Expression and characterization of active francisella tularensis FabI enzyme, a potential antibacterial target

Abstract

Francisella tularensis (F. tularensis) has been identified by the United States Centers for Disease Control (CDC) as one of the top six bacteria that could potentially be used as biological weapons. This research focuses on an enzyme of fatty acid biosynthesis in F. tularensis, namely FabI. FabI is an enoyl‐ACP reductase which catalyzes the last reduction step in each elongation cycle of type‐II fatty acid synthesis. The enzyme has been identified as a potential target for antibacterial drug development. In this research, the gene encoding of FabI was custom synthesized and cloned in pET15b expression vector. The enzyme was over‐expressed in E. coli, purified using Ni‐NTA affinity column, and its activity assayed spectrophotometrically. The pure FabI was tested with a set of indole‐2‐carboxylic acid compounds for their ability to inhibit FabI. We have discovered several new compounds with promising activity against F. tularensis FabI. The best result was obtained with indole‐2‐carboxylic acid analog WIUAKP‐031. The compound at 37.5 μM inhibited 98% of the FabI enzyme with IC50 value of 6 μM. The availability of the functional F. tularensis FabI will greatly aid in structural studies of this essential bacterial enzyme and facilitate the identification of small molecule inhibitors of type II fatty acid synthase.Supported in parts by seed grants from University Research Council, Western Illinois University.