Kinetics and structure of cold- and warm-adapted (𝘙�)-3-hydroxybutyrate dehydrogenase

Abstract

Temperature is a critical variable influencing the rate of chemical reactions and plays a key role in the evolution of enzymes. Considering that life likely arose in a hot environment and primordial enzymes had a thermophilic nature, most modern enzymes had to evolve to be efficient at lower temperatures. Psychrophilic enzymes offer opportunities in biocatalysis and biotechnology since they exhibit high catalytic rates, higher than their mesophilic counterparts, even when little kinetic energy is available in the system. The work documented in this thesis focuses on a side-by-side comparison between a psychrophilic and a mesophilic (𝘙)-3- hydroxybutyrate dehydrogenase (HBDH) in order to understand the differences in mechanism and structure between the two enzyme orthologues. HBDHs catalyse the NADH-dependent reduction of acetoacetate to (𝘙)-3-hydroxybutyrate, and are particularly interesting ketoreductases for biocatalysis, as they generate (𝘙)-3-hydroxycarboxylates, which are useful chiral precursors in the synthesis of carbapenem antibiotics and the monomeric components of biodegradable polyesters. Whilst probing the rate-limiting steps of the psychrohilic and mesophilic HBDHs this work identified that linear Eyring plots may conceal a change in rate- limiting step within enzyme-catalysed reactions, previously overlooked. Both enzymes display a concerted chemical mechanism, with the hydride- and proton-transfers happening within the same transition-state; a conserved tyrosine acts as the proton donor. At temperatures below zero, the chemical step is more optimised for the psychrophilic enzyme, whereas for the mesophilic enzyme chemistry is fully rate-limiting. At -5°C, the two enzymes exhibit very distinct transition states, while the psychrophilic enzyme exhibits a near-symmetric hydride transfer transition state, the mesophilic proceeds via an early or a late transition state. The active site of the two enzymes differs only in one single residue, that significantly effects catalysis. The psychrophilic enzyme is active with five unnatural substrates, however attempts to rationally engineer its activity were unsuccessful. Overall, this work provides the most detailed characterization of the chemical step of an HBDH-catalysed reaction reported to date.