Dissecting the Mechanism of (<i>R</i>)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry
Teresa F. G. Machado, Miha Purg, S.A. McMahon, Benjamin J. Read, Verena Oehler, Johan Åqvist, T.M. Gloster, Rafael G. da Silva
Abstract
:3-oxovalerate, illustrating the structural basis for the stereochemistry of the 3-hydroxycarboxylate products. QM/MM calculations using the crystal structures as a starting point predicted a higher activation energy for 3-oxovalerate reduction catalyzed by the mesophilic HBDH, in agreement with the higher reaction rate observed experimentally for the psychrophilic orthologue. Both transition states show concerted, albeit not synchronous, proton and hydride transfers to 3-oxovalerate. Setting the MM partial charges to zero results in identical reaction activation energies with both orthologues, suggesting the difference in activation energy between the reactions catalyzed by cold- and warm-adapted HBDHs arises from differential electrostatic stabilization of the transition state. Mutagenesis and phylogenetic analysis reveal the catalytic importance of His150 and Asn145 in the respective orthologues.