Steady-state Kinetics and Inhibitory Action of Antitubercular Phenothiazines onMycobacterium tuberculosisType-II NADH-Menaquinone Oxidoreductase (NDH-2)

Takahiro Yano, Lin-Sheng Li, Edward Weinstein, Jiah-Shin Teh, Harvey Rubin
2006 Journal of Biological Chemistry  
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
more » ... 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 IC 50 ‫؍‬ 12, 11, and 13 M, respectively. Trifluoperazine inhibition is non-competitive for NADH, whereas the inhibition kinetics is found to be uncompetitive in terms of Q2. The Gram-positive bacterium Mycobacterium tuberculosis (Mtb) 3 causes tuberculosis, one of the leading causes of morbidity and mortality in the world. Each year nine million active cases of the disease are diagnosed, accounting for three million deaths. Multidrug-resistant tuberculosis and the existence of "persistent" organisms that are tolerant to antibiotics exacerbate the problem, for which more effective and efficient treatments need to be urgently developed. Mtb is traditionally considered an obligate aerobe, yet during the normal course of events in the infectious cycle, the bacillus is able to survive in conditions of low oxygen and nutrient concentrations, such as those postulated to exist within granulomas. Mtb adapts its metabolic activity, cellular transcription, and protein expression accordingly (1). It is therefore of great importance to understand how Mtb generates ATP under a variety of environmental conditions. Type-II NADH-dehydrogenase (NDH-2) is a critical enzyme in the life cycle of Mtb. The enzyme has been purified from Saccharomyces cerevisiae (2), Escherichia coli (3, 4), Bacillus subtilis (5), Methyloccocus capsulatus (6) , Corynebacterium glutamicum (7, 8), Acidianus ambicalens (9, 10), and Sulfolobus metallicus (11) and is, in general, composed of a single polypeptide chain, which contains a flavin as a sole cofactor. It is noteworthy that this enzyme is not found in mitochondria. The essential role of NDH-2 in Mtb is supported by extensive evidence from biochemical (12) and transcriptional studies (13) , gene deletion analysis, investigation of bacterial growth in various media and under various culture conditions, and animal experiments (12). Mtb contains two copies of ndh genes (ndh and ndhA). The Mtb NDH-2 and NDH-2A share 67% sequence identity, and the genes are separated by 17 kb. Mtb NDH-2 is highly homologous to those of Mycobacterium leprae and Mycobacterium smegmatis with 91 and 81% amino acid sequence identity, respectively. A strain of Mtb in which ndh has been disrupted by transposon mutagenesis is nonviable (14) ; however, a ndhA deletion mutant of Mtb can be easily isolated (15) . We previously demonstrated that purified NDH-2A is a competent oxidoreductase (12). Therefore, we suggest that ndhA, although present in Mtb, is probably not expressed and cannot rescue mutations in Mtb ndh. NDH-2 is likely to be the sole NADH-dehydrogenase enzyme in the Mtb respiratory chain utilized for growth in an aerobic environment. Isoniazid (INH) and ethambutol are two of the standard anti-tuberculosis medications used throughout the world. Increasing resistance to these medications is recognized as a serious global public health threat. The discovery that a mechanism of INH drug resistance in Mtb is linked to mutations in Mtb NDH-2 that decrease its activity is profoundly important (16 -18). Although measured indirectly, INH-resistant mutant NADH oxidase activity is decreased 10 -50% compared with the wild-type level. It has been hypothesized that reduced NDH-2 activity in the mutants leads to an increase in the intracellular NADH/NAD ϩ balance and accounts for the mechanism of INH resistance (16, 18) . The present understanding of this effect is that increased concentrations of NADH decrease binding of activated INH adduct to InhA, which is an NADH-dependent enoyl-ACP reductase necessary for mycolic acid synthesis in Mtb. The anti-mycobacterial activity of phenothiazines has been reported for a number of years (19 -24). Trifluoperazine (TPZ) (Fig. 1) , for example, reduced in vitro ATP synthesis in M. leprae, suggesting that one of the target sites might be the electron transport pathway itself (25). TPZ is effective against a virulent Mtb strain H37Rv in a macrophage model of infection, and it is synergistic with both INH and rifampicin (26, 27) . In our previous investigation (12), we have shown that phenothiazines block NADH-dependent oxygen consumption by Mtb membranes. Furthermore, we have demonstrated that phenothiazines inhibit purified recombinant ndh and ndhA and hinder growth of Mtb both in culture and in a mouse model of tuberculosis. Although NDH-2 is essential for growth in Mtb and plays an important role in drug resistance, little is known about the catalytic reaction
doi:10.1074/jbc.m508844200 pmid:16469750 fatcat:fyt4ouqzongw5jwjyzifj3vhqy