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1.
An immunoadsorbent column chromatography procedure utilizing antibody to one component of a multienzyme complex has been utilized for purification of the second component of this complex. Rabbit immunoglobulin G (IgG) specific for the O-acetylserine sulfhydrylase component of the multienzyme complex cysteine synthetase was linked to Sepharose 4B resin. A crude preparation of cysteine synthetase was bound to a column of IgG-Sepharose, other proteins were removed by washing, and the serine acetyltransferase component of the complex was eluted with 50 mmO-acetylserine, which dissociates the complex of the two enzymes. This purification step produces about a 400-fold increase in specific activity. 相似文献
2.
Francesca Spyrakis Ratna Singh Pietro Cozzini Barbara Campanini Enea Salsi Paolo Felici Samanta Raboni Paolo Benedetti Gabriele Cruciani Glen E. Kellogg Paul F. Cook Andrea Mozzarelli 《PloS one》2013,8(10)
The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis. 相似文献
3.
Fumigation of both, cucurbit plants and cucurbit leaf homogenateswith hydrogen sulfide (H2S) resulted in an increase in solublethiol, mainly glutathione and cysteine. In leaf homogenatesthis increase was counteracted or prevented by the additionat 1 mM of inhibitors of pyridoxalphosphate dependent enzymesor of products of the cysteine desulfhydrase reaction. Thesecompounds inhibited cysteine desulfhydrase activity, but didnot severely affect O-acetylserine sulfhydrylase activity atthis concentration. These results provide circumstantial evidencethat cysteine desulfhydrase in its reverse reaction, but notO-acetylserine sulfhydrylase participates in the assimilationof atmospheric H2S. (Received February 4, 1991; Accepted April 30, 1991) 相似文献
4.
Crystal structure of serine acetyl transferase from Brucella abortus and its complex with coenzyme A
Sudhir Kumar Nitesh Kumar Neelima Alam Samudrala Gourinath 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(10):1741-1748
Brucella abortus is the major cause of premature foetal abortion in cattle, can be transmitted from cattle to humans, and is considered a powerful biological weapon. De novo cysteine biosynthesis is one of the essential pathways reported in bacteria, protozoa, and plants. Serine acetyltransferase (SAT) initiates this reaction by catalyzing the formation of O-acetylserine (OAS) using l-serine and acetyl coenzyme A as substrates. Here we report kinetic and crystallographic studies of this enzyme from B. abortus. The kinetic studies indicate that cysteine competitively inhibits the binding of serine to B. abortus SAT (BaSAT) and noncompetitively inhibits the binding of acetyl coenzyme A. The crystal structures of BaSAT in its apo state and in complex with coenzyme A (CoA) were determined to 1.96 Å and 1.87 Å resolution, respectively. BaSAT was observed as a trimer in a size exclusion column; however, it was seen as a hexamer in dynamic light scattering (DLS) studies and in the crystal structure, indicating it may exist in both states. The complex structure shows coenzyme A bound to the C-terminal region, making mostly hydrophobic contacts from the center of the active site extending up to the surface of the protein. There is no conformational difference in the enzyme between the apo and the complexed states, indicating lock and key binding and the absence of an induced fit mechanism. 相似文献
5.
Toru Nagasawa Gurdev Singh Dhillon Takafumi Ishii Hideaki Yamada 《Journal of biotechnology》1985,2(6):365-377
The regulatory properties of serine-O-transacetylase and O-acetylserine sulfhydrylase have been investigated with 3-chloro-l-alanine resistant Bacillus sphaericus L-118. The enhancement of O-acetylserine sulfhydrylase formation by 3-chloro-l-alanine was observed and this effect was counteracted by corepressor l-cysteine. O-Acetylserine sulfhydrylase occurring in B. sphaericus L-118 can catalyse β-replacement reaction of 3-chloro-l-alanine in the presence of a high concentration of sodium hydrosulfide to form l-cysteine. The optimal reaction conditions for l-cysteine production were studied using resting cells. Under optimal conditions, about 80% of the added 3-chloro-l-alanine could be converted to l-cysteine. The highest yield achieved was 70 mg of l-cysteine per 1.0 ml of the reaction mixture. 相似文献
6.
Cell-free extracts of Salmonella typhimurium synthesize a mutagenic azide metabolite from sodium azide and O-acetylserine. S. typhimurium mutant DW379 (O-acetylserine sulfhydrylase-deficient) extracts were neither able to carry out this reaction nor produce the mutagenic azide metabolite in vivo. The in vitro reaction was inhibited by sulfide but not by l-cysteine. The catalytic activity responsible for the mutagenic metabolite synthesis was stable to brief heating up to 55°C and had a pH optimum between 7–7.4. These results suggest that the enzyme O-acetylserine sulfhydrylase catalyzes the reaction of azide with O-acetylserine to form a mutagenic azide metabolite. 相似文献
7.
Hankuil Yi Sanghamitra Dey Sangaralingam Kumaran Soon Goo Lee Hari B. Krishnan Joseph M. Jez 《The Journal of biological chemistry》2013,288(51):36463-36472
Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase. Formation of the cysteine regulatory complex (CRC) is a critical biochemical control feature in plant sulfur metabolism. Here we present the 1.75–3.0 Å resolution x-ray crystal structures of soybean (Glycine max) SAT (GmSAT) in apoenzyme, serine-bound, and CoA-bound forms. The GmSAT-serine and GmSAT-CoA structures provide new details on substrate interactions in the active site. The crystal structures and analysis of site-directed mutants suggest that His169 and Asp154 form a catalytic dyad for general base catalysis and that His189 may stabilize the oxyanion reaction intermediate. Glu177 helps to position Arg203 and His204 and the β1c-β2c loop for serine binding. A similar role for ionic interactions formed by Lys230 is required for CoA binding. The GmSAT structures also identify Arg253 as important for the enhanced catalytic efficiency of SAT in the CRC and suggest that movement of the residue may stabilize CoA binding in the macromolecular complex. Differences in the effect of cold on GmSAT activity in the isolated enzyme versus the enzyme in the CRC were also observed. A role for CRC formation as a molecular chaperone to maintain SAT activity in response to an environmental stress is proposed for this multienzyme complex in plants. 相似文献
8.
A compound soluble in organic solvents and synthesized from [14C]acetate by isolated spinach chloroplasts incubated in the dark in the presence of dithiothreitol was shown to be O-acetyl dithiothreitol. The chloroplast system was required for the activation of acetate to acetyl CoA, but the transfer of the acetyl moiety to dithiothreitol was nonenzymatic. The first product of the reaction was shown to be S-acetyl dithiothreitol, but in the presence of an oxidant, simultaneous ring closure and migration of the acetyl group from the thiol to an adjacent hydroxyl group occurred to form an O-acetyl dithiothreitol.The acetyl transfer reaction involving acetyl CoA and dithiothreitol showed a marked pH dependence, being most active at about pH 9 and inoperative below pH 6. All acyl CoAs tested (C2-C18) rapidly labeled dithiothreitol; acetyl acyl carrier protein, and palmityl acyl carrier protein were much less reactive and free fatty acids were unreactive. The thiol reagents dithioerythritol, glutathione, and cysteine, in addition to dithiothreitol, reacted rapidly with acetyl CoA to form the corresponding acetyl mercaptans. 2-Mercaptoethanol was much less reactive; oxidized dithiothreitol was unreactive. The second-order rate constant for acetyl dithiothreitol synthesis was 12.3 m?1 min?1 at pH 8.5 and 30 °C. 相似文献
9.
The use of a sulfide ion selective electrode to study O-acetylserine sulfhydrylase from germinating rapeseed 总被引:1,自引:0,他引:1
P D That-Tjien-NgoShargool 《Analytical biochemistry》1973,54(1):247-261
O-Acetylserine sulfhydrylase was partially purified from germinating seeds of Rape (Brassica napus L. var Target), and a sulfide ion specific electrode used to study the properties of this enzyme. It was demonstrated that the sensitivity of the electrode to sulfide ion concentration was not affected by pH, or by various concentrations of cysteine, sulfate, sulfite, and acetate.Kinetic studies showed that the Km for O-acetyl-l-serine was 1.74 × 10?6m, while the Km for sulfide was 4.3 × 10?4m. The implications of the data, together with the methods used to obtain them, are discussed. 相似文献
10.
《Mutation Research Letters》1983,119(3-4):281-285
The ability of Arabidopsis, Drosophila and Neurospora to convert azide to its mutagenic metabolite was investigated. Cultures of these organisms all contained significant levels of O-acetylserine sulfhydrylase activity. Extracts from each organism produced a product from O-acetylserine and azide in vitro which was mutagenic in Salmonella typhimurium TA1530. 相似文献
11.
Summary A 1,2,4-triazole resistant mutant of S. typhimurium has been isolated, in which serine transacetylase activity is seven times higher than in wild type. Partially purified serine transacetylase from a strain carrying the trz-312 mutation has kinetic properties which are virtually identical to those of the wild type enzyme and binds to O-acetylserine sulfhydrylase A to form a cysteine synthetase complex which is also indistinguishable from that found in wild type. Thus the increased activity of serine transacetylase associated with trz-312 appears to result from increased quantities of a kinetically normal, enzyme protein. Resistance to 1,2,4-triazole is probably due to the ability of trz-312 strains to synthesize O-acetyl-l-serine at a rapid enough rate to compensate for that utilized by the O-acetylserine triazolylase reaction.Genetic mapping experiments, using P1-mediated transduction, show that trz-312 is 91–99% linked to cysE, the structural gene for serine transacetylase. The results of three point crosses indicate that this mutation is located at one extreme end of the cysE locus, as would be expected for a promotor mutation. 相似文献
12.
Regulation of Sulfate Assimilation in Tobacco Cells: EFFECT OF NITROGEN AND SULFUR NUTRITION ON SULFATE PERMEASE AND O-ACETYLSERINE SULFHYDRYLASE
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Smith IK 《Plant physiology》1980,66(5):877-883
The effect of nitrogen and sulfur nutrition on sulfate permease and O-acetylserine sulfhydrylase was studied in tobacco cells. 相似文献
13.
Role of o-acetylserine in hydrogen sulfide emission from pumpkin leaves in response to sulfate 总被引:2,自引:0,他引:2
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Rennenberg H 《Plant physiology》1983,73(3):560-565
In the presence of excess sulfate, cysteine synthesis in pumpkin (Cucurbita pepo) leaves is not limited by sulfate reduction, but by the availability of O-acetylserine. Feeding of O-acetylserine or its metabolic precursors S-acetyl-coenzyme-A and coenzyme A to leaf discs enhanced the incorportion of [35S]sulfate into reduced sulfur compounds, mainly into cysteine, at the cost of lowered H2S emission; the uptake and reduction of sulfate is not affected by these treatments. β-Fluoropyruvate, an inhibitor of the generation of S-acetyl-coenzyme A via pyruvate dehydrogenase, stimulated H2S emission in response to sulfate. This stimulation is overcompensated by addition of O-acetylserine, S-acetyl-coenzyme A, or coenzyme A. These results indicate that, in the presence of high amounts of sulfate, excess sulfur is reduced and emitted as H2S into the atmosphere. The H2S emitted seems to be produced by liberation from a precursor of cysteine rather than by cysteine desulfhydration. 相似文献
14.
M S Balakrishnan T R Sharp J J Villafranca 《Biochemical and biophysical research communications》1978,85(3):991-998
Oxygen-18 exchange out of [18O]Pi catalyzed by Mg2+-activated unadenylated glutamine synthetase from was followed by 31P-NMR in the presence of the other substrates, ADP and L-glutamine. The pattern of the in the species P18O4, P18O316O1, P18O216O2, P18O116O3, P16O4 during the exchange followed a binomial distribution consistent with indiscriminate removal of any of the four oxygens of Pi. The rate constant for exchange was 410±40 min?1 while the rate constant for net reaction (ATP formation) was 62±4 min?1. Thus exchange proceeds ~7 times faster than net reaction, a finding in accord with that of Stokes and Boyer ( (1976) , 5558) for the Mn2+-activated adenylylated glutamine synthetase. A model for the overall catalytic events first derived from rapid kinetic fluorescence experiments (Rhee and Chock, Proc. Natl. Acad. Sci. USA, (1976) , 476) was successfully used to fit the oxygen exchange data in this paper. 相似文献
15.
Sylvette Bas Elisabeth Imesch Daniel Ricquier Françoise Assimacopoulos-Jeannet Josiane Seydoux Jean-Paul Giacobino 《Life sciences》1983,32(18):2123-2130
The activities of the main enzymes involved in fatty acid utilization i.e. palmitoyl CoA synthetase as well as peroxisomal and mitochondrial β-oxidation were measured in brown adipose tissue homogenates of lean and / mice kept at 23°C or acclimated at 4°C. The proton conductance pathway, i.e. the number of purine nucleotide (GDP) binding sites and the percentage of 32,000 polypeptide in brown adipose tissue mitochondria were also measured. In the if/ mice at 23° C, the specific activities of the palmitoyl CoA synthetase and of the β-oxidation as well as the number of GDB binding sites were lower than in the lean mice by 26%, 43% and 37%, respectively. The percentage of 32, 000 polypeptide, however, was the same in both groups. In the / mice at 23° C, the lower homogenate β-oxidation specific activity was due to the fact that the peroxisomal and mitochondrial specific activities were 44% and 37% lower, respectively. Cold acclimation at 4° C was found to cause an increase of the palmitoyl CoA synthetase specific activity, of the palmitoyl CoA synthetase and peroxisomal β-oxidation total activities and of the number of GDP binding sites, in both lean and / mice. Cold acclimation increased the percentage of 32,000 polypeptide in the / mice only. 相似文献
16.
The role of acetyl coenzyme A: butyrate coenzyme A in the transferase uptake of butyrate by isolated membrane vesicles of Escherichia coli 总被引:6,自引:0,他引:6
F E Frerman 《Archives of biochemistry and biophysics》1973,159(1):444-452
The acetyl CoA:butyrate CoA transferase catalyzes the translocation of butyrate in membrane vesicles prepared from a strain of Escherichia coli which is depressed for the acetoacetate degradation operon. Butyrate accumulated in the membranes as butyryl CoA. The role of the transferase in uptake is supported by the following observations: (i) uptake is stimulated by acetyl CoA; (ii) the solubilized CoA transferase and uptake exhibit KmS for butyrate, pH optima and levels inhibition by N-ethylmaleimide that are virtually identical; (iii) significant amounts of the CoA transferase are found associated with the membranes and uptake is rapidly inhibited by butyryl CoA and acetate, the products of the CoA transferase-catalyzed reaction. The fact that butyrate uptake did not exhibit saturation kinetics with increasing concentrations of acetyl CoA suggested that the transferase is not localized on the outer surface of the membrane. The level of free butyrate in the vesicles, the fact that butyrate uptake exhibited saturation kinetics with increasing concentrations of butyrate, and the observation that radioactivity was not rapidly lost from the vesicles following addition of butyryl CoA or acetate to incubation mixtures indicated that butyrate is translocated rather than trapped by the CoA transferase. 相似文献
17.
L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Out of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium 相似文献
18.
David J.T. Porter Noel G. Rudie Harold J. Bright 《Archives of biochemistry and biophysics》1983,225(1):157-163
The reactivities of the nitro analogs of the substrates of adenylosuccinate synthetase and adenylosuccinate lyase, the enzymes which catalyze the penultimate and last step, respectively, in the pathway for AMP biosynthesis have been examined. Alanine-3-nitronate, an aspartate analog, was a substrate for the synthetase from Azotobacter vinelandii, having a which was ~30% that for aspartate. The product of this reaction was N6-(l-1-carboxy-2-nitroethyl)-AMP. Of nine other substrate analogs tested, only cysteine sulfinate (having 5.5% of the activity of aspartate) was reactive. These results demonstrate the strict requirement of the synthetase for a negatively charged substituent, with a carboxylate-like geometry, at the β-carbon of the α-amino acid substrate. The lyase, purified to homogeneity from brewer's yeast by a new procedure, did not utilize N6-(l-1-carboxy-2-nitroethyl)-AMP as a substrate. However, the nitronate form of this analog was a good inhibitor of the lyase ( when compared to adenylosuccinate), suggesting that it mimics a carbanionic intermediate in the reaction pathway. The avid binding of bromphenol blue by the lyase (i = 0.95 μM) was used for active site titrations and for displacement of the enzyme, in the purification protocol, from blue Sepharose. 相似文献
19.
20.
Rapid and reversible activation of acetyl CoA hydrolase in intact pineal cells by disulfide exchange
M.A.A. Namboodiri Joan L. Weller David C. Klein 《Biochemical and biophysical research communications》1980,96(1):188-195
Using intact pinealocytes in suspended cell culture it has been determined that acetyl CoA hydrolase activity can be rapidly increased by treatment with cystamine. Similar results are seen with diacetylcystamine, but not with GSSG, penicillamine disulfide, nor with oxidized DTT. The activation of acetyl CoA hydrolase by cystamine is reversible: after cystamine treatment is terminated, enzyme activity decreases slowly in cell culture. It is also possible to reverse the activation by treating homogenates of cystamine-treated cells with dithiothreitol. These observations are consistent with previous findings indicating that pineal acetyl CoA hydrolase activity can be regulated protein thiol: disulfide exchange. The observations presented in this report also indicate that conditions within the cell allow this type of reaction to take place, and raise the possibility that disulfide exchange mechanisms may be physiologically involved in the intracellular regulation of the activity of this and perhaps other enzymes. 相似文献