Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 281, Issue 17, Pages 11456-11463Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M508844200
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- NIAID NIH HHS [AI43420] Funding Source: Medline
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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 mu M, respectively. Trifluoperazine inhibition is non-competitive for NADH, whereas the inhibition kinetics is found to be uncompetitive in terms of Q2.
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