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J. Biol. Chem., Vol. 278, Issue 32, 29502-29508, August 8, 2003

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The Mechanism of Mycobacterium tuberculosis Alkylhydroperoxidase AhpD as Defined by Mutagenesis, Crystallography, and Kinetics^*

Aleksey Koshkin , Christine M. Nunn , Snezana Djordjevic  ¶ and Paul R. Ortiz de Montellano  ||

From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-2280 and Department of Biochemistry and Molecular Biology, University College, Gower Street, London WC1E 6BT, United Kingdom

AhpD, a protein with two cysteine residues, is required for physiological reduction of the Mycobacterium tuberculosis alkylhydroperoxidase AhpC. AhpD also has an alkylhydroperoxidase activity of its own. The AhpC/AhpD system provides critical antioxidant protection, particularly in the absence of the catalase-peroxidase KatG, which is suppressed in most isoniazid-resistant strains. Based on the crystal structure, we proposed recently a catalytic mechanism for AhpD involving a proton relay in which the Glu¹¹⁸ carboxylate group, via His¹³⁷ and a water molecule, deprotonates the catalytic residue Cys¹³³ (Nunn, C. M., Djordjevic, S., Hillas, P. J., Nishida, C., and Ortiz de Montellano, P. R. (2002) J. Biol. Chem. 277, 20033–20040). A possible role for His¹³² in subsequent formation of the Cys¹³³-Cys¹³⁰ disulfide bond was also noted. To test this proposed mechanism, we have expressed the H137F, H137Q, H132F, H132Q, E118F, E118Q, C133S, and C130S mutants of AhpD, determined the crystal structures of the H137F and H132Q mutants, estimated the pK_a values of the cysteine residues, and defined the kinetic properties of the mutant proteins. The collective results strongly support the proposed catalytic mechanism for AhpD.

Received for publication, April 10, 2003 , and in revised form, May 19, 2003.

^* This work was supported by National Institutes of Health Grant GM56531 and by Higher Education Funding Council for England UK. 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.

^¶ To whom correspondence may be addressed. Fax: 44-020-7679-7193; E-mail: snezana{at}biochemistry.ucl.ac.uk.

^|| To whom correspondence may be addressed: University of California, Mission Bay Genentech Hall, 600 16th St., San Francisco, CA 94143-2280. Fax: 415-502-4728; E-mail: ortiz{at}cgl.ucsf.edu.

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