Purification and characterization of the Mycobacterium tuberculosis FabD 2, a novel malonyl-CoA:AcpM transacylase of fatty acid synthase

Abstract

Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in fatty acid and mycolic acid biosynthesis of Mycobacterium tuberculosis. fabd 2 is a novel gene coding MCAT in M. tuberculosis besides another known fabd. In our study, fabd 2 was inserted into a bacterial expression vector pET28a resulting in a 6× Histidine-tag fabd 2 fusion gene construction. The protein was purified by nickel affinity chromatography and the characterizations of FabD 2 have been investigated. The molecular weight of FabD 2 was estimated to be 26 kDa by MALDI-TOF. Consistent with the biosynthesis specialty of reported MCATs, FabD 2 resulted in a typical activity of bacterial MCATs, which catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP. Some physical and chemical differences between FabD 2 and FabD also have been found. FabD 2 shows dissimilarity with FabD in secondary structure in different pH buffer and MCAT genes RT-PCR results reveal different transcript condition with each other. Furthermore, FabD 2 shows low similarity in protein sequence when alignment with other MCATs.