Escherichia coli acetyl-coenzyme A carboxylase: characterization and development of a high-throughput assay.
Bacterial acetyl-coenzyme A carboxylase (ACCase) is a multicomponent system composed of AccA, AccD, AccC, and AccB (also known as BCCP), which is required for fatty acid biosynthesis. It is essential for cell growth and has been chemically validated as a target for antimicrobial drug discovery. To identify ACCase inhibitors, a simple and robust assay that monitors the overall activity by measuring phosphate production at physiologically relevant concentrations of all protein components was developed. Inorganic phosphate production was demonstrated to directly reflect the coupled activities of AccC and AccA/D with BCCP cycling between the two half-reactions. The K(m) apparent values for ATP, acetyl-coenzyme A, and BCCP were estimated to be 60+/-14 microM, 18+/-4 microM, and 39+/-9 nM, respectively. The stoichiometry between the two half-reactions was measured to be 1:1. Carboxy-biotin produced in the first half-reaction was stable over the time course of the assay. The assay was adapted to a high-throughput screen (HTS) 384-well format using a modified published scintillation proximity method. The optimized HTS assay has acceptable Z' factor values and was validated to report inhibitions of either AccC or AccA/D. The assay is not susceptible to signal quenching due to colored compounds.
Soriano
Aileen
A
Schering-Plough Research Institute, Kenilworth, NJ 07033, USA. aileen.soriano@spcorp.com
Radice
Anthony D
AD
Herbitter
Amy H
AH
Langsdorf
Erik F
EF
Stafford
Jill M
JM
Chan
Sarah
S
Wang
Shihong
S
Liu
Yan-Hui
YH
Black
Todd A
TA
D000103:Acetyl-CoA Carboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology*; D010710:Phosphates / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D010710:Phosphates; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000103:Acetyl-CoA Carboxylase