Dissecting the Mechanism of (R)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry

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.

Transition States for Psychrophilic and Mesophilic (R)-3-Hydroxybutyrate Dehydrogenase-Catalyzed Hydride Transfer at Sub-zero Temperatures

“…The data displayed in Figure were utilized to calculate D k cat (Table ). For acetoacetate reduction, the D k cat of Pa HBDH is modest and temperature-independent, while the D k cat of Ab HBDH modestly increases from 2.2 at 5 °C, the same value found at 10 °C, to 2.8 at −5 °C. This points to chemistry remaining faster than other steps at low temperatures for Pa HBDH but becoming more rate-limiting for Ab HBDH at lower temperatures.…”

“…Deuterium-labeled d -glucose (1-D, 98%) was purchased from Cambridge Isotope Laboratories, and 3-oxovalerate was purchased from Carbosynth. NADH and 4 S -[4- 2 H]­NADH (NADD) were synthesized and purified as previously described , …”

“…Ground-state and transition-state structures for Pa HBDH- and Ab HBDH-catalyzed reduction of 3-oxovalerate were calculated via QM/MM at the M06-2X/ma-def2-SVP level of theory using ORCA 4.2.1 exactly as previously reported . Briefly, employing a micro/macro-iteration strategy (micro, M02-2X/ma-def2-SVP//CHARMM; macro, GFN-XTB2//CHARMM) we further optimized and characterized the reactant, product, and transition-state stationary points.…”

“…We have also used deuterium kinetic isotope effects, protein crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations to demonstrate that Pa HBDH and Ab HBDH catalysis proceeds via concerted hydride and proton transfers . Additionally, both enzymes show a highly conserved active site with a single diverging residue: Pa HBDH His150 is equivalent to Asn145 in Ab HBDH.…”

“…Additionally, both enzymes show a highly conserved active site with a single diverging residue: Pa HBDH His150 is equivalent to Asn145 in Ab HBDH. The mutant Asn145His- Ab HBDH has an only modestly decreased rate in comparison with that of the wild-type (WT) enzyme, but His150Asn- Pa HBDH displays drastically slowed catalysis in comparison with that of WT- Pa HBDH . Nevertheless, these kinetic studies were carried out at 10 °C, a temperature significantly higher than the range from −10 to −5 °C of the natural environment of P. arcticus …”