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J. Biol. Chem., Vol. 278, Issue 47, 47166-47170, November 21, 2003

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The Crystal Structure of 1-D-myo-Inosityl 2-Acetamido-2-deoxy--D-glucopyranoside Deacetylase (MshB) from Mycobacterium tuberculosis Reveals a Zinc Hydrolase with a Lactate Dehydrogenase Fold^*

Jason T. Maynes, Craig Garen, Maia M. Cherney, Gerald Newton¶, Dorit Arad||, Yossef Av-Gay||**, Robert C. Fahey¶, and Michael N. G. James, Canada Research Chair in Protein Structure and Function.

From the Canadian Institutes of Health Research, Group in Protein Structure and Function, Department of Biochemistry, Faculty of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada, the ^¶Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, and the ^||Department of Medicine, University of British Columbia, Vancouver, British Columbia V5Z 2J5, Canada

Mycothiol (1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy--D-glucopyranoside, MSH or AcCys-GlcN-inositol (Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc-Ins to yield GlcN-Ins. The deacetylase (MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn²⁺ metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x-ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N-terminal domain and a C-terminal domain consisting of two -sheets and two -helices. The zinc binding site is in the N-terminal domain occupying a position equivalent to that of the NAD⁺ co-factor of lactate dehydrogenase. The Zn²⁺ is 5 coordinate with 3 residues from MshB (His-13, Asp-16, His-147) and two water molecules. One water would be displaced upon binding of substrate (GlcNAc-Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp-15. In addition to the Zn²⁺ providing electrophilic assistance in the hydrolysis, His-144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S-conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol S-conjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol S-conjugate amidase based on the structure of MshB.

Received for publication, August 12, 2003 , and in revised form, September 3, 2003.

The atomic coordinates and structure factors (code 1Q74) 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 Grant MGP-37770 from the Canadian Institutes of Health Research (to M. N. G. J.) and by equipment funds from the Alberta Heritage Foundation for Medical Research. 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.

A Pfizer M.D./Ph.D Student. Supported by an M.D./Ph.D Training Award from the Canadian Institutes of Health Research and the Canadian Gene Cure Foundation and supplemental funding from the Alberta Heritage Foundation for Medical Research.

^** Supported by the British Columbia Lung Association. A British Columbia Lung-Canadian Institutes of Health Research Scholar.

To whom correspondence should be addressed. Tel.: 780-492-4550; Fax: 780-492-0886; E-mail: Michael.James{at}ualberta.ca.

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