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)

The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle, this protein engages in heterologous protein-protein interactions with three distinct partners, depending on its state of post-translational modification. Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the post-translational attachment of biotin to a single lysine residue on BCCP. Holo-BCCP then interacts with the biotin carboxylase subunit of acetyl-CoA carboxylase, which leads to the addition of the carboxylate group of bicarbonate to biotin. Finally, the carboxy-biotinylated form of BCCP interacts with transcarboxylase in the transfer of the carboxylate to acetyl-CoA to form malonyl-CoA. The determinants of protein-protein interaction specificity in this system are unknown. The NMR solution structure of the unbiotinylated form of an 87 residue C-terminal domain fragment (residue 70-156) of BCCP (holoBCCP87) and the crystal structure of the biotinylated form of a C-terminal fragment (residue 77-156) of BCCP from Escherichia coli acetyl-CoA carboxylase have previously been determined. Comparative analysis of these structures provided evidence for small, localized conformational changes in the biotin-binding region upon biotinylation of the protein. These structural changes may be important for regulating specific protein-protein interactions. Since the dynamic properties of proteins are correlated with local structural environments, we have determined the relaxation parameters of the backbone 15N nuclear spins of holoBCCP87, and compared these with the data obtained for the apo protein. The results indicate that upon biotinylation, the inherent mobility of the biotin-binding region and the protruding thumb, with which the biotin group interacts in the holo protein, are significantly reduced.
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PMID:Comparison of the backbone dynamics of the apo- and holo-carboxy-terminal domain of the biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase. 1004 24

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis 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 a system for site-directed mutagenesis of the biotin carboxylase component is described. The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from the mutant form of the enzyme which is coded on a plasmid. Separation of the two forms is accomplished using a histidine-tag attached to the amino terminus of the mutant form of the enzyme and nickel affinity chromatography. This system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to several different reactions catalyzed by biotin carboxylase. In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar results in all the different assays, suggesting that the mutated residues have a common function. The mutations did not affect the bicarbonate-dependent ATPase reaction. In contrast, the mutations decreased the maximal velocity of the biotin-dependent ATPase reaction 1000-fold but did not affect the Km for biotin. The activity of the ATP synthesis reaction catalyzed by biotin carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-fold by the mutations. The ATP synthesis reaction required biotin to stimulate the activity in the wild-type; however, biotin did not stimulate the activity of the mutant enzymes. The results showed that the mutations have abolished the ability of biotin to increase the activity of the enzyme. Thus, E211, E288, N290, and R292 were responsible, at least in part, for the substrate-induced synergism by biotin in biotin carboxylase.
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PMID:Mutations at four active site residues of biotin carboxylase abolish substrate-induced synergism by biotin. 1007 84

Plastidic acetyl-CoA carboxylase (ACCase; EC 6.4.1.2), which catalyses the synthesis of malonyl-CoA and is the regulatory enzyme of fatty acid synthesis, is activated by light, presumably under redox regulation. To obtain evidence of redox regulation in vivo, the activity of ACCase was examined in pea chloroplasts isolated from plants kept in darkness (dark-ACCase) or after exposure to light for 1 h (light-ACCase) in the presence or absence of a thiol-reducing agent, dithiothreitol (DTT). The protein level was similar for light-ACCase and dark-ACCase, but the activity of light-ACCase in the absence of DTT was approx. 3-fold that of dark-ACCase. The light-ACCase and dark-ACCase were activated approx. 2-fold and 6-fold by DTT respectively, indicating that light-ACCase was in a much more reduced, active form than the dark-ACCase. This is the first demonstration of the light-dependent reduction of ACCase in vivo. Measurement of the activities of ACCase, carboxyltransferase and biotin carboxylase in the presence and absence of DTT, and the thiol-oxidizing agent, 5, 5'-dithiobis-(2-nitrobenzoic) acid, revealed that the carboxyltransferase reaction, but not the biotin carboxylase reaction, was redox-regulated. The cysteine residue(s) responsible for redox regulation probably reside on the carboxyltransferase component. Measurement of the pH dependence of biotin carboxylase and carboxyltransferase activities in the ACCase suggested that both components affect the activity of ACCase in vivo at a physiological pH range. These results suggest that the activation of ACCase by light is caused partly by the pH-dependent activation of two components and by the reductive activation of carboxyltransferase.
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PMID:Light-dependent changes in redox status of the plastidic acetyl-CoA carboxylase and its regulatory component. 1021 91

We have examined whether chloroplast acetyl-CoA carboxylase is a phosphoprotein. Pea (Pisum sativum) chloroplasts were incubated in the presence of [gamma-33P]-ATP and radiolabeled proteins were examined after immunoprecipitation with antibodies against all four known subunits of heteromeric chloroplast acetyl-CoA carboxylase. The beta-subunit of the carboxyltransferase was found to be labeled by 33P. Phosphoamino acid analysis of the immunoprecipitated beta-subunit of the carboxyltransferase indicates that it is phosphorylated on serine residues. Incorporation of 33P into carboxyltransferase beta-subunit decreased in chloroplasts transferred to dark conditions after labeling in the light. Dephosphorylation of pea chloroplast extracts by an alkaline phosphatase-agarose conjugate reduced in vitro acetyl-CoA carboxylase activity by 67%. Furthermore, while acetyl-CoA carboxylase activity and its carboxyltransferase half-reaction were reduced in dephosphorylated extracts, the biotin carboxylase half-reaction was not inhibited. The evidence presented here points to the carboxyltransferase beta-subunit of chloroplast acetyl-CoA carboxylase as a candidate for regulation by protein phosphorylation/dephosphorylation.
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PMID:Phosphorylation of pea chloroplast acetyl-CoA carboxylase. 1041 2

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase activity, a biotin carboxyl carrier protein, and a carboxyltransferase activity. The C-terminal 87 amino acids of the biotin carboxyl carrier protein (BCCP87) form a domain that can be independently expressed, biotinylated, and purified (Chapman-Smith, A., Turner, D. L., Cronan, J. E., Morris, T. W., and Wallace, J. C. (1994) Biochem. J. 302, 881-887). The ability of the biotinylated form of this 87-residue protein (holoBCCP87) to act as a substrate for biotin carboxylase and carboxyltransferase was assessed and compared with the results with free biotin. In the case of biotin carboxylase holoBCCP87 was an excellent substrate with a K(m) of 0.16 +/- 0.05 mM and V(max) of 1000.8 +/- 182.0 min(-1). The V/K or catalytic efficiency of biotin carboxylase with holoBCCP87 as substrate was 8000-fold greater than with biotin as substrate. Stimulation of the ATP synthesis reaction of biotin carboxylase where carbamyl phosphate reacted with ADP by holoBCCP87 was 5-fold greater than with an equivalent amount of biotin. The interaction of holoBCCP87 with carboxyltransferase was characterized in the reverse direction where malonyl-CoA reacted with holoBCCP87 to form acetyl-CoA and carboxyholoBCCP87. The K(m) for holoBCCP87 was 0.45 +/- 0.07 mM while the V(max) was 2031.8 +/- 231.0 min(-1). The V/K or catalytic efficiency of carboxyltransferase with holoBCCP87 as substrate is 2000-fold greater than with biotin as substrate.
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PMID:The biotin domain peptide from the biotin carboxyl carrier protein of Escherichia coli acetyl-CoA carboxylase causes a marked increase in the catalytic efficiency of biotin carboxylase and carboxyltransferase relative to free biotin. 1054 97

Aryloxyphenoxypropionates, inhibitors of the plastid acetyl-CoA carboxylase (ACC) of grasses, also inhibit Toxoplasma gondii ACC. Clodinafop, the most effective of the herbicides tested, inhibits growth of T. gondii in human fibroblasts by 70% at 10 microM in 2 days and effectively eliminates the parasite in 2-4 days at 10-100 microM. Clodinafop is not toxic to the host cell even at much higher concentrations. Parasite growth inhibition by different herbicides is correlated with their ability to inhibit ACC enzyme activity, suggesting that ACC is a target for these agents. Fragments of genes encoding the biotin carboxylase domain of multidomain ACCs of T. gondii, Plasmodium falciparum, Plasmodium knowlesi, and Cryptosporidium parvum were sequenced. One T. gondii ACC (ACC1) amino acid sequence clusters with P. falciparum ACC, P. knowlesi ACC, and the putative Cyclotella cryptica chloroplast ACC. Another sequence (ACC2) clusters with that of C. parvum ACC, probably the cytosolic form.
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PMID:Growth of Toxoplasma gondii is inhibited by aryloxyphenoxypropionate herbicides targeting acetyl-CoA carboxylase. 1055 30

Acetyl-CoA carboxylase regulates the rate of fatty acid synthesis. This enzyme in plants is localized in plastids and is believed to be composed of biotin carboxyl carrier protein, biotin carboxylase, and carboxyltransferase made up of alpha and beta polypeptides, although the enzyme has not been purified yet. Accumulated evidence shows that pea plastidic acetyl-CoA carboxylase is activated by light and the activation is caused by light-dependent reduction of carboxyltransferase, but not of biotin carboxylase, via a redox cascade. To understand the reductive activation of carboxyltransferase at the molecular level here, we obtained the active enzyme composed of decahistidine-tagged (His tag) alpha and beta polypeptides through the expression of the pea plastidic carboxyltransferase gene in Escherichia coli. Gel filtration showed that the molecular size of the recombinant carboxyltransferase is in agreement with that of partially purified carboxyltransferase from pea chloroplasts. The catalytic activity of the recombinant enzyme was similar to that of native carboxyltransferase. These results indicate that the molecular structure and conformation of recombinant carboxyltransferase resemble those of its native counterpart and that native carboxyltransferase is indeed composed of alpha and beta polypeptides. This recombinant enzyme was activated by dithiothreitol, a known reductant of S-S bonds, with a profile similar to that of its native counterpart. The recombinant enzyme was activated by reduced thioredoxin-f, a signal transducer of redox potential in chloroplasts under irradiation. Thus, this enzyme was redox-regulated, like that of the native carboxyltransferase.
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PMID:Recombinant carboxyltransferase responsive to redox of pea plastidic acetyl-CoA carboxylase. 1074 68

Biotin carboxylase from Escherichia coli catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. For the carboxylation of biotin to occur, biotin must be deprotonated at its N1' position. Kinetic investigations, including solvent isotope effects and enzyme inactivation by N-ethylmaleimide, suggested a catalytic role for a cysteine residue and led to the proposal of a mechanism for the deprotonation of biotin. The proposed pathway suggests a catalytic base removes a proton from a nearby cysteine residue, forming a thiolate anion, which then abstracts the proton from biotin. Inactivation studies of pyruvate carboxylase, which has an analogous mode of action to biotin carboxylase, suggests the catalytic base in this reaction is a lysine residue. Using the crystal structure of biotin carboxylase, cysteine 230 and lysine 238 were identified as the likely active-site residues that act as this acid-base pair. To test the hypothesis that cysteine 230 and lysine 238 act as an acid-base pair to deprotonate biotin, site-directed mutagenesis was used to mutate cysteine 230 to alanine (C230A) and lysine 238 to glutamine (K238Q). Mutations at either residue resulted in a 50-fold increase in the K(m) for ATP. The C230A mutation had no effect on the formation of carboxybiotin, indicating that cysteine 230 does not play a role in the deprotonation of biotin. However, the K238Q mutation resulted in no formation of carboxybiotin, which showed that lysine 238 has a role in the carboxylation reaction. N-Ethylmaleimide was found to inactivate the C230A mutant but not the K238Q mutant, suggesting that N-ethylmaleimide is reacting with lysine 238 and not cysteine 230. The pH dependence of N-ethylmaleimide inactivation revealed that the pK value for lysine 238 was 9.4 or higher, suggesting lysine 238 is not a catalytic base. Thus, the results suggest that cysteine 230 and lysine 238 do not act as an acid-base pair in the deprotonation of biotin.
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PMID:Do cysteine 230 and lysine 238 of biotin carboxylase play a role in the activation of biotin? 1074 3

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In Escherichia coli, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. The biotin carboxylase component has served for many years as a paradigm for mechanistic studies devoted toward understanding more complicated biotin-dependent carboxylases. The three-dimensional x-ray structure of an unliganded form of E. coli biotin carboxylase was originally solved in 1994 to 2.4-A resolution. This study revealed the architecture of the enzyme and demonstrated that the protein belongs to the ATP-grasp superfamily. Here we describe the three-dimensional structure of the E. coli biotin carboxylase complexed with ATP and determined to 2.5-A resolution. The major conformational change that occurs upon nucleotide binding is a rotation of approximately 45(o) of one domain relative to the other domains thereby closing off the active site pocket. Key residues involved in binding the nucleotide to the protein include Lys-116, His-236, and Glu-201. The backbone amide groups of Gly-165 and Gly-166 participate in hydrogen bonding interactions with the phosphoryl oxygens of the nucleotide. A comparison of this closed form of biotin carboxylase with carbamoyl-phosphate synthetase is presented.
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PMID:Movement of the biotin carboxylase B-domain as a result of ATP binding. 1082 65

Strain BR54 of Clostridium beijerinckii was derived from the wild type strain, NCIMB 8052, by mutagenesis with Tn1545 and selection for butanol tolerance. It harbours a single copy of Tn 1545 in a 435 bp intergenic region separating two convergently transcribed genes, accC and gldA. The former encodes biotin carboxylase (E.C.6.3.4.14), a subunit of acetyl-CoA carboxylase and the latter encodes glycerol dehydrogenase (E.C.1.1.1.6). Since Tn1545 generates outwardly directed transcripts from its right end, we considered the possibility that the transposon inserted in strain BR54 might affect the expression of the adjacent gldA gene. RT-PCR experiments revealed that the mutant, but not the wild type, contains antisense RNA corresponding to the gldA gene. Correlated with this, the level of glycerol dehydrogenase activity in the mutant was only 25% of that in the wild type when bacteria were grown with either glucose or glycerol as the fermentable substrate. We conclude that transcripts emerging from the right end of the conjugative transposon, Tn1545, can reduce the expression of the adjacent gldA gene by the generation of antisense RNA and that this is associated with a butanol-tolerant phenotype.
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PMID:Butanol tolerance of Clostridium beijerinckii NCIMB 8052 associated with down-regulation of gldA by antisense RNA. 1093 92


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