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:6.2.1.1 (ACS)
78,556 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytosolic acetyl-CoA synthetase (AceCS1) activates acetate to supply the cells with acetyl-CoA for lipid synthesis. The cDNA for the mammalian AceCS1 has been isolated recently, and the mRNA was shown to be negatively regulated by sterols in cultured cells. In the current study, we describe the molecular mechanisms directing the sterol-regulated expression of murine AceCS1 by cloning and functional studies of the 5'-flanking region of the AceCS1 gene. An AceCS1 promoter-reporter gene (approximately 2.1 kilobase pairs) was negatively regulated when sterols were added to the medium of cultured cells, and the promoter was markedly induced by co-transfection of a plasmid that expresses the transcriptionally active nuclear form of either sterol regulatory element-binding protein (SREBP)-1a or -2 in HepG2 cells. Sequence analysis suggested that the AceCS1 promoter contains an E-box, two putative CCAAT-boxes, eight sterol regulatory element (SRE) motifs, and six GC-boxes. Gel shift assays demonstrated that all eight SRE motifs bound purified SREBP-1a in vitro with similar affinity. Luciferase reporter gene assays revealed that sterol regulation was critically dependent on three closely spaced SRE motifs and an adjacent GC-box. However, mutation of two putative upstream CCAAT-boxes did not affect SREBP dependent activation. Electrophoretic mobility "supershift" analyses confirmed that both Sp1 and Sp3 bound to the critical GC-box. In addition, transfection studies in Drosophila SL2 cells demonstrated that SREBP synergistically activated the AceCS1 promoter along with Sp1 or Sp3 but not with nuclear factor-Y.
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PMID:Transcriptional regulation of the murine acetyl-CoA synthetase 1 gene through multiple clustered binding sites for sterol regulatory element-binding proteins and a single neighboring site for Sp1. 1143 28

Pseudomonas aeruginosa ATCC 17933 uses a pyrroloquinoline quinone-dependent ethanol oxidation system. Two mutants of P. aeruginosa, unable to grow on ethanol and showing no acetyl-CoA synthetase (ACS) activity under standard test conditions, were complemented by cosmid pTB3018. Subcloning led to the isolation of a gene which encodes a protein with high similarity to acetyl-CoA synthetases. Interruption of the putative acsA gene by a kanamycin-resistance cassette resulted in a mutant also unable to grow on ethanol and with very low residual acetyl-CoA-forming activity. Complementation by the wild-type allele of the acsA gene restored growth and led to the expression of ACS activity in excess of that of wild-type cells. In wild-type P. aeruginosa, ACS activity was induced upon growth on ethanol, 2,3-butanediol, malonate and acetate. The wild-type and mutants defective in ACS activity showed an active acetate kinase (ACK) under the growth conditions used; however, phosphotransacetylase (PTA) could not be detected. The data indicate that P. aeruginosa requires active acsA gene product for growth on ethanol.
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PMID:The Pseudomonas aeruginosa acsA gene, encoding an acetyl-CoA synthetase, is essential for growth on ethanol. 1157 46

Acetyl coenzyme A (CoA) synthetase (ADP forming) (ACD) represents a novel enzyme of acetate formation and energy conservation (acetyl-CoA + ADP + P(i) right harpoon over left harpoon acetate + ATP + CoA) in Archaea and eukaryotic protists. The only characterized ACD in archaea, two isoenzymes from the hyperthermophile Pyrococcus furiosus, constitute 145-kDa heterotetramers (alpha(2), beta(2)). The coding genes for the alpha and beta subunits are located at different sites in the P. furiosus chromosome. Based on significant sequence similarity of the P. furiosus genes, five open reading frames (ORFs) encoding putative ACD were identified in the genome of the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus and one ORF was identified in the hyperthermophilic methanogen Methanococcus jannaschii. The ORFs constitute fusions of the homologous P. furiosus genes encoding the alpha and beta subunits. Two ORFs, AF1211 and AF1938, of A. fulgidus and ORF MJ0590 of M. jannaschii were cloned and functionally overexpressed in Escherichia coli. The purified recombinant proteins were characterized as distinctive isoenzymes of ACD with different substrate specificities. In contrast to the Pyrococcus ACD, the ACDs of Archaeoglobus and Methanococcus constitute homodimers of about 140 kDa composed of two identical 70-kDa subunits, which represent fusions of the homologous P. furiosus alpha and beta subunits in an alphabeta (AF1211 and MJ0590) or betaalpha (AF1938) orientation. The data indicate that A. fulgidus and M. jannaschii contains a novel type of ADP-forming acetyl-CoA synthetase in Archaea, in which the subunit polypeptides and their coding genes are fused.
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PMID:Novel type of ADP-forming acetyl coenzyme A synthetase in hyperthermophilic archaea: heterologous expression and characterization of isoenzymes from the sulfate reducer Archaeoglobus fulgidus and the methanogen Methanococcus jannaschii. 1179 Jul 32

CO dehydrogenase/acetyl-CoA synthase (CODH/ACS), a key enzyme in the Wood-Ljungdahl pathway of anaerobic CO(2) fixation, is a bifunctional enzyme containing CODH, which catalyzes the reversible two-electron oxidation of CO to CO(2), and ACS, which catalyzes acetyl-CoA synthesis from CoA, CO, and a methylated corrinoid iron-sulfur protein (CFeSP). ACS contains an active site nickel iron-sulfur cluster that forms a paramagnetic adduct with CO, called the nickel iron carbon (NiFeC) species, which we have hypothesized to be a key intermediate in acetyl-CoA synthesis. This hypothesis has been controversial. Here we report the results of steady-state kinetic experiments; stopped-flow and rapid freeze-quench transient kinetic studies; and kinetic simulations that directly test this hypothesis. Our results show that formation of the NiFeC intermediate occurs at approximately the same rate as, and its decay occurs 6-fold faster than, the rate of acetyl-CoA synthesis. Kinetic simulations of the steady-state and transient kinetic results accommodate the NiFeC species in the mechanism and define the rate constants for the elementary steps in acetyl-CoA synthesis. The combined results strongly support the kinetic competence of the NiFeC species in the Wood-Ljungdahl pathway. The results also imply that the methylation of ACS occurs by attack of the Ni(1+) site in the NiFeC intermediate on the methyl group of the methylated CFeSP. Our results indicate that CO inhibits acetyl-CoA synthesis by inhibiting this methyl transfer reaction. Under noninhibitory CO concentrations (below 100 microM), formation of the NiFeC species is rate-limiting, while at higher inhibitory CO concentrations, methyl transfer to ACS becomes rate-limiting.
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PMID:Rapid kinetic studies of acetyl-CoA synthesis: evidence supporting the catalytic intermediacy of a paramagnetic NiFeC species in the autotrophic Wood-Ljungdahl pathway. 1182 25

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD+ and NADP+ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C4-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C2-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.
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PMID:[Peculiarities of ethanol metabolism in an Acinetobacter sp. mutant strain defective in exopolysaccharide synthesis]. 1202 23

In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha(2)beta(2) tetrameric 310 kDa bifunctional enzyme. ACS catalyzes the reversible reduction of CO(2) to CO and the synthesis of acetyl-CoA from CO (or CO(2) in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains seven metal-sulfur clusters of four different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kDa beta subunit, while the A-cluster, a Ni-X-Fe(4)S(4) cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kDa alpha subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire beta subunit was removed, as long as CO, rather than CO(2) and a low-potential reductant, was used as a substrate. Truncating an approximately 30 kDa region from the N-terminus of the alpha subunit yielded a 49 kDa protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO-binding properties. Further truncation afforded a 23 kDa protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of [Fe(4)S(4)](2+) clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 and 82 kDa fragments of the alpha subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multicentered metalloenzymes and to generate new complex metalloenzymes with interesting properties.
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PMID:Genetic construction of truncated and chimeric metalloproteins derived from the alpha subunit of acetyl-CoA synthase from Clostridium thermoaceticum. 1212 Nov 9

The acsABCDE genes in the Clostridium thermoaceticum genome are used for autotrophic acetyl-CoA synthesis using the Wood-Ljungdahl pathway. A 2.8-kb region between acsC and acsD was cloned and sequenced. Two open reading frames, orf7 (approximately 1.9 kb) and acsF (approximately 0.7 kb) were identified. orf7 appears to encode an Fe-S protein, in that it contains five conserved cysteine residues, three of which are present in a motif (CGGXXXCGXC) commonly used to coordinate Fe-S clusters. However, Orf7 is probably not involved in autotrophic acetyl-CoA synthesis, as homologous genes are present in organisms that do not utilize this pathway and are absent in many that do. In contrast, acsF is probably involved in this pathway. Sequence alignment of AcsF and eleven homologs reveals a number of conserved regions, including a P-loop that binds nucleoside triphosphates and catalyzes their hydrolysis. One homolog is CooC, an ATPase/GTPase that inserts Ni into a precursor form of the C-cluster of the carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum. Purified AcsF lacked Ni and Fe, and slowly catalyzed the hydrolysis of ATP. Such similarities to CooC suggest that AcsF may function to insert Ni into a Ni-deficient form of the bifunctional acetyl-CoA synthase/CODH from C. thermoaceticum (ACS(Ct)). However, this could not be established, as expression of acsF did not effect activation of recombinant AcsAB expressed in E. coli. Also, E. coli cells defective in hypB retained the ability to synthesize active recombinant AcsAB. Rather, the concentration of extracellular Ni(2+) ions was critical to activation.
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PMID:Identification and preliminary characterization of AcsF, a putative Ni-insertase used in the biosynthesis of acetyl-CoA synthase from Clostridium thermoaceticum. 1253 50

The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood-Ljungdahl pathway of autotrophic CO(2) fixation. A recent structure of the Moorella thermoacetica enzyme revealed that the ACS active site contains a [4Fe-4S] cluster bridged to a binuclear Cu-Ni site. Here, biochemical and x-ray absorption spectroscopic (XAS) evidence is presented that the copper ion at the M. thermoacetica ACS active site is essential. Depletion of copper correlates with reduction in ACS activity and in intensity of the "NiFeC" EPR signal without affecting either the activity or the EPR spectroscopic properties associated with CODH. In contrast, Zn content is negatively correlated with ACS activity without any apparent relationship to CODH activity. Cu is also found in the methanogenic CODH/ACS from Methanosarcina thermophila. XAS studies are consistent with a distorted Cu(I)-S(3) site in the fully active enzyme in solution. Cu extended x-ray absorption fine structure analysis indicates an average Cu-S bond length of 2.25 A and a metal neighbor at 2.65 A, consistent with the Cu-Ni distance observed in the crystal structure. XAS experiments in the presence of seleno-CoA reveal a Cu-S(3)Se environment with a 2.4-A Se-Cu bond, strongly implicating a Cu-SCoA intermediate in the mechanism of acetyl-CoA synthesis. These results indicate an essential and functional role for copper in the CODH/ACS from acetogenic and methanogenic organisms.
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PMID:Functional copper at the acetyl-CoA synthase active site. 1258 21

SIR2 proteins have NAD(+)-dependent histone deacetylase activity, but no metabolic role has been assigned to any of these proteins. In Salmonella enterica, SIR2 function was required for activity of the acetyl-CoA synthetase (Acs) enzyme. A greater than two orders of magnitude increase in the specific activity of Acs enzyme synthesized by a sirtuin-deficient strain was measured after treatment with homogeneous S. enterica SIR2 protein. Human SIR2A and yeast SIR2 proteins restored growth of SIR2-deficient S. enterica on acetate and propionate, suggesting that eukaryotic cells may also use SIR2 proteins to control the synthesis of acetyl-CoA by the level of acetylation of acetyl-CoA synthetases. Consistent with this idea, growth of a quintuple sir2 hst1 hst2 hst3 hst4 mutant strain of the yeast Saccharomyces cerevisiae on acetate or propionate was severely impaired. The data suggest that the Hst3 and Hst4 proteins are the most important for allowing growth on these short-chain fatty acids.
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PMID:Short-chain fatty acid activation by acyl-coenzyme A synthetases requires SIR2 protein function in Salmonella enterica and Saccharomyces cerevisiae. 1261 94

The crystal structure of the tetrameric alpha2beta2 acetyl-coenzyme A synthase/carbon monoxide dehydrogenase from Moorella thermoacetica has been solved at 1.9 A resolution. Surprisingly, the two alpha subunits display different (open and closed) conformations. Furthermore, X-ray data collected from crystals near the absorption edges of several metal ions indicate that the closed form contains one Zn and one Ni at its active site metal cluster (A-cluster) in the alpha subunit, whereas the open form has two Ni ions at the corresponding positions. Alternative metal contents at the active site have been observed in a recent structure of the same protein in which A-clusters contained one Cu and one Ni, and in reconstitution studies of a recombinant apo form of a related acetyl-CoA synthase. On the basis of our observations along with previously reported data, we postulate that only the A-clusters containing two Ni ions are catalytically active.
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PMID:Ni-Zn-[Fe4-S4] and Ni-Ni-[Fe4-S4] clusters in closed and open subunits of acetyl-CoA synthase/carbon monoxide dehydrogenase. 1266 Jul 15


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