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dc.contributor.authorMachado, Teresa F. G.
dc.contributor.authorPurg, Miha
dc.contributor.authorÅqvist, Johan
dc.contributor.authorda Silva, Rafael G.
dc.identifier.citationMachado , T F G , Purg , M , Åqvist , J & da Silva , R G 2021 , ' Transition states for psychrophilic and mesophilic (R)-3-hydroxybutyrate dehydrogenase-catalyzed hydride transfer at sub-zero temperatures ' , Biochemistry , vol. 60 , no. 27 , pp. 2186–2194 .
dc.identifier.otherRIS: urn:983598083E268C2A112CE1DD59EE034D
dc.identifier.otherORCID: /0000-0002-1308-8190/work/96817607
dc.descriptionThis work was supported by the Engineering and Physical Sciences Research Council (EPSRC) (Grant EP/L016419/1) via a CRITICAT Centre for Doctoral Training studentship to T.F.G.M., and by the Swedish Research Council and KAW Foundation grants to J.Å.en
dc.description.abstract(R)-3-Hydroxybutyrate dehydrogenase (HBDH) catalyzes the NADH-dependent reduction of 3-oxocarboxylates to (R)-3-hydroxycarboxylates. The active sites of a pair of cold- and warm-adapted HBDHs are identical except for a single residue, yet kinetics evaluated at −5, 0, and 5 °C show a much higher steady-state rate constant (kcat) for the cold-adapted than for the warm-adapted HBDH. Intriguingly, single-turnover rate constants (kSTO) are strikingly similar between the two orthologues. Psychrophilic HBDH primary deuterium kinetic isotope effects on kcat (Dkcat) and kSTO (DkSTO) decrease at lower temperatures, suggesting more efficient hydride transfer relative to other steps as the temperature decreases. However, mesophilic HBDH Dkcat and DkSTO are generally temperature-independent. The DkSTO data allowed calculation of intrinsic primary deuterium kinetic isotope effects. Intrinsic isotope effects of 4.2 and 3.9 for cold- and warm-adapted HBDH, respectively, at 5 °C, supported by quantum mechanics/molecular mechanics calculations, point to a late transition state for both orthologues. Conversely, intrinsic isotope effects of 5.7 and 3.1 for cold- and warm-adapted HBDH, respectively, at −5 °C indicate the transition state becomes nearly symmetric for the psychrophilic enzyme, but more asymmetric for the mesophilic enzyme. His-to-Asn and Asn-to-His mutations in the psychrophilic and mesophilic HBDH active sites, respectively, swap the single active-site position where these orthologues diverge. At 5 °C, the His-to-Asn mutation in psychrophilic HBDH decreases Dkcat to 3.1, suggesting a decrease in transition-state symmetry, while the His-to-Asn mutation in mesophilic HBDH increases Dkcat to 4.4, indicating an increase in transition-state symmetry. Hence, temperature adaptation and a single divergent active-site residue may influence transition-state geometry in HBDHs.
dc.subjectHydride transferen
dc.subjectRedox reactionsen
dc.subjectHydrogen isotopesen
dc.subjectQD Chemistryen
dc.titleTransition states for psychrophilic and mesophilic (R)-3-hydroxybutyrate dehydrogenase-catalyzed hydride transfer at sub-zero temperaturesen
dc.typeJournal articleen
dc.contributor.institutionUniversity of St Andrews. Biomedical Sciences Research Complexen
dc.contributor.institutionUniversity of St Andrews. School of Biologyen
dc.description.statusPeer revieweden

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