Western Illinois University, Chemistry, Macomb, IL 61455
Francisella tularensis is an extremely infectious airborne pathogen, and has long been considered as a potential biological weapon. The enzymes responsible for bacterial fatty acid synthesis (FAS) pathway are attractive targets for the development of new antibacterial agents because of their biosynthesis pathway differences between bacteria and mammals. The intent of this research is to study a key regulatory enzyme of type II FAS of Francisella tularensis, namely FabH, as a novel drug target against tularemia. FabH, also known as (3-oxoacyl-acyl carrier protein synthase III), catalyzes the initial condensation and elongation reactions in fatty acid synthesis. The condensation reaction requires malonyl-acyl carrier protein (ACP) as an elongator unit of the growing acyl chain. Synthesis of malonyl-ACP by transacylation of ACP with malonate is catalyzed by FabD.
Genes encoding the Francisella tularensis of FabD and FabH were custom synthesized and inserted in pET15b expression vector by Celtek Genes. Each recombinant His-tagged fusion protein was overexpressed by IPTG induction. The success of induction was demonstrated by SDS-polyacrylamide gel electrophoresis. Both the recombinant FabD and FabH proteins were purified by affinity chromatography on a Ni-NTA column. FabH activity was measured by monitoring the conversion of [14C]-acetyl-CoA and malonyl-ACP substrates (produced by FabD in the coupled assay) to a radiolabeled acetoacetyl-ACP. The active enzyme has been successfully used in screening a class of 46 newly synthesized indole-2-carboxylic acid derivatives for its inhibition. Three compounds have shown IC50 values in the range of 2–6 µM against Francisella tularensis FabH.
This project is supported in parts by University Research Council, Western Illinois University.
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