Development of a Scintillation Proximity Assay for β-Ketoacyl-acyl Carrier Protein Synthase III

✍ Scribed by Xin He; John P. Mueller; Kevin A. Reynolds

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 170 KB
• Volume: 282
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4594

✦ Synopsis

Assays of ␤-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 carrier protein (MACP). The radiolabeled acetoacetyl-ACP product is precipitated and separated from the substrate before quantitation. We have developed a scintillation proximity assay (SPA) where use of biotinylated MACP (BMACP) allows the generation of a biotinylated acetoacetyl-ACP. This product, when captured by the streptavidin-coated scintillant-impregnated microspheres, generates an SPA signal. A BMACP K m of 7.1 M was determined using this SPA with the Streptomyces glaucescens FabH. A similar MACP K m (6 M) was determined in a precipitation assay, demonstrating that BMACP is an effective substrate for FabH. IC 50 values of 15.2 M (SPA) and 24.8 M were obtained with iodoacetamide and the S. glaucescens FabH. Comparable IC 50 values of 160 M (SPA) and 125 M were also obtained with the antibiotic thiolactomycin and the Escherichia coli FabH. These observations demonstrate that FabH inhibitors can be readily detected using a SPA with BMACP and that 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 novel antibiotics.