MenD, or 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate (SEPHCHC) synthase, is involved in menaquinone (Vitamin K2) biosynthesis in Escherichia coli. It catalyzes a thiamin diphosphate (ThDP) - and Mg²⁺-dependent reaction of isochorismate with α-ketoglutarate to form SEPHCHC and to release CO₂ through a unique tetrahedral intermediate (11). The tetrahedral intermediate shows distinct conformation which has never been observed in previous research and indicates a novel mode of ThDP-dependent catalysis. To gain insights into this new catalytic mode, both steady-state kinetics and rapid kinetics were investigated for E. coli MenD. Real-time monitoring of the cofactor structure with circular dichroism spectroscopy showed that a new post-decarboxylation intermediat...[ Read more ]

MenD, or 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate (SEPHCHC) synthase, is involved in menaquinone (Vitamin K2) biosynthesis in Escherichia coli. It catalyzes a thiamin diphosphate (ThDP) - and Mg²⁺-dependent reaction of isochorismate with α-ketoglutarate to form SEPHCHC and to release CO₂ through a unique tetrahedral intermediate (11). The tetrahedral intermediate shows distinct conformation which has never been observed in previous research and indicates a novel mode of ThDP-dependent catalysis. To gain insights into this new catalytic mode, both steady-state kinetics and rapid kinetics were investigated for E. coli MenD. Real-time monitoring of the cofactor structure with circular dichroism spectroscopy showed that a new post-decarboxylation intermediate is formed from a multiple-step process rate-limited by binding of the α-ketoglutarate substrate before being quickly converted to the characterized tetrahedral intermediate. To understand the roles of two highly conserved arginine residues (R395 and R413) in this process, mutant proteins R395K, R395A, R413K, and R413A were overexpressed, purified and characterized like the wild-type enzyme and their structures in complex with the reaction intermediate were solved by protein crystallography. Both kinetic and structural results show that, when the interaction between the C2-succinyl group of the intermediate and two conserved arginine residues is moderately weakened by amino acid substitutions, the resulting proteins are in lower catalytic efficiency without significant changes of the intermediate conformation. However, when the interaction is significantly weakened in the R413A mutant, the C2-succinyl group in the intermediate flips 180° to an alternative site with a total activity loss. Surprisingly, the residues in the alternative site are also highly conserved and a computational stimulation result indicates that the α-ketoglutarate analog can bind the alternative site tightly when native binding pocket was weakened. Finding of this non-native site not only sheds new lights on the new ThDP-dependent catalytic mechanism but also provides an opportunity for design of substrate analogs or other inhibitors to develop new antibacterial agents against the essential enzyme in vitamin K biosynthesis. On the other hand，we are characterizing another ThDP-dependent enzyme involved in prodigiosin biosynthesis, namely PigD, that catalyzes a similar Stetter reaction like MenD. Using similar kinetic and structural methods, we aim to show that the new ThDP-dependent catalytic mode is not limited to MenD but adopted generally by enzymes catalyzing a similar reaction.