Specificity and reactivity in menaquinone biosynthesis: the structure of Escherichia coli MenD (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase)

Abstract

The thiamine diphosphate (ThDP) and metal-ion-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase, or MenD, catalyze the Stetter-like conjugate addition of alpha-ketoglutarate with isochorismate to release 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate and carbon dioxide. This reaction represents the first committed step for biosynthesis of menaquinone, or vitamin K2, a key cofactor for electron transport in bacteria and a metabolite for posttranslational modification of proteins in mammals. The medium-resolution structure of MenD from Escherichia coli (EcMenD) in complex with its cofactor and Mn2+ has been determined in two related hexagonal crystal forms. The subunit displays the typical three-domain structure observed for ThDP-dependent enzymes in which two of the domains bind and force the cofactor into a configuration that supports formation of a reactive ylide. The structures reveal a stable dimer-of-dimers association in agreement with gel filtration and analytical ultracentrifugation studies and confirm the classification of MenD in the pyruvate oxidase family of ThDP-dependent enzymes. The active site, created by contributions from a pair of subunits, is highly basic with a pronounced hydrophobic patch. These features, formed by highly conserved amino acids, match well to the chemical properties of the substrates. A model of the covalent intermediate formed after reaction with the first substrate alpha-ketoglutarate and with the second substrate isochorismate positioned to accept nucleophilic attack has been prepared. This, in addition to structural and sequence comparisons with putative MenD orthologues, provides insight into the specificity and reactivity of MenD and allows a two-stage reaction mechanism to be proposed.

Fig. 1

The reactions catalyzed by MenD and MenH and tautomeric forms of the pyrimidine moiety of the cofactor. (a) MenD catalyzes the reaction of isochorismate (5S,6S)-5-[(1-carboxylatoethenyl)oxy]-6-hydroxycyclohexa-1,3-diene-1-carboxylate with α-ketoglutarate to produce SEPHCHC and carbon dioxide. MenH catalyzes the cleavage of pyruvate from SEPHCHC to release SHCHC. This reaction can also occur spontaneously. M²⁺ represents Mg²⁺ or Mn²⁺. (b) Chemical structure and numbering scheme for ThDP. Three tautomeric forms of the pyrimidine moiety are implicated in ylide generation. PP represents the cofactor diphosphate.

Fig. 2

The primary and assigned secondary structure of EcMenD. α-Helices are shown as cylinders and β-strands are shown as arrows, colored according to the domain to which they have been assigned. Residues with a black background are conserved in 90% of MenD sequences listed in UniProt, which have been filtered to remove those with ≥90% sequence identity level (78 sequences in total). The ThDP interacting and metal binding residues are indicated by black stars and potential substrate binding residues are denoted by yellow circles.

Fig. 3

The secondary, tertiary, and dimer structure of EcMenD. (a) A ribbon diagram of the EcMenD subunit showing the elements of secondary structure. ThDP is depicted in stick representation colored according to atom type: C, black; N, blue; O, red; S, yellow; and P, orange. The N- and C-terminal positions are labeled, as are the elements of secondary structure using the scheme employed in Fig. 2. A black star marks the position of ThDP C2. (b) Ribbon diagram of the dimer. Subunit A is red and in the same orientation as in (a); subunit B is blue and the position of the dimer twofold (NCS) is marked. The cofactor is depicted as in (a).

Fig. 4

The EcMenD tetramer. The tetramer is shown as a van der Waals surface with subunits labeled and colored differently. The dimer of subunits A and B is in the same orientation as in Fig. 3b.

Fig. 5

The cofactor-binding site. The cofactor is shown as in Fig. 3, and C atoms of the protein residues are colored green. Mn²⁺ and a water molecule are shown as purple and red spheres, respectively. Hydrogen bonds are depicted as black broken lines, and interactions between Mn²⁺ and O ligands are shown as purple lines. In the view selected, the interaction between the cation and Gly471 is obscured. The difference density omit map (black chicken wire) for ThDP calculated with (F_o-F_c) and α_c coefficients and contoured at the 3 σ level. F_o represents the observed structure factors, F_c denotes the calculated structure factors, and α_c indicates the phases calculated without any contributions from ThDP atoms.

Fig. 6

A network of interactions at the dimer interface between two cofactor-binding sites. A similar color scheme to Fig. 5 is used with the addition that C atoms of residues in subunit B are gray.

Fig. 7

A stereoview of the model for substrates binding to EcMenD. The post-decarboxylation covalent adduct of ThDP and α-ketoglutarate (ThDP*) is shown, and the reactive C atoms of this intermediate and isochorismate are 1.6 Å apart. This separation is indicated by a magenta broken line. The polypeptide main chain is shown as a gray ribbon and atomic positions are colored as in the previous figures except that the C atoms of the second substrate, isochorismate, are yellow. A calculated electrostatic surface potential (blue, positive; red, negative; white, neutral) showing the active-site cleft. The figure was produced using PyMOL with Adaptive Poisson–Boltzmann Solver with electrostatic potential isocontours set at +5 kT/e (blue) and −5 kT/e (red).

Fig. 8

A two-stage mechanism for catalysis by EcMenD. An asterisk marks the isochorismate C2, which is attacked by the carbanion intermediate.