title: Cloning, expression, and enzymatic activity of Acinetobacter baumannii and Klebsiella pneumoniae acetyl-coenzyme A carboxylases.
abstract: Pathogenic Gram-negative bacteria are a major public health concern because they are causative agents of life-threatening hospital-acquired infections. Due to the increasing rates of resistance to available antibiotics, there is an urgent need to develop new drugs. Acetyl-coenzyme A carboxylase (ACCase) is a promising target for the development of novel antibiotics. We describe here the expression, purification, and enzymatic activity of recombinant ACCases from two clinically relevant Gram-negative pathogens, Acinetobacter baumannii and Klebsiella pneumoniae. Recombinant ACCase subunits (AccAD, AccB, and AccC) were expressed and purified, and the holoenzymes were reconstituted. ACCase enzyme activity was monitored by direct detection of malonyl-coenzyme A (malonyl-CoA) formation by liquid chromatography tandem mass spectrometry (LC-MS/MS). Steady-state kinetics experiments showed similar k(cat) and K(M) values for both enzymes. In addition, similar IC(50) values were observed for inhibition of both enzymes by a previously reported ACCase inhibitor. To provide a higher throughput assay suitable for inhibitor screening, we developed and validated a luminescence-based ACCase assay that monitors ATP depletion. Finally, we established an enzyme activity assay for the isolated AccAD (carboxyltransferase) subunit, which is useful for determining whether novel ACCase inhibitors inhibit the biotin carboxylase or carboxyltransferase site of ACCase. The methods described here could be applied toward the identification and characterization of novel inhibitors.
authors.affiliation: Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA.
authors.forename: Juliano
authors.initials: J
authors.lastname: Alves
authors.forename: Lucas
authors.initials: L
authors.lastname: Westling
authors.forename: Eric C
authors.initials: EC
authors.lastname: Peters
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Harris
authors.forename: John W
authors.initials: JW
authors.lastname: Trauger
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000103:Acetyl-CoA Carboxylase / Q000235:genetics* / Q000378:metabolism*; D040981:Acinetobacter baumannii / Q000201:enzymology*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D055162:Biocatalysis; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D003001:Cloning, Molecular; D005470:Fluorometry; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008316:Malonyl Coenzyme A / Q000378:metabolism; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D008316:Malonyl Coenzyme A; D000105:Acetyl Coenzyme A; D000251:Adenosine Triphosphatases; D019731:Carbon-Nitrogen Ligases; C019791:biotin carboxylase; D000103:Acetyl-CoA Carboxylase