A high-throughput screening assay for the carboxyltransferase subunit of acetyl-CoA carboxylase.
One consequence of the dramatic rise of antibiotic-resistant pathogenic bacteria is the need for new targets for antibiotics. Because membrane lipid biogenesis is essential for bacterial growth, enzymes of the fatty acid biosynthetic pathway offer attractive possibilities for the development of new antibiotics. Acetyl-coenzyme A carboxylase (ACC) catalyzes the first committed and regulated step in fatty acid biosynthesis in bacteria and thus is a prime target for development of antibiotics. ACC is a multifunctional enzyme composed of three separate proteins. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin. The biotin carboxyl carrier protein features a biotin molecule covalently attached at Lys122 of the Escherichia coli enzyme. The carboxyltransferase subunit catalyzes the transfer of a carboxyl group from biotin to acetyl-coenzyme A (acetyl-CoA) to form malonyl-CoA. The objective of this study was to develop an assay for high-throughput screening for inhibitors of the carboxyltransferase subunit. The carboxyltransferase reaction was assayed in the reverse direction in which malonyl-CoA reacts with biocytin (an analog of the biotin carboxyl carrier protein) to form acetyl-CoA and carboxybiotin. The production of acetyl-CoA was coupled to citrate synthase, which produced citrate and coenzyme A. The amount of coenzyme A formed was detected using 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). The assay has been developed for use in both 96- and 384-well microplate formats and was validated using a known bisubstrate analog inhibitor of carboxyltransferase. The spectrophotometric readout in the visible absorbance range used in this assay does not generate the number of false negatives associated with frequently used NAD/NADH assay systems that rely on detection of NADH using UV absorbance.
Santoro
Nicholas
N
Antibacterial Pharmacology, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA.
Brtva
Teresa
T
Roest
Steve Vander
SV
Siegel
Karen
K
Waldrop
Grover L
GL
D000103:Acetyl-CoA Carboxylase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000166:Acridines / Q000378:metabolism / Q000494:pharmacology; D001952:Bridged-Ring Compounds / Q000378:metabolism / Q000494:pharmacology; D019878:Carboxyl and Carbamoyl Transferases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D021122:Protein Subunits / Q000032:analysis* / Q000737:chemistry; D013053:Spectrophotometry / Q000379:methods*; D013997:Time Factors
D000166:Acridines; C451420:BisA compound; D001952:Bridged-Ring Compounds; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D019878:Carboxyl and Carbamoyl Transferases; D000103:Acetyl-CoA Carboxylase