Mycobacterial MenG: Partial Purification, Characterization, and Inhibition

Menaquinone (MK) is an essential component of the electron transport chain (ETC) in the gram-variable Mycobacterium tuberculosis and many Gram-positive pathogens. Three genes in the M. tuberculosis genome were annotated as methyltransferases involved in lipoquinone synthesis in mycobacteria. Heterologous expression of Rv0558 complemented an ubiE (the quinone C-methyltransferase involved in ubiquinone and menaquinone synthesis) deletion in Escherichia coli, and expression in a wild-type E. coli strain increased quinone C-methyltransferase specific activity by threefold. Rv0558 encodes a canonical C-methyltransferase or, more specifically, a S-adenosylmethionine/demethylmenaquinol methyltransferase. Partially purified recombinant protein catalyzed the formation of MK from demethylmenaquinone (DMK), although the activity of the recombinant protein was low and appeared to require a cofactor or intact membrane structure for activity. Membrane preparations from irradiated M. tuberculosis also showed poor activity; however, membrane preparations from wild-type Mycobacterium smegmatis showed robust, substrate-dependent activity. The apparent K_m values for demethylmenaquinone and SAM were 14 ± 5.0 and 17 ± 7.0 μM, respectively. Interestingly, addition of dithiothreitol, dithionite, NADH, or other substrates of primary dehydrogenases to reaction mixtures containing membrane preparations stimulated the activity. Thus, these observations strongly suggest that demethylmenaquinol is the actual substrate of MenG. Ro 48-8071, previously reported to inhibit mycobacterial MK synthesis and growth, inhibited Rv0558 activity with an IC₅₀ value of 5.1 ± 0.5 μM, and DG70 (GSK1733953A), first described as a respiration inhibitor in M. tuberculosis, inhibits MenG activity with an IC₅₀ value of 2.6 ± 0.6 μM.

S-adenosylmethionine/demethylmenaquinol methyltransferase; bacteria; bacterial metabolism; demethylmenaquinol; demethylmenaquinone; electron transport; enzyme kinetics; menaquinone; mycobacteria.