Functional expression of Francisella tularensis FabH and FabI, potential antibacterial targets.
Francisella tularensis is an extremely infectious airborne pathogen that has long been considered as a potential biological weapon. Enzymes of fatty acid synthesis (FAS) pathway are attractive targets for the development of new antibacterial agents because of differences between the biosynthesis pathways of bacteria and mammals. We report here the first expression of three functional enzymes in F. tularensis FAS-II pathway: FabH (3-oxoacyl-acyl carrier protein synthase III) which initiates elongation in FAS-II; FabD (Malonyl-CoA-acyl carrier protein transacylase) which catalyzes the transfer of a malonyl moiety from malonyl-CoA to ACP generating malonyl-ACP, and FabI (enoyl-ACP reductase) which catalyzes the reduction of enoyl-acyl-ACP derivatives. The genes encoding the FabD, FabH, and FabI were custom synthesized and cloned in pET15b expression vector. Each recombinant His-tagged fusion protein was overexpressed by IPTG induction, and then purified by affinity chromatography on a Ni-NTA column. The purified FabH and FabI have been used as targets for new drug development. Screening of a class of indole-2-carboxylic acid compounds has led to the discovery of several new compounds with promising activity against F. tularensis FabH or FabI enzymes. For example, indole derivative WIUAKP-001 inhibited 80% the FabH enzyme at 40 microM with IC(50) value of 2 microM whereas WIUAKP-031 inhibited 98% the FabI enzyme at 37.5 microM with IC(50) value of 6 microM. These compounds hold great promise for future development of new indole derivatives as inhibitors of type II FAS enzymes, and as potential new treatment for tularemia.
Wen
Lisa
L
Department of Chemistry, Western Illinois University, One University Circle, Macomb, IL 61455, USA. L-Wen@wiu.edu
Chmielowski
Jennifer N
JN
Bohn
Kelsey C
KC
Huang
Jenq-Kuen
JK
Timsina
Yam N
YN
Kodali
Phanichand
P
Pathak
Ashish K
AK
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D054045:Biological Warfare Agents; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005604:Francisella tularensis / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D054045:Biological Warfare Agents; D011994:Recombinant Proteins; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)