Single‐Turnover Kinetics Reveal a Distinct Mode of Thiamine Diphosphate‐Dependent Catalysis in Vitamin K Biosynthesis

Abstract

MenD, or (1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase, uses a thiamine diphosphate (ThDP)-dependent tetrahedral Breslow intermediate rather than a canonical enamine for catalysis in the biosynthesis of vitamin K. By real-time monitoring of the cofactor chemical state with circular dichroism spectroscopy, we found that a new post-decarboxylation intermediate was formed from a multistep process that was rate limited by binding of the α-ketoglutarate substrate before it quickly relaxed to the characterized tetrahedral Breslow intermediate. In addition, the chemical steps leading to the reactive post-decarboxylation intermediates were not affected by the electrophilic substrate, isochorismate, whereas release of the product was found to limit the whole catalytic process. Moreover, these intermediates are likely kinetically stabilized owing to the low biological availability of isochorismate under physiological conditions, in contrast to the tight coupling of enamine formation with binding of the electrophilic acceptor in some other ThDP-dependent enzymes. Together with the unusual tetrahedral structure of the intermediates, these findings strongly support a new ThDP-dependent catalytic mode distinct from canonical enamine chemistry.

Chorismate- and isochorismate converting enzymes: versatile catalysts acting on an important metabolic node

“…106,107 In addition to mostly hydrophobic amino acids contributing to the active site, usually five basic amino acid residues provide a suitable chemical environment to promote 1,4-conjugate addition and stabilisation of the product. 104,105,[107][108][109] The most important residues that contribute to the process are: (i) a highly conserved glutamate activating ThDP, (ii) two conserved arginines that are suggested to be crucial for substrate binding and catalysis, and (iii) two polar amino acids (Gln and Ser) that finally enable product formation (Fig. 4I).…”

“…This is in contrast to other ThDP-dependent enzymes where the enamine form can be detected as well. [102][103][104][105][107][108][109] ThDP and the C2a-decarboxylated 2-oxoglutarate are proposed to be connected in a unique rigid conformation with a defined stereochemistry by two conserved arginine residues keeping…”

“…Unported Licence.intermediate I in the proper orientation for nucleophilic attack at isochorismate, which proceeds with formation of a novel C-C single bond, culminating in intermediate II 105,[107][108][109]. Finally, the interplay of two polar amino acids (Gln and Ser), the isochorismate moiety, and the C2a-hydroxyl group of intermediate II, as well as the aminopyrimidine ring of ThDP, in a complex hydrogen-bond network enables product formation.…”

Enzymatic Stetter Reaction: Computational Study of the Reaction Mechanism of MenD

“…17,18 It was suggested that this arose from the transfer of a proton from N4′ of ThDP to the C2α-carbanion form of Int2. 17,23 Scheme 4 shows that such a transfer would produce Int2-Td, that is, the cofactor would be in its N4′ imino (IP) form. We have evaluated this possibility and found the IP form to be ca.…”

“…22 This structure was later combined with stopped-flow and mutagenesis experiments to prompt the suggestion that MenD may operate in a novel catalytic mode, one quite distinct from the canonical enamine chemistry. 23 Despite the plethora of X-ray structures, mutagenesis, and kinetic investigations, the MenD reaction mechanism has not been fully established. A number of questions are still open, the most important of which relates to the catalytic competence of the post-decarboxylation tetrahedral species.…”