HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 37, 32539-32547, September 16, 2005

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 280/37/32539    most recent M413216200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brown, A. K. Articles by Besra, G. S. Search for Related Content PubMed PubMed Citation Articles by Brown, A. K. Articles by Besra, G. S. Social Bookmarking                      What's this?

Probing the Mechanism of the Mycobacterium tuberculosis -Ketoacyl-Acyl Carrier Protein Synthase III mtFabH

FACTORS INFLUENCING CATALYSIS AND SUBSTRATE SPECIFICITY^*

Alistair K. Brown1, Sudharsan Sridharan¶1, Laurent Kremer||, Sandra Lindenberg, Lynn G. Dover, James C. Sacchettini¶, and Gurdyal S. Besra, A Lister-Jenner Research Fellow2

From the School of Cellular and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United Kingdom, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom, the ^¶Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, and ^||Laboratoire de Dynamique Moléculaire des Interactions Membranaires, CNRS-UMR 5539, Université de Montpellier II, Case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France

Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These -alkyl, -hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C₂₄-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 -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 Cys¹²².

Received for publication, November 23, 2004 , and in revised form, July 21, 2005.

The atomic coordinates and structure factors (codes 1M1M, 2AHB, and 2AJ9) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by The Medical Research Council, The Wellcome Trust, and the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 44-121-415-8125; Fax: 44-121-414-5925; E-mail: g.besra{at}bham.ac.uk.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				V. Bhowruth, A. K. Brown, and G. S. Besra Synthesis and biological evaluation of NAS-21 and NAS-91 analogues as potential inhibitors of the mycobacterial FAS-II dehydratase enzyme Rv0636 Microbiology, July 1, 2008; 154(7): 1866 - 1875. [Abstract] [Full Text] [PDF]

				A. K. Brown, A. Bhatt, A. Singh, E. Saparia, A. F. Evans, and G. S. Besra Identification of the dehydratase component of the mycobacterial mycolic acid-synthesizing fatty acid synthase-II complex Microbiology, December 1, 2007; 153(12): 4166 - 4173. [Abstract] [Full Text] [PDF]

				A. K. Brown, A. Papaemmanouil, V. Bhowruth, A. Bhatt, L. G. Dover, and G. S. Besra Flavonoid inhibitors as novel antimycobacterial agents targeting Rv0636, a putative dehydratase enzyme involved in Mycobacterium tuberculosis fatty acid synthase II Microbiology, October 1, 2007; 153(10): 3314 - 3322. [Abstract] [Full Text] [PDF]

				V. Molle, A. K. Brown, G. S. Besra, A. J. Cozzone, and L. Kremer The Condensing Activities of the Mycobacterium tuberculosis Type II Fatty Acid Synthase Are Differentially Regulated by Phosphorylation J. Biol. Chem., October 6, 2006; 281(40): 30094 - 30103. [Abstract] [Full Text] [PDF]

				Y.-M. Zhang, J. Hurlbert, S. W. White, and C. O. Rock Roles of the Active Site Water, Histidine 303, and Phenylalanine 396 in the Catalytic Mechanism of the Elongation Condensing Enzyme of Streptococcus pneumoniae J. Biol. Chem., June 23, 2006; 281(25): 17390 - 17399. [Abstract] [Full Text] [PDF]