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J. Biol. Chem., Vol. 281, Issue 17, 11456-11463, April 28, 2006

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Steady-state Kinetics and Inhibitory Action of Antitubercular Phenothiazines on Mycobacterium tuberculosis Type-II NADH-Menaquinone Oxidoreductase (NDH-2)^*

Takahiro Yano¹, Lin-Sheng Li, Edward Weinstein, Jiah-Shin Teh, and Harvey Rubin²

From the Departments of Medicine and Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Type-II NADH-menaquinone oxidoreductase (NDH-2) is an essential respiratory enzyme of the pathogenic bacterium Mycobacterium tuberculosis (Mtb) that plays a pivotal role in its growth. In the present study, we expressed and purified highly active Mtb NDH-2 using a Mycobacterium smegmatis expression system, and the steady-state kinetics and inhibitory actions of phenothiazines were characterized. Purified NDH-2 contains a non-covalently bound flavin adenine dinucleotide cofactor and oxidizes NADH with quinones but does not react with either NADPH or oxygen. Ubiquinone-2 (Q2) and decylubiquinone showed high electron-accepting activity, and the steady-state kinetics and the NADH-Q2 oxidoreductase reaction were found to operate by a ping-pong reaction mechanism. Phenothiazine analogues, trifluoperazine, Compound 1, and Compound 2 inhibit the NADH-Q2 reductase activity with IC₅₀ = 12, 11, and 13 µM, respectively. Trifluoperazine inhibition is non-competitive for NADH, whereas the inhibition kinetics is found to be uncompetitive in terms of Q2.

Received for publication, August 10, 2005 , and in revised form, February 8, 2006.

^* This work was supported by National Institutes of Health Grant AI43420 (to H. 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 Current address: ERATO ATP Systems, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan.

² To whom correspondence should be addressed: Div. of Infectious Diseases, Dept. of Medicine, 522 Johnson Pavilion, 36th and Hamilton Walk, University of Pennsylvania, Philadelphia, PA 19104. Tel.: 215-662-6475; Fax: 215-573-1240; E-mail: rubinh{at}mail.med.upenn.edu.

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