysis suggested the relatively ubiquitous and preferential expression in the hypocotyls of etiolated seedlings. Immunoblot analysis indicated the accumulation of SATase predominantly in etiolated plants. L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the SATase in an allosteric manner, indicating the regulatory function of SATase in this metabolic pathway, whereas beta-(pyrazole-1-yl)-L-alanine, a secondary metabolite formed partly through the cysteine biosynthetic pathway, showed no inhibitory effect. A multi-enzyme complex was formed from recombinant proteins of SATase and cysteine synthase (O-acetylserine(thiol)-lyase) from watermelon, suggesting efficient metabolic channeling from serine to cysteine, preventing the diffusion of intermediary O-acetyl-L-serine.
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PMID:Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory role in cysteine biosynthesis from watermelon. 760

When cells of Escherichia coli are grown on lactate (or other carbon sources), an addition of serine to the medium causes growth inhibition. This growth inhibition is caused by inhibition by serine of homoserine dehydrogenase I, which is involved in threonine-isoleucine biosynthesis [Hama, H., Sumita, Y., Kakutani, Y., Tsuda, M., & Tsuchiya, T. (1990) Biochem. Biophys. Res. Commun. 168, 1211-1216]. We have cloned and sequenced genes which enhance the serine-sensitivity. Two open reading frames were found and designated as sseA and sseB. Introduction of either sseA or sseB gene, or both, into E. coli cells enhanced the serine-sensitivity. The sseA gene elicited stronger enhancement than sseB. The deduced amino acid sequence of SseA showed considerable similarity with that of bovine liver rhodanese, which catalyzes sulfur transfer from thiosulfate. We observed a twofold increase in rhodanese activity in E. coli cells harboring a plasmid carrying the sseA gene. The position of sseA in the genetic map is around 52'. However, sseA is different from cysM, which codes for O-acetylserine sulfhydrylase-B, an enzyme catalyzing sulfur transfer from thiosulfate to O-acetylserine, the map position of which is also around 52'.
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PMID:Enhancement of serine-sensitivity by a gene encoding rhodanese-like protein in Escherichia coli. 798 94

The responsibility of cysteine synthase (EC 4.2.99.8) from watermelon (Citrullus vulgaris) for the formation of beta-(pyrazole-1-yl)-L-alanine, a non-protein amino acid specifically accumulated in Curcubitaceae plants, was confirmed in vitro and in vivo by the cloned cDNA on expression vectors, pCCS11 and pCEN1. The cDNA sequence derived from pCCS11, an expression vector driven by the lacZ promoter, was placed under the transcriptional control of strong T7 promoter of pET3d to yield an over-expression vector, pCEN1, in Escherichia coli. The concentration of the exogenous cysteine synthase protein was increased up to approximately 10% of the total soluble protein of E. coli cells by the expression of cDNA on pCEN1. beta-(Pyrazole-1-yl)-L-alanine was formed in vitro from O-acetyl-L-serine and pyrazole by the action of cysteine synthase expressed in E. coli carrying pCCS11 or pCEN1. To confirm the responsibility of cysteine synthase for the formation of beta-(pyrazole-1-yl)-L-alanine in vivo, the feeding experiments of pyrazole and serine or O-acetyl-L-serine were carried out using the transformed E. coli culture. beta-(Pyrazole-1-yl)-L-alanine was produced in vivo by feeding the substrates to the culture of E. coli carrying pCEN1. These results provide the confirming evidence that the cloned cysteine synthase of watermelon catalyzes the formation of beta-(pyrazole-1-yl)-L-alanine, indicating that beta-pyrazolealanine synthase is identical with cysteine synthase in Cucurbitaceae plants.
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PMID:Evidence for identity of beta-pyrazolealanine synthase with cysteine synthase in watermelon: formation of beta-pyrazole-alanine by cloned cysteine synthase in vitro and in vivo. 828 Jan 25

The reaction of the substrate O-acetyl-L-serine (OAS) with the pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase-A (OASS-A) proceeds via the transient formation of an external aldimine absorbing at 420 nm and a stable alpha-aminoacrylate intermediate absorbing at 330 and 465 nm. Stable external aldimine species are obtained by reaction of the enzyme with either the reaction product L-cysteine or the product analog L-serine. Static and time-resolved fluorescence emission properties of the coenzyme in the above catalytic intermediates have been used to directly probe the active site conformation at different stages of the catalytic pathway. Upon excitation at either 420 or 330 nm, the external aldimines with L-cysteine and L-serine exhibit a structured emission centered at 490 nm with a shoulder at 530 nm. Fluorescence decays upon excitation at 420 nm are best fitted using two components with lifetimes of 1.1 and 3.8 ns, with the fractional intensity of the slow component being 0.92 with L-cysteine and 0.75 with L-serine, respectively. The fast component, emitting at 530 nm, is attributed to a dipolar species formed in the excited state by proton dissociation, and the slow component, emitting at 490 nm, is attributed to a ketoenamine tautomer of the external aldimine. The slow component for external aldimine fluorescence decay is characterized by the same lifetime value as that of the internal aldimine with an increased fractional intensity, indicating that the distribution between the ketoenamine and the dipolar species is shifted toward the ketoenamine tautomer in the external aldimine, compared to the internal aldimine. Differences in equilibrium distribution of ketoenamine and enolimine tautomers can also account for differences in the emission properties of the external aldimines of L-cysteine and L-serine. The alpha-aminoacrylate species is characterized by a relatively weak emission. Upon excitation at 330 nm, the emission exhibits two bands centered at 420 and 540 nm, whereas upon excitation at 420 nm the emission bands are centered at 500 and 540 nm, and upon excitation at 465 nm, the main absorbance peak of the alpha-aminoacrylate species, the emission spectrum shows a band at 540 nm. The fluorescence decays, upon excitation at 330 nm, are best fitted using three components with lifetime values similar to those found for the internal aldimine, with the slow component predominating. Species-associated spectra, collected between 400 and 520 nm upon excitation at 350 nm, indicate the presence of a fast component overlapping the slow component on the blue side of the emission spectrum, as detected for the internal aldimine. When the excitation wavelength is 420 nm, there are only two components with the fast one predominating. A further increase in the fractional intensity of the fast component is observed upon excitation at 465 nm. The weak emission and the short lifetime of the emission excited at 465 nm indicate that this alpha-aminoacrylate tautomer interacts significantly with neighboring groups of the protein matrix and may be endowed with a higher mobility than the external aldimine.
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PMID:Time-resolved fluorescence of O-acetylserine sulfhydrylase catalytic intermediates. 939 72

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


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