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Biochemical Characterization of Acyl Carrier Protein (AcpM) and Malonyl-CoA:AcpM Transacylase (mtFabD), Two Major Components of Mycobacterium tuberculosis Fatty Acid Synthase II

Lookup NU author(s): Dr Laurent Kremer, Dr Madhavan Nampoothiri, Dr Lynn Dover, Professor David Minnikin, Professor Del Besra

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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.


Publication metadata

Author(s): Kremer L, Nampoothiri KM, Lesjean S, Dover LG, Graham S, Betts J, Brennan PJ, Minnikin DE, Locht C, Besra GS

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 2001

Volume: 276

Issue: 30

Pages: 27967-27974

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

Publisher: American Society for Biochemistry and Molecular Biology, Inc

URL: http://dx.doi.org/10.1074/jbc.M103687200

DOI: 10.1074/jbc.M103687200

PubMed id: 11373295


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