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

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...

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Published inThe Journal of biological chemistry Vol. 281; no. 17; pp. 11456 - 11463
Main Authors Yano, Takahiro, Li, Lin-Sheng, Weinstein, Edward, Teh, Jiah-Shin, Rubin, Harvey
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 28.04.2006
American Society for Biochemistry and Molecular Biology
Subjects
Antitubercular Agents - pharmacology
Binding, Competitive
Enzyme Inhibitors - pharmacology
Flavin-Adenine Dinucleotide - metabolism
Kinetics
Mycobacterium smegmatis
Mycobacterium tuberculosis
Mycobacterium tuberculosis - enzymology
NAD - metabolism
Phenothiazines - pharmacology
Quinone Reductases - antagonists & inhibitors
Quinone Reductases - metabolism
Quinones - chemistry
Ubiquinone - analogs & derivatives
Ubiquinone - metabolism
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Summary: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.
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
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ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M508844200