Structure and Reactivity of Bacillus subtilis MenD Catalyzing the First Committed Step in Menaquinone Biosynthesis

Abstract

The first committed step in the classical biosynthetic route to menaquinone (vitamin K2) is a Stetter-like conjugate addition of α-ketoglutarate with isochorismate. This reaction is catalyzed by the thiamine diphosphate and metal-ion-dependent 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD). The medium-resolution (2.35 Å) crystal structure of Bacillus subtilis MenD with cofactor and Mn2+ has been determined. Based on structure–sequence comparisons and modeling, a two-stage mechanism that is primarily driven by the chemical properties of the cofactor is proposed. Hypotheses for the molecular determinants of substrate recognition were formulated. Five basic residues (Arg32, Arg106, Arg409, Arg428, and Lys299) are postulated to interact with carboxylate and hydroxyl groups to align substrates for catalysis in combination with a cluster of non-polar residues (Ile489, Phe490, and Leu493) on one side of the active site. The powerful combination of site-directed mutagenesis, where each of the eight residues is replaced by alanine, and steady-state kinetic measurements has been exploited to address these hypotheses. Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of α-ketoglutarate. Arg32 and in particular Arg106 are critical for recognition of isochorismate. Mutagenesis of Phe490 and Ile489 has the most profound influence on catalytic efficiency, indicating that these two residues are important for binding of isochorismate and for stabilizing the cofactor position. These data allow for a detailed description of the structure–reactivity relationship that governs MenD function and refinement of the model for the catalytic intermediate that supports the Stetter-like conjugate addition.

A Differential Fluorescence-Based Genetic Screen Identifies Listeria monocytogenes Determinants Required for Intracellular Replication

“…As the corresponding gene has been designated pepP in the previously sequenced L. monocytogenes strain M7 (32), we have designated LMRG_00804 pepP. menD (LMRG_01292) encodes the menaquinone biosynthetic enzyme catalyzing the addition of 2-oxoglutarate to isochorismate to generate 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (33), and hepT (LMRG_01077) encodes the heptaprenyl diphosphate synthase component II, a protein involved in the synthesis of the heptaprenyl moiety that anchors menaquinone to the plasma membrane (34,35). Interestingly, L. monocytogenes lacks the genes necessary for ubiquinone biosynthesis and therefore requires menaquinone to complete a functional electron transport chain.…”

Characterization of 1,4-dihydroxy-2-naphthoyl-coenzyme A synthase (MenB) in phylloquinone biosynthesis of Synechocystis sp. PCC 6803

“…Most of the biosynthetic enzymes in the bacterial menaquinone biosynthesis have been scrutinized structurally and mechanistically [9,11,[23][24][25][26][27][28][29][30]. 1,4-Dihydroxy-2-naphthoylCoA (DHNA-CoA) synthase, or MenB, is one of these enzymes, which catalyzes a multiple-step intramolecular Claisen condensation of o-succinylbenzoyl-CoA (OSB-CoA).…”

Menaquinone as a potential target of antibacterial agents

“…The classical pathway involves enzymes, namely MenF, MenD, MenH, MenC, MenE, MenB, yfbB (MenI), MenA and MenG ( Figure 2) (3,(14)(15)(16)(17). Targeting these enzymes that exist in bacteria and not in humans, can open up an avenue for novel antimicrobial agents with therapeutic potential.…”