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J. Biol. Chem., Vol. 278, Issue 3, 1735-1743, January 17, 2003
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From the Establishment or maintenance of a
persistent infection by Mycobacterium tuberculosis requires
the glyoxylate pathway. This is a bypass of the tricarboxylic
acid cycle in which isocitrate lyase and malate synthase (GlcB)
catalyze the net incorporation of carbon during growth of
microorganisms on acetate or fatty acids as the primary carbon source.
The glcB gene from M. tuberculosis, which
encodes malate synthase, was cloned, and GlcB was expressed in
Escherichia coli. The influence of media conditions
on expression in M. tuberculosis indicated that this enzyme
is regulated differentially to isocitrate lyase. Purified GlcB had
Km values of 57 and 30 µM for its
substrates glyoxylate and acetyl coenzyme A, respectively, and was
inhibited by bromopyruvate, oxalate, and phosphoenolpyruvate. The GlcB
structure was solved to 2.1-Å resolution in the presence of glyoxylate
and magnesium. We also report the structure of GlcB in complex with the
products of the reaction, coenzyme A and malate, solved to 2.7-Å
resolution. Coenzyme A binds in a bent conformation, and the details of
its interactions are described, together with implications on the
enzyme mechanism.
The atomic coordinates and structure factors (codes 1N8I and
1N8W) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New
Brunswick, NJ
(http://www.rcsb.org/).
Biochemical and Structural Studies of Malate Synthase from
Mycobacterium tuberculosis*
,§,
**,, and§§
Department of Biochemistry and Biophysics,
Texas A & M University, College Station, Texas 77843-2128, Department of Microbiology and Immunology, College of Veterinary
Medicine, Cornell University, Ithaca, New York 14853, and
¶ Department of Medical Microbiology, University of Szeged,
Szeged POB 8-6701, Hungary
*
This work was supported by the Robert A. Welch Foundation,
National Institutes of Health Grant AI46392, and GlaxoSmithKline.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.
To whom correspondence may be addressed. Tel.: 979-862-7636;
Fax: 979-862-7638; E-mail: sacchett@tamu.edu.
§§
Present address: Tulane University School of Medicine, 1430 Tulane Ave., SL38, New Orleans, LA 70112.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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