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J. Biol. Chem., Vol. 283, Issue 10, 6193-6200, March 7, 2008

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The Tuberculosis Prodrug Isoniazid Bound to Activating Peroxidases^*

Clive Metcalfe, Isabel K. Macdonald¹, Emma J. Murphy, Katherine A. Brown, Emma Lloyd Raven, and Peter C. E. Moody^¶2

From the Departments of Chemistry and ^¶Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN and Department of Life Sciences, Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, Imperial College, Exhibition Road, London SW7 2AZ, United Kingdom

Isoniazid (INH, isonicotinic acid hydrazine) is one of only two therapeutic agents effective in treating tuberculosis. This prodrug is activated by the heme enzyme catalase peroxidase (KatG) endogenous to Mycobacterium tuberculosis but the mechanism of activation is poorly understood, in part because the binding interaction has not been properly established. The class I peroxidases ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP) have active site structures very similar to KatG and are also capable of activating isoniazid. We report here the first crystal structures of complexes of isoniazid bound to APX and CcP. These are the first structures of isoniazid bound to any activating enzymes. The structures show that isoniazid binds close to the -heme edge in both APX and CcP, although the precise binding orientation varies slightly in the two cases. A second binding site for INH is found in APX at the -heme edge close to the established ascorbate binding site, indicating that the -heme edge can also support the binding of aromatic substrates. We also show that in an active site mutant of soybean APX (W41A) INH can bind directly to the heme iron to become an inhibitor and in a different mode when the distal histidine is replaced by alanine (H42A). These structures provide the first unambiguous evidence for the location of the isoniazid binding site in the class I peroxidases and provide rationalization of isoniazid resistance in naturally occurring KatG mutant strains of M. tuberculosis.

Received for publication, September 4, 2007 , and in revised form, October 29, 2007.

The atomic coordinates and structure factors (codes 2V23, 2V2E, 2VCF, 2VCN, and 2VCS) 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 Biotechnology and Biological Sciences Research Council Grants BBC/0011841 (to E. L. R. and P. C. E. M.), 91/B19083 (to E. L. R.), and BBS/S/A/2004/12421 (to E. L. R.). 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.

¹ Present address: The Edward Jenner Inst. for Vaccine Research, Compton, Newbury, Berkshire RG20 7NN, UK.

² To whom correspondence should be addressed. Tel.: 44-116-229-7097; Fax: 44-116-229-7084; E-mail: peter.moody{at}le.ac.uk.

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