Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA : AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II

Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, inc luding Mycobacterium tuberculosis. MCAT catalyzes the transacylation of mal onate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, whic h is an elongation substrate in fatty acid biosynthesis. To clarify the rol es of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-COA: AcpM transacylase) from M. tuberculosis. Accordin g to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry. The mtfabD gene encoding a p utative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displayi ng strong MCAT activity in vitro. Enzymatic studies using different ACP sub strates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function rel ationship of mtFabD, different mutant proteins were generated. All mutation s (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT acti vity in vitro, thus underlining the importance of these residues in transac ylation. The generation and characterization of the AcpM forms and mtFabD o pens the way for further studies relating to fatty acid and mycolic acid bi osynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug ta rget waiting to be exploited.