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J. Biol. Chem., Vol. 276, Issue 30, 27967-27974, July 27, 2001
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From 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.
Biochemical Characterization of Acyl Carrier Protein (AcpM) and
Malonyl-CoA:AcpM Transacylase (mtFabD), Two Major Components of
Mycobacterium tuberculosis Fatty Acid Synthase
II*
§,,
,,,, and INSERM U447, Institut Pasteur de Lille, 1 rue du
Pr. Calmette, BP245-59019 Lille Cedex, France, the ¶ Department
of Microbiology & Immunology, University of Newcastle, Newcastle upon
Tyne NE2 4HH, United Kingdom, GlaxoSmithKine Research and
Development, Stevenage SG1 2NY, United Kingdom, the ** Department of
Microbiology, Colorado State University, Fort Collins, Colorado
80523-1677, and the Department of
Chemistry, University of Newcastle,
Newcastle upon Tyne NE1 7RU, United Kingdom
*
This work was supported in part by Grants 49343 and 49338 from the Medical Research Council; by Grant AI-38087 from the National Cooperative Drug Discovery Groups for the Treatment of Opportunistic Infections, NIAID, National Institutes of Health; and by INSERM, Institut Pasteur de Lille, Région Nord Pas-de-Calais, and
Ministere de la Recherche.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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