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• Characterization of a bifunctional archaeal acyl coenzyme A carboxylase.

  Chuakrut S., Arai H., Ishii M., Igarashi Y.

  Acyl coenzyme A carboxylase (acyl-CoA carboxylase) was purified from Acidianus brierleyi. The purified enzyme showed a unique subunit structure (three subunits with apparent molecular masses of 62, 59, and 20 kDa) and a molecular mass of approximately 540 kDa, indicating an alpha(4)beta(4)gamma(4) ... >> More

  Acyl coenzyme A carboxylase (acyl-CoA carboxylase) was purified from Acidianus brierleyi. The purified enzyme showed a unique subunit structure (three subunits with apparent molecular masses of 62, 59, and 20 kDa) and a molecular mass of approximately 540 kDa, indicating an alpha(4)beta(4)gamma(4) subunit structure. The optimum temperature for the enzyme was 60 to 70 degrees C, and the optimum pH was around 6.4 to 6.9. Interestingly, the purified enzyme also had propionyl-CoA carboxylase activity. The apparent K(m) for acetyl-CoA was 0.17 +/-0.03 mM, with a V(max) of 43.3 +/-2.8 U mg(-1), and the K(m) for propionyl-CoA was 0.10 +/-0.008 mM, with a V(max) of 40.8 +/-1.0 U mg(-1). This result showed that A. brierleyi acyl-CoA carboxylase is a bifunctional enzyme in the modified 3-hydroxypropionate cycle. Both enzymatic activities were inhibited by malonyl-CoA, methymalonyl-CoA, succinyl-CoA, or CoA but not by palmitoyl-CoA. The gene encoding acyl-CoA carboxylase was cloned and characterized. Homology searches of the deduced amino acid sequences of the 62-, 59-, and 20-kDa subunits indicated the presence of functional domains for carboxyltransferase, biotin carboxylase, and biotin carboxyl carrier protein, respectively. Amino acid sequence alignment of acetyl-CoA carboxylases revealed that archaeal acyl-CoA carboxylases are closer to those of Bacteria than to those of Eucarya. The substrate-binding motifs of the enzymes are highly conserved among the three domains. The ATP-binding residues were found in the biotin carboxylase subunit, whereas the conserved biotin-binding site was located on the biotin carboxyl carrier protein. The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit. << Less

  J Bacteriol 185:938-947(2003) [PubMed] [EuropePMC]

• Identification and characterization of the first class of potent bacterial acetyl-CoA carboxylase inhibitors with antibacterial activity.

  Freiberg C., Brunner N.A., Schiffer G., Lampe T., Pohlmann J., Brands M., Raabe M., Haebich D., Ziegelbauer K.

  The multisubunit acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid biosynthesis, is broadly conserved among bacteria. Its rate-limiting role in formation of fatty acids makes this enzyme an attractive target for the design of novel broad-spectrum antibacterials. Howeve ... >> More

  The multisubunit acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid biosynthesis, is broadly conserved among bacteria. Its rate-limiting role in formation of fatty acids makes this enzyme an attractive target for the design of novel broad-spectrum antibacterials. However, no potent inhibitors have been discovered so far. This report describes the identification and characterization of highly potent bacterial acetyl-CoA carboxylase inhibitors with antibacterial activity for the first time. We demonstrate that pseudopeptide pyrrolidine dione antibiotics such as moiramide B inhibit the Escherichia coli enzyme at nanomolar concentrations. Moiramide B targets the carboxyltransferase reaction of this enzyme with a competitive inhibition pattern versus malonyl-CoA (K(i) value = 5 nm). Inhibition at nanomolar concentrations of the pyrrolidine diones is also demonstrated using recombinantly expressed carboxyltransferases from other bacterial species (Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa). We isolated pyrrolidine dione-resistant strains of E. coli, S. aureus, and Bacillus subtilis, which contain mutations within the carboxyltransferase subunits AccA or AccD. We demonstrate that such mutations confer resistance to pyrrolidine diones. Inhibition values (IC(50)) of >100 microm regarding an eukaryotic acetyl-CoA carboxylase from rat liver indicate high selectivity of pyrrolidine diones for the bacterial multisubunit enzyme. The natural product moiramide B and synthetic analogues show broad-spectrum antibacterial activity. The knowledge of the target and the availability of facile assays using carboxyltransferases from different pathogens will enable evaluation of the antibacterial potential of the pyrrolidine diones as a promising antibacterial compound class acting via a novel mode of action. << Less

  J. Biol. Chem. 279:26066-26073(2004) [PubMed] [EuropePMC]

• The structure of the carboxyltransferase component of acetyl-coA carboxylase reveals a zinc-binding motif unique to the bacterial enzyme.

  Bilder P., Lightle S., Bainbridge G., Ohren J., Finzel B., Sun F., Holley S., Al-Kassim L., Spessard C., Melnick M., Newcomer M., Waldrop G.L.

  Acetyl-coA carboxylase (ACC) is a central metabolic enzyme that catalyzes the committed step in fatty acid biosynthesis: biotin-dependent conversion of acetyl-coA to malonyl-coA. The bacterial carboxyltransferase (CT) subunit of ACC is a target for the design of novel therapeutics that combat seve ... >> More

  Acetyl-coA carboxylase (ACC) is a central metabolic enzyme that catalyzes the committed step in fatty acid biosynthesis: biotin-dependent conversion of acetyl-coA to malonyl-coA. The bacterial carboxyltransferase (CT) subunit of ACC is a target for the design of novel therapeutics that combat severe, hospital-acquired infections resistant to the established classes of frontline antimicrobials. Here, we present the structures of the bacterial CT subunits from two prevalent nosocomial pathogens, Staphylococcus aureus and Escherichia coli, at a resolution of 2.0 and 3.0 A, respectively. Both structures reveal a small, independent zinc-binding domain that lacks a complement in the primary sequence or structure of the eukaryotic homologue. << Less

  Biochemistry 45:1712-1722(2006) [PubMed] [EuropePMC]

• A tale of two functions: enzymatic activity and translational repression by carboxyltransferase.

  Meades G. Jr., Benson B.K., Grove A., Waldrop G.L.

  Acetyl-CoA Carboxylase catalyzes the first committed step in fatty acid synthesis. Escherichia coli acetyl-CoA carboxylase is composed of biotin carboxylase, carboxyltransferase and biotin carboxyl carrier protein functions. The accA and accD genes that code for the alpha- and beta-subunits, respe ... >> More

  Acetyl-CoA Carboxylase catalyzes the first committed step in fatty acid synthesis. Escherichia coli acetyl-CoA carboxylase is composed of biotin carboxylase, carboxyltransferase and biotin carboxyl carrier protein functions. The accA and accD genes that code for the alpha- and beta-subunits, respectively, are not in an operon, yet yield an alpha(2)beta(2) carboxyltransferase. Here, we report that carboxyltransferase regulates its own translation by binding the mRNA encoding its subunits. This interaction is mediated by a zinc finger on the beta-subunit; mutation of the four cysteines to alanine diminished nucleic acid binding and catalytic activity. Carboxyltransferase binds the coding regions of both subunit mRNAs and inhibits translation, an inhibition that is relieved by the substrate acetyl-CoA. mRNA binding reciprocally inhibits catalytic activity. Preferential binding of carboxyltransferase to RNA in situ was shown using fluorescence resonance energy transfer. We propose an unusual regulatory mechanism by which carboxyltransferase acts as a 'dimmer switch' to regulate protein production and catalytic activity, while sensing the metabolic state of the cell through acetyl-CoA concentration. << Less

  Nucleic Acids Res. 38:1217-1227(2010) [PubMed] [EuropePMC]