Abstract
Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis. MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis. To clarify the roles 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. According 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 putative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro. Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated. All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation. The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.
Highlights
§ These authors contributed to this work within the hostile environment of host macrophages that produce microbicidal molecules, usually sufficient to kill other bacteria
In contrast to fatty acid synthase (FAS)-I, FAS-II consists of dissociable enzyme components, which act upon a substrate bound to an acyl carrier protein (ACP), recently designated as AcpM in M. tuberculosis [12]
It has been previously shown that FabD is a critical enzyme involved in fatty acid biosynthesis in all bacteria, by catalyzing the transacylation reaction of malonate from malonyl-coenzyme A (CoA) to holo-ACP; malonyl-ACP is a key substrate for elongation of fatty acids
Summary
We have recently shown that mtFabH, a -ketoacyl-ACP synthase, forms a pivotal link between the type I and type II FAS elongation systems in M. tuberculosis. Analysis of the M. tuberculosis genome [15] revealed that acpM (Rv2244) is genetically linked to Rv2243 whose product is homologous to FabD proteins from various microorganisms (16 –18) Both acpM and mtfabD belong to the same transcriptional unit that includes kasA and kasB. The relative abundance of each form could be modulated by the growth conditions, a useful feature in generating valuable substrates (holo-AcpM and palmitoylated-AcpM) for future studies in fatty acid and mycolic acid biosynthesis in M. tuberculosis.. We provide evidence that the product encoded by mtfabD catalyzes MCAT activity both in vivo and in vitro using E. coli holo-ACP and M. tuberculosis holo-AcpM, respectively, as substrates for transacylation The relative abundance of each form could be modulated by the growth conditions, a useful feature in generating valuable substrates (holo-AcpM and palmitoylated-AcpM) for future studies in fatty acid and mycolic acid biosynthesis in M. tuberculosis. we provide evidence that the product encoded by mtfabD catalyzes MCAT activity both in vivo and in vitro using E. coli holo-ACP and M. tuberculosis holo-AcpM, respectively, as substrates for transacylation
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