Characterization of MenA (isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate isoprenyltransferase) from Mycobacterium tuberculosis

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 K m 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.

Allosteric regulation of menaquinone (vitamin K2) biosynthesis in the human pathogen Mycobacterium tuberculosis

“…The ability of DHNA to act as a regulatory signal in the pathogen M. tuberculosis is in line with both the biological significance of DHNA and the importance of regulating menaquinone levels within the bacteria. As the last nonprenylated soluble metabolite in the MK biosynthetic pathway, DHNA sits at the point where the pathway moves from an aqueous cytosolic location to a lipophilic membrane-immersed one (25) and has the potential to provide feedback on the catalytic status of MenA (and perhaps the downstream MK pool).…”

“…The classical MK biosynthesis pathway ( Fig. 1A) starts with the synthesis of the naphthoquinone headgroup precursor (1,4dihydroxy-2-naphthoic acid (DHNA)) in the cytosol (22,23), followed by prenylation and methylation by membrane-bound enzymes to produce the lipid-soluble MK (24,25). In addition to its electron transport role, the prenyl tail of MK can be further modified, and these modified quinones have been shown to regulate virulence of Mtb infection (7,26,27).…”

Allosteric Regulation of Vitamin K2 Biosynthesis in a Human Pathogen

“…The classical MK biosynthesis pathway ( Figure 1A) starts with the synthesis of the napthoquinone head-group precursor (DHNA) in the cytosol [22,23], followed by prenylation and methylation by membrane bound enzymes to produce the lipid soluble MK [24,25]. In addition to its electron transport role, the prenyl tail of MK can be further modified and these modified quinones have been shown to regulate virulence of the Mtb infection [7,26,27].…”

“…The ability of DHNA to act as a regulatory signal in the pathogen M. tuberculosis is in line with both the biological significance of DHNA and the importance of regulating menaquinone levels within the bacteria. As the last non-prenylated soluble metabolite in the MK biosynthetic pathway, DHNA sits at the point where the pathway moves from an aqueous cytosolic location to a lipophilic membrane-immersed one [25] and has the potential to provide feedback on the catalytic status of MenA (and perhaps the downstream MK pool). DHNA is also the first metabolite in the pathway with a complete redox-capable napthoquinone ring [39], and has the capacity in its own right to catalyze redox reactions [40].…”

Self-control of vitamin K2 production captured in the crystal

“…After solving the three-dimensional structures, a clear extra electron density corresponding to 1,4-dihydroxy-2-napthoic acid (DHNA) was found in a cleft of domain II. DHNA is the substrate of MenA, which converts DHNA to demethylmenaquinone (5). In MenD, the DHNA binding site is distant by at least 20 Å from the active site and characterized by the presence of an "arginine cage" composed of three arginine residues, namely Arg-97, Arg-277, and Arg-303 ( Fig.…”