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Probing the mechanism of the Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthase III mtFabH: Factors influencing catalysis and substrate specificity

Lookup NU author(s): Dr Alistair Brown

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Abstract

Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These α-alkyl, β-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C 24-C26 fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The β-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg46 revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg161 → Ala substitution. Our structural studies suggested that His258, previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys 122. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.


Publication metadata

Author(s): Brown AK, Sridharan S, Kremer L, Lindenberg S, Dover LG, Sacchettini JC, Besra GS

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 2005

Volume: 280

Issue: 37

Pages: 32539-32547

Print publication date: 16/09/2005

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

Publisher: American Society for Biochemistry and Molecular Biology

DOI: 10.1074/jbc.M413216200

PubMed id: 16040614


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