Wen Lisa, Chmielowski Jennifer N, Bohn Kelsey C, Huang Jenq-Kuen, Timsina Yam N, Kodali Phanichand, Pathak Ashish K
Department of Chemistry, Western Illinois University, One University Circle, Macomb, IL 61455, USA.
Protein Expr Purif. 2009 May;65(1):83-91. doi: 10.1016/j.pep.2008.11.011. Epub 2008 Dec 6.
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.
土拉弗朗西斯菌是一种极具传染性的空气传播病原体，长期以来一直被视为潜在的生物武器。由于细菌和哺乳动物的生物合成途径存在差异，脂肪酸合成（FAS）途径的酶是开发新型抗菌剂的有吸引力的靶点。我们在此报告土拉弗朗西斯菌FAS-II途径中三种功能酶的首次表达：启动FAS-II延伸的FabH（3-氧代酰基-酰基载体蛋白合酶III）；催化丙二酰部分从丙二酰辅酶A转移到酰基载体蛋白（ACP）生成丙二酰-ACP的FabD（丙二酰辅酶A-酰基载体蛋白转酰基酶），以及催化烯酰-ACP衍生物还原的FabI（烯酰-ACP还原酶）。编码FabD、FabH和FabI的基因经定制合成并克隆到pET15b表达载体中。每种重组His标签融合蛋白通过异丙基-β-D-硫代半乳糖苷（IPTG）诱导进行过表达，然后通过镍-氮三乙酸（Ni-NTA）柱上的亲和层析进行纯化。纯化后的FabH和FabI已被用作新药开发的靶点。对一类吲哚-2-羧酸化合物的筛选导致发现了几种对土拉弗朗西斯菌FabH或FabI酶具有有前景活性的新化合物。例如，吲哚衍生物WIUAKP-001在40微摩尔时对FabH酶的抑制率为80%，半数抑制浓度（IC50）值为2微摩尔，而WIUAKP-031在37.5微摩尔时对FabI酶的抑制率为98%，IC50值为6微摩尔。这些化合物作为II型FAS酶抑制剂以及作为兔热病潜在新疗法的新型吲哚衍生物的未来开发具有很大潜力。
