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Engineering of Thermostable ?-Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines.

ABSTRACT: The ?-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-?HAD), which catalyzes the NADP⁺ -dependent oxidation of ?-hydroxyacids, was engineered to accept imines as substrates. The catalytic activity of the proton-donor variant K189D was further increased by the introduction of two nonpolar flanking residues (N192?L, N193?L). Engineering the putative alternative proton donor (D258S) and the gate-keeping residue (F250?A) led to a switched substrate specificity as compared to the single and triple variants. The two most active Ta-?HAD variants were applied to biocatalytic asymmetric reductions of imines at elevated temperatures and enabled enhanced product formation at a reaction temperature of 50?°C.

SUBMITTER: Stockinger P

PROVIDER: S-EPMC7756219 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Engineering of Thermostable β-Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines.

Stockinger Peter P Schelle Luca L Schober Benedikt B Buchholz Patrick C F PCF Pleiss Jürgen J Nestl Bettina M BM

Chembiochem : a European journal of chemical biology 20200916 24

The β-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-βHAD), which catalyzes the NADP<sup>+</sup> -dependent oxidation of β-hydroxyacids, was engineered to accept imines as substrates. The catalytic activity of the proton-donor variant K189D was further increased by the introduction of two nonpolar flanking residues (N192 L, N193 L). Engineering the putative alternative proton donor (D258S) and the gate-keeping residue (F250 A) led to a switched substrate specificity as compared to the sin ...[more]

PMID: 32939899

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Project description:Ketone bodies, including acetoacetate, 3-hydroxybutyrate, and acetone, are produced in the liver of animals during glucose starvation. Enzymes for the metabolism of (R)-3-hydroxybutyrate have been extensively studied, but little is known about the metabolism of its enantiomer (S)-3-hydroxybutyrate. Here, we report the characterization of a novel pathway for the degradation of (S)-3-hydroxybutyrate in anaerobic bacteria. We identify and characterize a stereospecific (S)-3-hydroxylbutyrate dehydrogenase (3SHBDH) from Desulfotomaculum ruminis, which catalyzes the reversible NAD(P)H-dependent reduction of acetoacetate to form (S)-3-hydroxybutyrate. 3SHBDH also catalyzes oxidation of d-threonine (2R, 3S) and l-allo-threonine (2S, 3S), consistent with its specificity for β-(3S)-hydroxy acids. Isothermal calorimetry experiments support a sequential mechanism involving binding of NADH prior to (S)-3-hydroxybutyrate. Homologs of 3SHBDH are present in anaerobic fermenting and sulfite-reducing bacteria, and experiments with Clostridium pasteurianum showed that 3SHBDH, acetate CoA-transferase (YdiF), and (S)-3-hydroxybutyryl-CoA dehydrogenase (Hbd) are involved together in the degradation of (S)-3-hydroxybutyrate as a carbon and energy source for growth. (S)-3-hydroxybutyrate is a human metabolic marker and a chiral precursor for chemical synthesis, suggesting potential applications of 3SHBDH in diagnostics or the chemicals industry. IMPORTANCE (R)-3-hydroxybutyrate is well studied as a component of ketone bodies produced by the liver and of bacterial polyesters. However, the biochemistry of its enantiomer (S)-3-hydroxybutyrate is poorly understood. This study describes the identification and characterization of a stereospecific (S)-3-hydroxylbutyrate dehydrogenase and its function in a metabolic pathway for the degradation of (S)-3-hydroxybutyrate as a carbon and energy source in anaerobic bacteria. (S)-3-hydroxybutyrate is a mammalian metabolic marker and a precursor for chemical synthesis and bioplastics, suggesting potential applications of these enzymes in diagnostics and biotechnology.

Dataset Information

Engineering of Thermostable ?-Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines.

Publications

Engineering of Thermostable β-Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines.

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