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

Abstract

Menaquinone (MQ) is an essential component of the respiratory chains of many pathogenic organisms, including Mycobacterium tuberculosis (Mtb). The first committed step in MQ biosynthesis is catalyzed by 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD), a thiamin diphosphate (ThDP)-dependent enzyme. Catalysis proceeds through two covalent intermediates as the substrates 2-oxoglutarate and isochorismate are successively added to the cofactor before final cleavage of the product. We have determined a series of crystal structures of Mtb-MenD that map the binding of both substrates, visualizing each step in the MenD catalytic cycle, including both intermediates. ThDP binding induces a marked asymmetry between the coupled active sites of each dimer, and possible mechanisms of communication can be identified. The crystal structures also reveal conformational features of the two intermediates that facilitate reaction but prevent premature product release. These data fully map chemical space to inform early-stage drug discovery targeting MenD.

Alteration of the Route to Menaquinone towards Isochorismate‐Derived Metabolites

“…Although Ec MenD and Mtb MenD share low sequence identity (30 %), some residues that interact with 2 are conserved and are positioned in a similar manner concerning this substrate, namely Ile474, Leu478, Phe475, Arg107, and Gln118. In particular, Arg107 was shown to bind the C1‐carboxyl and C6‐hydroxy groups, and Gln118 the C6‐hydroxy group, as predicted for Ec MenD in our model . Regarding the loop that interacts with the enolpyruvyl moiety, in Mtb MenD the side chain of Arg282 is involved in a bidentate interaction, while this residue is not conserved in Ec MenD.…”

“…However, as 2 has two carboxylic acid moieties, it is difficult to unequivocally judge which of the carboxylates binds to each Arg residue. Recently, M. tuberculosis MenD ( Mtb MenD) was crystallized with bound 2 . Although Ec MenD and Mtb MenD share low sequence identity (30 %), some residues that interact with 2 are conserved and are positioned in a similar manner concerning this substrate, namely Ile474, Leu478, Phe475, Arg107, and Gln118.…”

Allosteric Regulation of Vitamin K2 Biosynthesis in a Human Pathogen

“…The binding site for DHNA (Figure 2A) is essentially the same in all four structures. The DHNA molecule occupies an 'arginine cage' formed by three arginine residues, Arg97, Arg277 and Arg303, arranged such that the side chains of Arg277 and Whether there is any connectivity between the four allosteric DHNA binding sites in the Mtb-MenD tetramer (as there is between the active sites [13]) is unclear.…”

“…Like other members of the ThDP-dependent pyruvate oxidase (POX) family, which are dimers or tetramers (comprising two interfacing dimers), MenD is tetrameric, with each monomer comprising three domains [11,12]. Domains I and III have known roles in catalytic function, domain I from one monomer in the dimer pairs with domain III of the other monomer (and vice versa) to form two paired active sites per dimer, with residues from both domains contributing to each active site [13][14][15]. Domain II, however, is much less conserved and does not appear to participate in cofactor or substrate binding [11,15].…”

“…We previously determined a series of crystal structures of Mtb-MenD showing each step in the MenD catalytic cycle, as substrates a-ketoglutarate and isochorismate are successively added to the ThDP cofactor before the final product is released [13].…”