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:6.4.1.2 (acetyl-CoA carboxylase)
2,876 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have cloned a DNA fragment from a genomic library of Myxococcus xanthus using an oligonucleotide probe representing conserved regions of biotin carboxylase subunits of acetyl coenzyme A (acetyl-CoA) carboxylases. The fragment contained two open reading frames (ORF1 and ORF2), designated the accB and accA genes, capable of encoding a 538-amino-acid protein of 58.1 kDa and a 573-amino-acid protein of 61.5 kDa, respectively. The protein (AccA) encoded by the accA gene was strikingly similar to biotin carboxylase subunits of acetyl-CoA and propionyl-CoA carboxylases and of pyruvate carboxylase. The putative motifs for ATP binding, CO(2) fixation, and biotin binding were found in AccA. The accB gene was located upstream of the accA gene, and they formed a two-gene operon. The protein (AccB) encoded by the accB gene showed high degrees of sequence similarity with carboxyltransferase subunits of acetyl-CoA and propionyl-CoA carboxylases and of methylmalonyl-CoA decarboxylase. Carboxybiotin-binding and acyl-CoA-binding domains, which are conserved in several carboxyltransferase subunits of acyl-CoA carboxylases, were found in AccB. An accA disruption mutant showed a reduced growth rate and reduced acetyl-CoA carboxylase activity compared with the wild-type strain. Western blot analysis indicated that the product of the accA gene was a biotinylated protein that was expressed during the exponential growth phase. Based on these results, we propose that this M. xanthus acetyl-CoA carboxylase consists of two subunits, which are encoded by the accB and accA genes, and occupies a position between prokaryotic and eukaryotic acetyl-CoA carboxylases in terms of evolution.
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PMID:Molecular cloning and characterization of two genes for the biotin carboxylase and carboxyltransferase subunits of acetyl coenzyme A carboxylase in Myxococcus xanthus. 1098 50

RNA editing is an important post-transcriptional process in chloroplasts and is thought to be functionally significant. Here we show a requirement of RNA editing for a functional enzyme. In peas, acetyl-CoA carboxylase (ACCase), a key enzyme of fatty acid synthesis, is composed of biotin carboxylase with the biotin carboxyl carrier protein and carboxyltransferase (CT). CT is composed of the nuclear-encoded alpha polypeptide and the chloroplast-encoded beta polypeptide in peas. One nucleotide of the beta polypeptide mRNA, which is edited in pea chloroplasts, converts the serine codon to the leucine codon. We show that this RNA editing is required for functional CT by comparing the unedited and edited recombinant enzymes. In plants not having a leucine codon at the same position, editing was shown to take place so as to create the leucine codon, indicating that editing is needed for in vivo CT activity and therefore for ACCase. To our knowledge, ACCase is an essential enzyme, suggesting that the chloroplast RNA editing is necessary for these plants.
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PMID:Chloroplast RNA editing required for functional acetyl-CoA carboxylase in plants. 1107 38

Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first step in the synthesis of fatty acids in Lactobacillus plantarum L137, were cloned and characterized. We identified six potential open reading frames, namely, manB, fabH, accB, accC, accD, and accA, in that order. Nucleotide sequence analysis suggested that fabH encoded beta-ketoacyl-acyl carrier protein synthase III, that the accB, accC, accD, and accA genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the beta and alpha subunits of carboxyltransferase, respectively, and that these genes were clustered. The organization of acc genes was different from that reported for Escherichia coli, for Bacillus subtilis, and for Pseudomonas aeruginosa. E. coli accB and accD mutations were complemented by the L. plantarum accB and accD genes, respectively. The predicted products of all five genes were confirmed by using the T7 expression system in E. coli. The gene product of accB was biotinylated in E. coli. Northern and primer extension analyses demonstrated that the five genes in L. plantarum were regulated polycistronically in an acc operon.
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PMID:Molecular characterization of Lactobacillus plantarum genes for beta-ketoacyl-acyl carrier protein synthase III (fabH) and acetyl coenzyme A carboxylase (accBCDA), which are essential for fatty acid biosynthesis. 1113 75

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multimeric protein complex consisting of three distinct and separate components: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source and has a distinct architecture that is characteristic of the ATP-grasp superfamily of enzymes. Included in this superfamily are d-Ala d-Ala ligase, glutathione synthetase, carbamyl phosphate synthetase, N(5)-carboxyaminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide transformylase, all of which have known three-dimensional structures and contain a number of highly conserved residues between them. Four of these residues of biotin carboxylase, Lys-116, Lys-159, His-209, and Glu-276, were selected for site-directed mutagenesis studies based on their structural homology with conserved residues of other ATP-grasp enzymes. These mutants were subjected to kinetic analysis to characterize their roles in substrate binding and catalysis. In all four mutants, the K(m) value for ATP was significantly increased, implicating these residues in the binding of ATP. This result is consistent with the crystal structures of several other ATP-grasp enzymes, which have shown specific interactions between the corresponding homologous residues and cocrystallized ADP or nucleotide analogs. In addition, the maximal velocity of the reaction was significantly reduced (between 30- and 260-fold) in the 4 mutants relative to wild type. The data suggest that the mutations have misaligned the reactants for optimal catalysis.
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PMID:Site-directed mutagenesis of ATP binding residues of biotin carboxylase. Insight into the mechanism of catalysis. 1134 47

Acetyl-CoA carboxylase catalyses the synthesis of malonyl-CoA, the first intermediate in fatty acid (hence phospholipid) synthesis. The accBC operon from Bacillus subtilis codes for two subunits of acetyl-CoA carboxylase, biotin carboxyl-carrier and biotin carboxylase. In this work, the 5'-end of the accBC mRNA was determined by primer-extension, and transcriptional fusion analysis of the accBC promoter was carried out under a variety of growth conditions. A direct correlation between the levels of transcription of the accBC genes and the rate of cellular growth is reported. Consistent results were also obtained in nutritional upshift and downshift experiments.
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PMID:Growth-rate regulation of the Bacillus subtilis accBC operon encoding subunits of acetyl-CoA carboxylase, the first enzyme of fatty acid synthesis. 1135 16

Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis. The Escherichia coli biotin carboxylase is readily isolated from the other components of the acetyl-CoA carboxylase complex such that enzymatic activity is retained. The three-dimensional structure of biotin carboxylase, determined by x-ray crystallography, demonstrated that the enzyme is a homodimer consisting of two active sites in which each subunit contains a complete active site. To understand how each subunit contributes to the overall function of biotin carboxylase, we made hybrid molecules in which one subunit had a wild-type active site, and the other subunit contained an active site mutation known to significantly affect the activity of the enzyme. One of the two genes encoded a poly-histidine tag at its N terminus, whereas the other gene had an N-terminal FLAG epitope tag. The two genes were assembled into a mini-operon that was induced to give high level expression of both enzymes. "Hybrid" dimers composed of one subunit with a wild-type active site and a second subunit having a mutant active site were obtained by sequential chromatographic steps on columns of immobilized nickel chelate and anti-FLAG affinity matrices. In vitro kinetic studies of biotin carboxylase dimers in which both subunits were wild type revealed that the presence of the N-terminal tags did not alter the activity of the enzyme. However, kinetic assays of hybrid dimer biotin carboxylase molecules in which one subunit had an active site mutation (R292A, N290A, K238Q, or E288K) and the other subunit had a wild-type active site resulted in 39-, 28-, 94-, and 285-fold decreases in the activity of these enzymes, respectively. The dominant negative effects of these mutant subunits were also detected in vivo by monitoring the rate of fatty acid biosynthesis by [(14)C]acetate labeling of cellular lipids. Expression of the mutant biotin carboxylase genes from an inducible arabinose promoter resulted in a significantly reduced rate of fatty acid synthesis relative to the same strain that expressed the wild type gene. Thus, both the in vitro and in vivo data indicate that both subunits of biotin carboxylase are required for activity and that the two subunits must be in communication during enzyme function.
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PMID:Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer. 1139 Apr 6

Fatty acid synthesis in pea chloroplasts is regulated by light/dark. The regulatory enzyme acetyl-CoA carboxylase is modulated by light/dark, presumably under redox regulation. Acetyl-CoA carboxylase is a multienzyme complex composed of biotin carboxylase and carboxyltransferase (CT). To demonstrate the redox regulation of CT, composed of the nuclear-encoded alpha and the chloroplast-encoded beta subunits, we identified the cysteine residues involved in such regulation. We expressed the recombinant CT in Escherichia coli and found that the partly deleted CT was, like the full-length CT, sensitive to a redox state. Site-directed mutagenesis of the deleted CT showed that replacement by alanine of the cysteine residue 267 in the alpha polypeptide or 442 in the beta polypeptide resulted in redox-insensitive CT and broke the intermolecular disulfide bond between the alpha and beta polypeptides. Similar results were confirmed in the full-length CT. These results indicate that the two cysteines in recombinant CT are involved in redox regulation by intermolecular disulfide-dithiol exchange between the alpha and beta subunits. Immunoblots of extract from plants incubated in the light or dark supported that such a disulfide-dithiol exchange is relevant in vivo. A covalent bond between a nuclear-encoded polypeptide and a chloroplast-encoded polypeptide probably regulates the enzyme activity in response to light.
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PMID:Thiol-disulfide exchange between nuclear-encoded and chloroplast-encoded subunits of pea acetyl-CoA carboxylase. 1154 65

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report, the synthesis of a bisubstrate analog inhibitor of carboxyltransferase is described. The inhibitor was synthesized by covalently linking biotin to coenzyme A via an acyl bridge between the sulfur of coenzyme A and the 1'-N of biotin. The steady-state kinetics of carboxyltransferase are characterized in the reverse direction, in which malonyl-CoA reacts with biocytin to form acetyl-CoA and carboxybiocytin. The inhibitor exhibited competitive inhibition versus malonyl-CoA and noncompetitive inhibition versus biocytin, with a slope inhibition constant (K(is)) of 23 +/- 2 microM. The bisubstrate analog has an affinity for carboxyltransferase 350 times higher than biotin. This suggests the inhibitor will be useful in structural studies, as well as aid in the search for chemotherapeutic agents that target acetyl-CoA carboxylase.
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PMID:A bisubstrate analog inhibitor of the carboxyltransferase component of acetyl-CoA carboxylase. 1188 46

The committed step for de novo fatty acid biosynthesis is the carboxylation of acetyl-CoA catalyzed by acetyl-CoA carboxylase (ACCase). Plastidial ACCase from most plants is a multisubunit complex composed of multiple copies of four different polypeptides, biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), and carboxyltransferase (alpha-CT and beta-CT). Immunoblot analyses revealed these four proteins were mostly (69% of total) associated with a 17,000 g insoluble fraction from lysed pea chloroplasts. Under the same conditions only 8% of ribulose-1,5-bisphosphate carboxylase was associated with this insoluble fraction. BCCP and biotin carboxylase BC subunits freely dissociated from 17 kg insoluble fractions under high ionic strength conditions, whereas alpha-CT and beta-CT subunits remained tightly associated. Both CT subunits were highly enriched in envelope versus stroma and thylakoid preparations whereas BC and BCCP subunits were predominantly stromal-localized due to partial dissociation. Rapid solubilization of intact chloroplasts with Triton X-100 followed by centrifugation at 30 kg resulted in a pellet that was up to 8-fold enriched in ACCase activity and 21-fold enriched in BC activity. Triton-insoluble 30 kg pellets were reduced in lipid and chlorophyll content but enriched in chloroplast DNA due to the isolation of nucleoid particles. However, ACCase was not directly associated with nucleoids since enzymatic digestion of DNA or RNA had no effect on the association with Triton-insoluble matter. The amount of Triton-insoluble ACCase was similar in chloroplasts isolated from dark- or light-adapted leaves suggesting transitory starch granules were also not involved in this association. It is proposed that ACCase is associated with envelope membranes through interactions with an unidentified integral membrane protein.
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PMID:The multisubunit acetyl-CoA carboxylase is strongly associated with the chloroplast envelope through non-ionic interactions to the carboxyltransferase subunits. 1205 35

The biotin carboxylase family is comprised of a group of enzymes that utilize a covalently bound prosthetic group, biotin, as a cofactor. These enzymes, which include acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, geranoyl-CoA carboxylase, oxaloacetate decarboxylase, methylmalonyl-CoA decarboxylase, transcarboxylase and urea amidolyase, are found in diverse biosynthetic pathways in both pro-karyotes and eukaryotes. The reactions catalyzed by most members of this group of enzymes share two common features: (1) carboxylation of biotin, apparently via the formation of a carboxyphosphate intermediate, followed by (2) transcarboxylation of CO(2) from biotin to specific acceptor molecules to yield different products. Structural determinations by NMR and X-ray crystallography, complemented by mutagenesis studies, have identified some motifs that are structurally or catalytically important. Analysis of the amino acid sequences of a number of biotin carboxylases not only shows remarkable similarities within certain domains but also that there appears to have been domain rearrangements between groups of carboxylases. Acyl-coenzyme A derivatives, which bind either as substrates or as allosteric regulators of the biotin carboxylases, do not appear to share any of the CoA binding motifs that have been identified in other CoA-SH/acyl-CoA binding proteins. Further comparisons of biotin-dependent carboxylases with other groups of enzymes in the protein data bank reveal that this family of biotin enzymes has strong similarities in specific domains to a number of ATP-utilizing enzymes and to the lipoyl-containing enzymes. These structural homologies are so extensive as to be highly suggestive of evolutionary relationships between biotin carboxylases and these other enzymes.
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PMID:The biotin enzyme family: conserved structural motifs and domain rearrangements. 1276 20


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