Journal:Acta Cryst F:S2053230X25006181

spinqualitylabelsall models LmoMenD monomer (PDB entry 9e9b) with PYR (green), TH3 (orange) and PP (blue) domains. Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D The structures of Listeria monocytogenes MenD in ThDP-bound and in-crystallo captured intermediate I-bound forms Michelle Bailey, Fiona M. Given, Ngoc Anh Thu Ho, F. Grant Pearce, Timothy M. Allison and Jodie M. Johnston Molecular Tour The ThDP-dependent enzyme MenD (2-succinyl-5-enolpyruvyl-6- hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase is involved in bacterial menaquinone biosynthesis. Menaquinones (vitamin K2), a family of redox-active small lipophilic molecules, are important to many bacteria acting as vital electron carriers in electron transport and energy generation. Menaquinones have also been implicated in a range of other roles variously linked to environmental adaptation, virulence, persistence, biofilm formation and sporulation in different bacteria. Humans don’t make menaquinone (though they get it from their diet and use if for very different uses as a cofactor for enzymes involved in blood clotting and bone health). Thus, the bacterial enzymes that make menaquinone have become of interest as antimicrobial drug targets. Bacteria contain either one of two pathways to make menaquinone, the classical or futalosine pathways, and MenD catalyses the first irreversible step in the classical pathway. It takes two substrates, 2-oxoglutarate and isochorismate and converts them to SEPHCHC via a series of reactions involving covalent ThDP-bound intermediates. We have solved several structures of MenD from the listeria-causing pathogen Listeria monocytogenes. Analysis of the structures show a typical ThDP-dependent three-domain (PP, PYR, TH3 domain) fold similar to other MenD enzymes. Our first structure, captured in the ThDP cofactor-bound form has enabled us to visualise and understand how the co-factor binds. By comparing this structure to one without ThDP-bound (apo) in the protein databank we can see how the enzyme active site partially-closes around the co-factor. In our second structure we were able to capture reactivity within the crystal with an in-crystallo formed covalent ThDP-intermediate (Intermediate I) bound in the active site. Studying the shape and interactions of this intermediate helps us understand more about the chemistry of the enzyme. In additional studies we used our structural analysis combined with other methods (size-exclusion chromatography, mass photometry and small angle X-ray scattering analysis) to understand the oligomeric state of the enzyme. Showing that like other MenD’s, Listeria monocytogenes MenD has a homotetrameric quaternary structure. We also undertook enzyme kinetics to show the enzyme was active, and that there was weak inhibition of enzyme activity in the presence of 1,4- dihydroxy-2-naphthoic acid, a downstream metabolite in the menaquinone-biosynthesis pathway that has previously been shown to be a potent allosteric regulator of Mycobacterium tuberculosis MenD. References

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