Structure-based Mutational Studies of D-3-hydroxybutyrate Dehydrogenase for Substrate Recognition of Aliphatic Hydroxy Acids with a Variable Length of Carbon Chain

Lee, Hoe-Suk; Na, Jeong-Geol; Lee, Jinwon; Yeon, Young Joo

doi:10.1007/s12257-019-0135-1

Cited by 5 publications

(5 citation statements)

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“…Given the clustering of all HBDHs from the Asn145 group in the phylogenetic tree and the distinct catalytic effects of Asn145 and His150 mutations on Ab HBDH and Pa HBDH, respectively, it is possible to hypothesize that this loop region may underpin the differences in catalytic rate constants observed between Ab HBDH and Pa HBDH and that these differences perhaps extend to Asn-145-group and His150-group HBDHs in general. For instance, P. lemoignei and Alcaligenes faecalis HBDHs , belong to the His150 group and have catalytic rates of acetoacetate reduction much higher than that of Ab HBDH . Thus, the phylogenetic analysis could lay the foundation for future research into other HBDH orthologues from both groups to test this hypothesis and elucidate the exact role of the Gly149-Phe150-Gly152/Ala154-Ser155 loops.…”

Section: Results and Discussionmentioning

confidence: 99%

“… 8 Accordingly, recent publications highlight several ongoing HBDH engineering efforts to improve production of its native and non-native products via biocatalysis and synthetic biology. 9 − 11 …”

Section: Introductionmentioning

confidence: 99%

“…For instance, the native product of the HBDH reaction, ( R )-3-hydroxybutyrate, is a precursor in the synthesis of carbapenem antibiotics, and ( R )-3-hydroxybutyrate, ( R )-3-hydroxyvalerate, and ( R )-3-hydroxyhexanoate, all products of HBDH-catalyzed reduction of 3-oxocarboxylates, are the monomeric components of biodegradable polyesters . Accordingly, recent publications highlight several ongoing HBDH engineering efforts to improve production of its native and non-native products via biocatalysis and synthetic biology. − …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Results and Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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“…One approach to engineering enzymes is to introduce mutations at their active sites when the structures are available, in particular for the residues to interact with substrates (Yagonia et al, 2015;Lee et al, 2019). In addition, understanding the catalytic mechanism may provide useful insights into how a particular amino acid residue functions in enzymatic reactions.…”

Section: Engineering Of Coenzyme B 12 -Dependent Gdht Improvement In mentioning

confidence: 99%

Recent Progress in the Understanding and Engineering of Coenzyme B12-Dependent Glycerol Dehydratase

Nasır

Ashok

Shim

et al. 2020

Front. Bioeng. Biotechnol.

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Coenzyme B 12-dependent glycerol dehydratase (GDHt) catalyzes the dehydration reaction of glycerol in the presence of adenosylcobalamin to yield 3-hydroxypropanal (3-HPA), which can be converted biologically to versatile platform chemicals such as 1,3-propanediol and 3-hydroxypropionic acid. Owing to the increased demand for biofuels, developing biological processes based on glycerol, which is a byproduct of biodiesel production, has attracted considerable attention recently. In this review, we will provide updates on the current understanding of the catalytic mechanism and structure of coenzyme B 12-dependent GDHt, and then summarize the results of engineering attempts, with perspectives on future directions in its engineering.

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“…The products of the HBDH reaction, ( R )-3-hydroxycarboxylates, are chiral precursors in the synthesis of value-added compounds such as antibiotics and also form the monomeric units of biodegradable polyesters . Thus, recent attempts have been made to harness the catalytic properties of HBDH for the production of its native and non-native products via biocatalysis and synthetic biology. − …”

mentioning

confidence: 99%

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

et al. 2021

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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 steadystate rate constant (k cat ) for the cold-adapted than for the warmadapted HBDH. Intriguingly, single-turnover rate constants (k STO ) are strikingly similar between the two orthologues. Psychrophilic HBDH primary deuterium kinetic isotope effects on k cat ( D k cat ) and k STO ( D k STO ) decrease at lower temperatures, suggesting more efficient hydride transfer relative to other steps as the temperature decreases. However, mesophilic HBDH D k cat and D k STO are generally temperature-independent. The D k STO 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 D k cat to 3.1, suggesting a decrease in transition-state symmetry, while the Histo-Asn mutation in mesophilic HBDH increases D k cat 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.

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Structure-based Mutational Studies of D-3-hydroxybutyrate Dehydrogenase for Substrate Recognition of Aliphatic Hydroxy Acids with a Variable Length of Carbon Chain

Cited by 5 publications

References 28 publications

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

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

Recent Progress in the Understanding and Engineering of Coenzyme B12-Dependent Glycerol Dehydratase

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

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