Structures of Listeria monocytogenes MenD in ThDP-bound and in-crystallo captured intermediate I-bound forms

Abstract

Menaquinones (vitamin K₂) are a family of redox-active small lipophilic molecules that serve as vital electron carriers in many bacterial electron-transport pathways. The ThDP-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase (MenD) catalyses the first irreversible step in bacterial classical menaquinone biosynthesis via a series of reactions involving covalent ThDP-bound intermediates. We report structures of MenD from the pathogen Listeria monocytogenes (LmoMenD) in its ThDP cofactor-bound and in-crystallo captured intermediate I-bound forms. Analysis of the structures revealed that LmoMenD adopts the typical three-domain ThDP-dependent fold observed for MenD orthologs, while a combination of structure, size-exclusion chromatography, mass photometry and small-angle X-ray scattering analysis showed that the enzyme has a homotetrameric quaternary structure. While both of the ligand-bound structures reported here were very similar, comparison with an apo form from the PDB revealed a closing down of the active site in the ligand-bound forms, with more complete models suggesting lower levels of disorder around key regions of the active site that interface with ThDP or the captured intermediate. Enzyme kinetics characterization showed the enzyme was active and enabled allosteric inhibition to be measured. There was weak inhibition of enzyme activity in the presence of 1,4-dihydroxy-2-naphthoic acid, an allosteric regulator of Mycobacterium tuberculosis MenD and downstream metabolite in the menaquinone-biosynthesis pathway.

Keywords: Listeria monocytogenes; MenD; SEPHCHC synthase; intermediate I capture; menaquinone biosynthesis; thiamine diphosphate-dependent enzyme.

Figure 1

MenD catalytic cycle illustrating the formation of several ThDP-bound covalent intermediates from sequential reaction with the substrates 2-oxo­glutarate (intermediate I) and isochorismate (intermediate II), resulting ultimately in regeneration of the ThDP and release of the product SEPHCHC. Note that the aminopyrimidine ring is shown in its AP (4′-aminopyrimidine) tautomer form in ThDP and intermediate I and its IP (1′,4′-imino­pyrimidine) tautomer form in intermediate II; shuttling of tautomer states is important during the catalytic cycle.

Figure 2

(a) LmoMenD monomer (PDB entry 9e9b) with PYR (green), TH3 (orange) and PP (blue) domains. (b) Dimer-of-dimers LmoMenD tetramer (PDB entry 9e9b; dimer 1, blue and green; dimer 2, orange and rose).

Figure 3

(a) ThDP and Mg²⁺ (yellow) binding in the LmoMenD (PDB entry 9e9b) active site. (b) Intermediate I (IntI; rose) formation in the LmoMenD (PDB entry 9mnn) active site. The residues comprising the active site originate from the PYR (green) and PP (blue) domains, with the catalytic glutamate (Glu56) highlighted (*). Water molecules are depicted as orange spheres and polar contacts as black dashes. (c) ThDP (PDB entry 9e9b; yellow) and intermediate I (PDB entry 9mnn; rose) in their respective 2mF_o − DF_c electron-density maps (contoured at 1σ; grey). (d) Overlay of the apo (PDB entry 3lq1; grey), ThDP-bound (PDB entry 9e9b; green) and intermediate I-bound (PDB entry 9mnn; blue) active sites, with residues differing between the structures shown as sticks. (e) Overlay of the apo (PDB entry 3lq1; grey) and ThDP-bound (PDB entry 9e9b; green) LmoMenD monomers, with regions becoming ordered upon cofactor binding highlighted in orange. (f) Overlay of the apo (PDB entry 3lq1; grey) and ThDP-bound (PDB entry 9e9b; PYR domain, green; PP domain, blue) active sites, with regions becoming ordered upon cofactor binding highlighted in orange and ThDP in the active site shown as yellow sticks.

Figure 4

(a) Overlay of the putative LmoMenD (PDB entry 9e9b; green) and MtbMenD (PDB entry 6o0j; grey) DHNA (orange) binding sites. Residues hypothesized to fulfil equivalent functions to a particular M. tuberculosis arginine-cage constituent are in bold, with LmoMenD Lys325 potentially able to fulfil the function of MtbMenD Arg277 or Arg303. (b) Surface representation of the MtbMenD (PDB entry 6o0j; grey) allosteric site showing the DHNA (orange) binding pocket. (c) Surface representation of the LmoMenD (PDB entry 9mnn; green) putative allosteric site, highlighting the requirement of induced-fit conformational changes to accommodate DHNA (orange) binding.