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 malonyl-acyl 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 microM was determined using this SPA with the Streptomyces glaucescens FabH. A similar MACP K(m) (6 microM) was determined in a precipitation assay, demonstrating that BMACP is an effective substrate for FabH. IC(50) values of 15.2 microM (SPA) and 24.8 microM were obtained with iodoacetamide and the S. glaucescens FabH. Comparable IC(50) values of 160 microM (SPA) and 125 microM 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.
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Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia, 23219, USA.
Mueller
J P
JP
Reynolds
K A
KA
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000032:analysis* / Q000737:chemistry; D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000213:Acyl Carrier Protein / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019921:Biotinylation; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007460:Iodoacetamide / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis*; D007700:Kinetics; D008863:Microspheres; D008954:Models, Biological; D011487:Protein Conformation; D012588:Scintillation Counting; D013302:Streptomyces / Q000737:chemistry; D013876:Thiophenes / Q000494:pharmacology; D013997:Time Factors
D000213:Acyl Carrier Protein; D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D013876:Thiophenes; D000105:Acetyl Coenzyme A; C035068:thiolactomycin; C078182:beta-ketoacyl-acyl carrier protein synthase I; C077402:3-ketoacyl-acyl carrier protein synthase III; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D007460:Iodoacetamide