Using Substrate Analogues To Probe the Kinetic Mechanism and Active Site of Escherichia coli MenD

Abstract

MenD catalyzes the thiamin diphosphate-dependent decarboxylative carboligation of α-ketoglutarate and isochorismate. The enzyme is essential for menaquinone biosynthesis in many bacteria and has been proposed to be an antibiotic target. The kinetic mechanism of this enzyme has not previously been demonstrated because of the limitations of the UV-based kinetic assay. We have reported the synthesis of an isochorismate analogue that acts as a substrate for MenD. The apparent weaker binding of this analogue is advantageous in that it allows accurate kinetic experiments at substrate concentrations near K(m). Using this substrate in concert with the dead-end inhibitor methyl succinylphosphonate, an analogue of α-ketoglutarate, we show that MenD follows a ping-pong kinetic mechanism. Using both the natural and synthetic substrates, we have measured the effects of 12 mutations of residues at the active site. The results give experimental support to previous models and hypotheses and allow observations unavailable using only the natural substrate.

Menaquinone biosynthesis inhibition: a review of advancements toward a new antibiotic mechanism

“…1,14 Investigations into inhibitions of this enzyme have been conducted by Fang et al 13 by using acylphosphonate derivatives as false substrates of a-ketoglutarate. Of the eleven compounds that they synthesised, two compounds were effective inhibitors.…”

Identification of Point Mutations in Clinical Staphylococcus aureus Strains That Produce Small-Colony Variants Auxotrophic for Menadione

“…In the majority of Gram-positive bacteria, menaquinone is the only quinone in the electron transport chain, making the enzymes that synthesize this compound promising drug targets for multidrug-resistant pathogens such as S. aureus and Mycobacterium tuberculosis (34). In this regard, we note that growth inhibition has been reported for compounds that target the gene products of menA (34,36), menD (16), menE (18), and menB (37). The initial findings for this new class of antimicrobial molecules highlight the importance of the work presented here.…”

“…Many of the typical SCV characteristics (e.g., low growth rate, lack of pigmentation, increased aminoglycoside resistance, and decreased alpha-toxin production) can be linked to electron transport deficiencies (reviewed in reference 11). Most studies investigating SCVs have used laboratory strains containing stable mutations in the menD or hemB gene, resulting in menadione-and hemin-auxotrophic strains, respectively, the two most common SCV phenotypes (16)(17)(18).…”

Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms

“…These involve the side chain of Gln118, which is essential for activity but whose role is not understood. Mutation of the analogous Gln residue abolishes activity in Ec-MenD (Fang et al, 2011) and severely impairs activity in another ThDP-dependent carboligase enzyme acetohydroxy acid synthase (Vyazmensky et al, 2011). In Mtb-MenD, the rehybridization of the IC ring when intermediate II is formed enables Gln118 to be positioned such that its OE1 atom forms a short hydrogen bond (2.3 Å ) with the C2a-OH and a C-H.O hydrogen bond (3.1 Å ) with the IC carbon C1 of IC, while its NE2 atom hydrogen bonds to the peptide oxygen of Ser79.…”