Engineering of Thermostable β‐Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines

Abstract

The β-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-βHAD), which catalyzes the NADP +-dependent oxidation of βhydroxyacids, 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-βHAD variants were applied to biocatalytic asymmetric reductions of imines at elevated temperatures and enabled enhanced product formation at a reaction temperature of 50°C.

Engineering of a Reductive Aminase to Enable the Synthesis of a Key Intermediate to a CDK 2/4/6 Inhibitor

“…The IR007 active site is characterized by residue Y165 that projects down from the interdomain helix into the active site in close proximity to the nicotinamide ring of the cofactor. Equivalent tyrosine residues have been implicated in catalysis in IREDs, especially those for which the selectivity for the model substrate 2-methylpyrroline is ( S ). ,, Mutation of these tyrosine residues to F or A can render the enzymes inactive. ,, The helix containing Y165 also contributes L169, F172, and L176 to the left-hand wall of the active site, as seen in Figure b, together providing a largely hydrophobic environment for substrate binding. The front of the active site, which is formed in the closed conformation of the enzyme, is dominated by two methionine residues from the “B” chain, M232 and M233, the side chains of which project toward the nicotinamide ring.…”

“…Equivalent tyrosine residues have been implicated in catalysis in IREDs, especially those for which the selectivity for the model substrate 2-methylpyrroline is (S). 19,21,22 Mutation of these tyrosine residues to F or A can render the enzymes inactive. 19,21,22 The helix containing Y165 also contributes L169, F172, and L176 to the left-hand wall of the active site, as seen in Figure 6b, together providing a largely hydrophobic environment for substrate binding.…”

“…19,21,22 Mutation of these tyrosine residues to F or A can render the enzymes inactive. 19,21,22 The helix containing Y165 also contributes L169, F172, and L176 to the left-hand wall of the active site, as seen in Figure 6b, together providing a largely hydrophobic environment for substrate binding. The front of the active site, which is formed in the closed conformation of the enzyme, is dominated by two methionine residues from the "B" chain, M232 and M233, the side chains of which project toward the nicotinamide ring.…”

Redox Out of the Box: Catalytic Versatility Across NAD(P)H‐Dependent Oxidoreductases

“…The approach was transferable to other β-HAD scaffolds (Scheme 5B). [43] In addition to IREDs, the reduction of C=O bonds was also observed with RedAms. With α-fluoroacetophenone derivatives, and under reductive amination conditions, the conversion was dependent on the type of amine present and the degree of fluorination, matching the electrophilic strength of the substrate.…”

Redox mal anders: katalytische Vielseitigkeit bei NAD(P)H‐abhängigen Oxidoreduktasen

“…Diese Herangehensweise ist auch auf andere β-HADs als Grundgerüste übertragbar (Abbildung 5B). [43] Neben IREDs wurde die Reduktion von C=O-Bindungen auch mit RedAms beobachtet. Mit α-Fluoracetophenon-Derivaten und unter Bedingungen der reduktiven Aminierung hing der Umsatz von der Art des eingesetzten Amins…”