Mycobacterial MenJ: An Oxidoreductase Involved in Menaquinone Biosynthesis

Abstract

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 K 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 K values were determined to be 59 ± 14 and 64 ± 15 μM. The K 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.

Predicting nitroimidazole antibiotic resistance mutations in Mycobacterium tuberculosis with protein engineering

“…It should be noted that the catalytic rate is likely to be higher in the crowded native environment of the M. tuberculosis cell where Ddn and menaquinone are co-localised at the cell membrane [24]. The enzyme kinetics of Ddn are comparable to another mycobacterial menaquinone oxidoreductase, MenJ (Rv0561c), which is found in the menaquinone biosynthetic pathway (k cat /K M of~10 4 M -1 s -1 and K M of 30 μM [25]). The K M value is significantly lower than the median K M of enzymes with their native substrates (~130 μM), and the k cat /K M value is within the broad distribution of known enzymes (10 3 −10 7 M -1 s -1 ), albeit at the lower end of the range [26,27].…”

SAR Analysis of Small Molecules Interfering with Energy-Metabolism in Mycobacterium tuberculosis

“…These enzymes catalyze the oxidation of NADH to NAD + and succinate to fumarate, respectively, followed by the menaquinone/menaquinol reduction. The menaquinone/menaquinol redox pool then transfers the electrons to a final acceptor (oxygen in aerobic conditions and nitrate or fumarate in anaerobic conditions) via terminal respiratory oxidases, while protons are pumped across the membrane, generating the PMF [ 17 , 18 , 19 ]. Despite an emerging role of fumarate reductase and nitrate reductase under hypoxia, in such conditions, the redox mediated by these enzymes is poorly understood, and a major focus is still given to the cytochrome bc1-aa3 “supercomplex” and the cytochrome bd oxidase, acknowledged to play a vital role in aerobic conditions.…”

“…It is well known that menaquinone (vitamin K2) plays an important role in producing the electrochemical gradient required for the production of ATP in ETC by shuttling electrons to terminal reductases. Therefore, each step involved in menaquinone biosynthesis, catalyzed by Men enzymes (MenA-J), is a potential chemotherapeutic target; besides, unlike bacteria, human cells are unable to produce menaquinone ex novo , as it is obtained only through the diet [ 17 , 18 , 19 ]. The most promising results come from inhibitors of Men-enzymes that function downstream of the synthesis pathway (MenA, MenB, MenG), of which DG70 is the most recent compound.…”

Demethylmenaquinone Methyl Transferase Is a Membrane Domain-Associated Protein Essential for Menaquinone Homeostasis in Mycobacterium smegmatis

“…MenH/UbiE), forming menaquinone (Dhiman et al, 2009). Finally, the double bond in the β-isoprene unit of the polyprenyl chain is reduced by the reductase MenJ to form the mature product, such as MK-9 (II-H 2 ) (Upadhyay et al, 2015, 2018). Our comparative proteomic analysis of the IMD and the PM-CW suggested that MenG and MenJ are enriched in the IMD, while MenA was not detected in either the IMD or the PM-CW (Hayashi et al, 2016).…”