Dissecting the mechanism of (R)-3-hydroxybutyrate dehydrogenase by kinetic isotope effects, protein crystallography, and computational chemistry

DOI: 10.1021/acscatal.0c04736

Authors: Teresa F. G. Machado (University of St Andrews) , Miha Purg (Uppsala University) , Stephen A. Mcmahon (University of St Andrews) , Benjamin J. Read (University of St Andrews) , Verena Oehler (University of St Andrews) , Johan Åqvist (Uppsala University) , Tracey M. Gloster (University of St Andrews) , Rafael G. Da Silva (University of St Andrews)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: December 2020

Abstract: The enzyme (R)-3-hydroxybutyrate dehydrogenase (HBDH) catalyzes the enantioselective reduction of 3-oxocarboxylates to (R)-3-hydroxycarboxylates, the monomeric precursors of biodegradable polyesters. Despite its application in asymmetric reduction, which prompted several engineering attempts of this enzyme, the order of chemical events in the active site, their contributions to limit the reaction rate, and interactions between the enzyme and non-native 3-oxocarboxylates have not been explored. Here, a combination of kinetic isotope effects, protein crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations were employed to dissect the HBDH mechanism. Initial velocity patterns and primary deuterium kinetic isotope effects establish a steady-state ordered kinetic mechanism for acetoacetate reduction by a psychrophilic and a mesophilic HBDH, where hydride transfer is not rate limiting. Primary deuterium kinetic isotope effects on the reduction of 3-oxovalerate indicate that hydride transfer becomes more rate limiting with this non-native substrate. Solvent and multiple deuterium kinetic isotope effects suggest hydride and proton transfers occur in the same transition state. Crystal structures were solved for both enzymes complexed to NAD+:acetoacetate and NAD+: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.

Journal Keywords: 3-hydroxybutyrate dehydrogenase; isotope effects; hydride transfer; quantum mechanics/molecular mechanics; enzyme catalysis

Diamond Keywords: Enzyme

Subject Areas: Chemistry, Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography

Added On: 14/12/2020 10:17

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Organic Chemistry Biophysics Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)