Abstract
The menaquinone biosynthetic pathway presents a promising drug target against Mycobacterium tuberculosis and potentially other Gram-positive pathogens. In the present study, the essentiality, steady state kinetics of MenA from M. tuberculosis and the mechanism of MenA inhibition by Ro 48–8071 were characterized. MenA [isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate (DHNA) isoprenyltransferase] catalyzes a critical reaction in menaquinone biosynthesis that involves the conversion of cytosolic DHNA, to membrane bound demethylmenaquinone by transferring a hydrophobic 45-carbon isoprenoid chain (in the case of mycobacteria) to the ring nucleus of DHNA. Rv0534c previously identified as the gene encoding MenA in M. tuberculosis complemented a menA deletion in E. coli and an E. coli host expressing Rv0534c exhibited an eight-fold increase in MenA specific activity over the control strain harboring empty vector under similar assay conditions. Expression of Rv0534c is essential for mycobacterial survival and the native enzyme from M. tuberculosis H37Rv was characterized using membrane preparations as it was not possible to solubilize and purify the recombinant enzyme. The enzyme is absolutely dependent on the presence of a divalent cation for optimal activity with Mg+2 being the most effective and is active over a wide pH range, with pH 8.5 being optimal. The apparent Km values for DHNA and farnesyl diphosphate were found to be 8.2 and 4.3 μM, respectively. Ro 48–8071, a compound previously reported to inhibit mycobacterial MenA activity, is non-competitive with regard to DHNA and competitive with regard to the isoprenyldiphosphate substrate.
Highlights
The electron transport chain (ETC) involved in oxidative phosphorylation, consists of dehydrogenases and terminal reductases that are linked by isoprenoid lipoquinones
The M. tuberculosis genome contains a single open reading frame (Rv0534c) encoding a protein with similarity to MenA from E. coli. This gene, located near other genes annotated as Characterization and inhibition of MenA from M. tuberculosis being involved in menaquinone synthesis on the chromosome, was confirmed to encode functional MenA [10]
The protein encoded by Rv0534c was not predicted to be essential by high density transposon mutagenesis [29] but subsequent high-resolution phenotypic profiling suggested that the enzyme is essential for bacterial growth [30]
Summary
The electron transport chain (ETC) involved in oxidative phosphorylation, consists of dehydrogenases and terminal reductases that are linked by isoprenoid lipoquinones. The nature of the isoprenoid lipoquinones found in the ETC varies from organism to organism. Eukaryotic cells synthesize and utilize ubiquinone, a benzoquinone, in their ETC, whereas many prokaryotes, such as Gram-negative Enterobacteriaceae tend to contain ubiquinones, menaquinones (naphthaquinones, MK), demethylmenaquinones (DMK) or a combination of these compounds, and strictly aerobic Gram-negatives generally synthesize only ubiquinones. Mycobacterium spp. synthesize MK with isoprenoid chains of nine isoprene units, with one of these being saturated reviewed in [2] and is designated MK-9(H2) here. The chain-length and degree of saturation of the isoprenoid moiety is used to identify these compounds; for example, menaquinone from Escherichia coli is designated MK-8 [menaquinone with eight isoprene units (40 carbon atoms)]
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