MenD, or 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase, is a thiamine diphosphate and Mg²⁺ -dependent enzyme catalyzing an essential 1,4-addition reaction of 2-ketoglutarate to isochorismate in vitamin K₂ biosynthesis. The conserved amino acid residues Arg413 and Arg395 in Escherichia coli (E. coli) MenD have been found to be vital for the binding and orientation of substrates during the catalysis. Mutations of these conserved residues have been found to form an inactive tetrahedral post-decarboxylation intermediate in an alternate binding site to disable the catalysis. Not only the tetrahedral intermediate indicates a new binding mode in ThDP-dependent catalysis but also the potential inhibition ability in the new discovered alternate binding site s...[ Read more ]

MenD, or 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase, is a thiamine diphosphate and Mg²⁺ -dependent enzyme catalyzing an essential 1,4-addition reaction of 2-ketoglutarate to isochorismate in vitamin K₂ biosynthesis. The conserved amino acid residues Arg413 and Arg395 in Escherichia coli (E. coli) MenD have been found to be vital for the binding and orientation of substrates during the catalysis. Mutations of these conserved residues have been found to form an inactive tetrahedral post-decarboxylation intermediate in an alternate binding site to disable the catalysis. Not only the tetrahedral intermediate indicates a new binding mode in ThDP-dependent catalysis but also the potential inhibition ability in the new discovered alternate binding site since it has led to a dead end in the enzymatic catalysis in ecMenD R413A mutant combined with the results in steady state kinetics data. The presence of alternate binding site has inspired a new target for development of potential inhibitors to inhibit MenD in vitamin K₂ biosynthetic pathway.

To investigate this unique mechanism, MenD and its mutants of different orthologues including Escherichia coli, Bacillus subtilis, Mycobacterium tuberculosis and Staphylococcus aureus were overexpressed, purified and characterized by in-vitro kinetics assay. In addition, the interactions between the ThDP adduct and one of the MenD mutants, bsMenD R409A were investigated by X-ray crystallography. The solved structure showed a tetrameter of bsMenD with four active sites, in which the ThDP adduct flips to the alternate binding sites. These results have provided evidence and consistency with the previous solved structure of ecMenD to provide further insights into the catalytic mechanism on the basis of kinetics, molecular interactions and conformational changes for potential drug development against bacterial pathogens.

To develop potential inhibitor against MenD catalysis, three analogs of the 2-ketoglutarate, including 5-amino-2,5-dioxopentanoic acid, 5-methyl-2,5-dioxopentanoic acid and 5-(methylamino)-2,5-dioxopentanoic acid were designed, synthesized and characterized as potential suicide inhibitors to MenD orthologues of E. coli, B. subtilis, M. tuberculosis and S. aureus. They were indeed found to inactivate MenD in a time-dependent manner. Not only the rate constant of formation of enzyme-inhibitor complex has been determined, but also the role of thiamine diphosphate (ThDP) to protect the formation of enzyme-inhibitor complex has been observed. Most importantly, bacterial growth inhibition assay has as well been carried out to test the inhibition activities of the analogs towards a multitude of Gram-positive bacteria including B. subtilus, E. faecalis, S. aureus and MRSA. Among the candidates, 5-amino-2,5-dioxopentanoic acid shows the most potent activities to inhibit the growth of Gram-positive bacteria, showing a certain extent of bactericidal activities. The substrate analog could as well hinder the growth of Gram-positive bacteria with antibiotic resistance. The results would pave the way for developing novel antibiotics against bacterial pathogens with developed resistance.