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 CAT in M. tuberculosis besides another known fabd. In our study, fabd(2) was inserted into a bacterial expression vector pET28a resulting in a 6x Histidine-tagfabd(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. (c) 2005 Elsevier Inc. All rights reserved.