How Chorismatases Regulate Distinct Reaction Channels in a Single Conserved Active Pocket: Mechanistic Analysis with QM/MM (ONIOM) Investigations

The FkbO and Hyg5 subfamilies of chorismatases share the same active-site architectures, but perform distinct reaction mechanisms, that is, FkbO employs a hydrolysis reaction whereas Hyg5 proceeds through an intramolecular mechanism. Despite extensive research efforts, the detailed mechanism of the product selectivity in chorismatases need to be further unmasked. In this study, the effects of the A/G residue group (A244^FkbO/G240^Hyg5) and the V/Q residue group (V209^FkbO/Q201^Hyg5) on the catalytic mechanisms are investigated by employing molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations of the two wild-type models (FkbO/CHO and Hyg5/CHO; CHO=chorismate) and four mutants models (A244G-FkbO/CHO and G240A-Hyg5/CHO; V209Q-FkbO/CHO and Q201V-Hyg5/CHO). Our results showed that the A/G residue group mentioned by previous works would cause changes in the binding states of the substrate and the orientation of the catalytic glutamate, but only these changes affect the product selectivity in chorismatases limitedly. Interestingly, the distal V/Q residue group, which determines the internal water self-regulating ability at the active site, has significant impact on the selectivity of the catalytic mechanisms. The V/Q residue group is suggested to be an important factor to control the catalytic activities in chorismatases. The results are consistent with biochemical and structural experiments, providing novel insight into the mechanism of product selectivity in chorismatases.