Mycobacterial MenJ: An Oxidoreductase Involved in Menaquinone Biosynthesis.

MenJ, annotated as an oxidoreductase, was recently demonstrated to catalyze the reduction (saturation) of a single double bond in the isoprenyl side-chain of mycobacterial menaquinone. This modification was shown to be essential for bacterial survival in J774A.1 macrophage-like cells, suggesting that MenJ may be a conditional drug target in Mycobacterium tuberculosis and other pathogenic mycobacteria. Recombinant protein was expressed in a heterologous host, and the activity was characterized. Although highly regiospecific in vivo, the activity is not absolutely regiospecific in vitro; in addition, the enzyme is not specific for naphthoquinones vs benzoquinones. Coenzyme Q-1 (a benzoquinone, UQ-1) was used as the lipoquinone substrate, and NADH oxidation was followed spectrophotometrically as the activity readout. NADPH could not be substituted for NADH in the reaction mixture. The enzyme contains a FAD binding site that was 72% occupied in the purified recombinant protein. Enzyme activity was maximal at 37 °C and pH 7.0; addition of divalent cations, EDTA, and reducing agents such as dithiothreitol to the reaction mixture had no effect on activity. The addition of detergents did not stimulate activity, and addition of saturating levels of FAD had relatively little effect on the observed kinetic parameters. These properties allowed the development of a facile assay needed to study this potential drug target, which is also amenable to high throughput screening. The Km values for UQ-1 using recombinant MenJ from Mycobacterium smegmatis or M. tuberculosis without saturating concentrations of FAD were found to be 52 ± 9.6 and 44 ± 4.8 μM, respectively, while the KmNADH values were determined to be 59 ± 14 and 64 ± 15 μM. The Km for MK-1, the menaquinone analogue of UQ-1, using recombinant MenJ from M. tuberculosis without saturating concentrations of FAD but in the presence of 0.5% Tween 80 was shown to be 30 ± 2.9 μM. Thus, this is the first report of a kinetic characterization of a member of the geranylgeranyl reductase family of enzymes.