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J. Biol. Chem., Vol. 279, Issue 41, 43098-43106, October 8, 2004

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Catalase-peroxidases (KatG) Exhibit NADH Oxidase Activity^*

Rahul Singh, Ben Wiseman, Taweewat Deemagarn, Lynda J. Donald, Harry W. Duckworth, Xavi Carpena, Ignacio Fita¶, and Peter C. Loewen||

From the Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada, the Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada, and the ^¶Consejo Superior de Investigacione Cientifícas, Parc Cientific, Josep Samitier 1-5, 08028 Barcelona, Spain

Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD⁺ and either H₂O₂ or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (K_m = 12 µM), but the oxidase reaction is slow (k_cat = 0.54 min^-1) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H₂O₂-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD⁺ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD⁺ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD⁺ and INH more efficiently than B. pseudomallei KatG.

Received for publication, June 8, 2004 , and in revised form, July 22, 2004.

^* This work was supported by Grant OGP9600 from the Natural Sciences and Engineering Research Council of Canada (to P. C. L.), by the Canadian Research Chair Program (to P. C. L.), and by Fellowship EX-2003-0866 from the Ministerio de Educación Cultura y Deporte, Spain (to X. C.). 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 should be addressed: Dept. of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. Tel.: 204-474-8334; Fax: 204-474-7603; E-mail: peter_loewen{at}umanitoba.ca.

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