Development of a scintillation proximity assay for beta-ketoacyl-acyl carrier protein synthase III

Assays of beta-ketoacyl-acyl carrier protein synthases III (KASIII; FabH), a key enzyme initiating bacterial type II fatty acid biosynthesis, usually involve incubation of radiolabeled acetyl-coenzyme A and malonylacyl carrie r protein (MACP). The radiolabeled acetoacetyl-ACP product is precipitated and separated from the substrate before quantitation. We have developed a s cintillation proximity assay (SPA) where use of biotinylated MACP (BMACP) a llows the generation of a biotinylated acetoacetyl-ACP, This product, when captured by the streptavidin-coated scintillant-impregnated microspheres, g enerates an SPA signal. A BMACP K-m of 7.1 mu M was determined using this S PA with the Streptomyces glaucescens FabH. A similar MACP K-m (6 mu M) was determined in a precipitation assay, demonstrating that BMACP is an effecti ve substrate for FabH. IC50 values of 15.2 mu M (SPA) and 24.8 mu M were ob tained with iodoacetamide and the S. glaucescens FabH, Comparable IC50 valu es of 160 mu M (SPA) and 125 mu M were also obtained with the antibiotic th iolactomycin and the Escherichia coli FabH. These observations demonstrate that FabH inhibitors can be readily detected using a SPA with BMACP and tha t the effectiveness of inhibitors in the SPA is comparable to that obtained using MACP and a standard TCA precipitation assay. A FabH SPA adaptable to high-throughput screening should facilitate the discovery of potential nov el antibiotics. (C) 2000 Academic Press.