Engineering the Active Site of an (S)‐Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines

Abstract

A protein engineering approach for expanding the substrate scope of the (S)‐selective Chromobacterium violaceum amine transaminase is presented. Amino acid residues in the small binding pocket of the active site were targeted in order to increase the pocket size for acceptance of primary amines bearing two bulky groups. A highly sensitive fluorescence assay was then used to evaluate the generated enzyme variants for their activity towards propyl‐ and benzyl‐substituted screening substrates. The best variant, L59A/F88A, was successfully applied in the kinetic resolution of 1,2‐diphenylethylamine using different conditions and substrate loadings. The variant L59A/F88A generated enantiomerically pure (R)‐1,2‐diphenylethylamine with ee>99% under all tested conditions. The variant also holds great promise for synthesis of hydrophobic compounds as it shows optimum activity when 20‐30% (v/v) DMSO is applied as cosolvent. The variant L59A/F88A provides a great addition to the available catalyst toolbox for synthesis of chiral amines, as it is the first published (S)‐selective amine transaminase showing activity towards benzyl‐substituted primary amines.

ω-Transaminase-Mediated Asymmetric Synthesis of (S)-1-(4-Trifluoromethylphenyl)Ethylamine

“…The TR 8 activity decreased by 49% in the presence of 50% (v/v) DMSO. Similar stability patterns have been observed using other ω-TAs during the production of chiral amines [45,46]. A concentration of 25% (v/v) DMSO was thus selected for subsequent experiments for bioprocess development.…”

Computational Redesign of an ω-Transaminase from Pseudomonas jessenii for Asymmetric Synthesis of Enantiopure Bulky Amines

“…Similarly, the best variant of CvTA possessed the corresponding mutation F88L and exhibited 99% conversion in the synthesis of 1b. Other small residues that improved activity in the synthesis of bulky amines when replacing Phe86 are Ala 11,18,60 and Val. 15 The computational approach presented in this work avoids multiple design iterations and instead uses the Rosetta energy function in a single dock-and-design step.…”

“… 10 This approach of rational design based on structural analysis was employed to redesign an ω-TA from Chromobacterium violaceum ( Cv TA) (PDB 4A6T) to obtain derivatives that are more suitable for the kinetic resolution of 1,2-diphenylethylamine. 11 The active site architecture was investigated, and two positions in the small binding pocket were identified as potential targets for mutagenesis. A double mutant with 30-fold higher activity than the wild-type enzyme was identified.…”

“…Examination of crystal structures or homology models of ω-TAs can be used to identify target sites for mutagenesis and reduce the amount of screening required for finding desired variants . This approach of rational design based on structural analysis was employed to redesign an ω-TA from Chromobacterium violaceum ( Cv TA) (PDB 4A6T) to obtain derivatives that are more suitable for the kinetic resolution of 1,2-diphenylethylamine . The active site architecture was investigated, and two positions in the small binding pocket were identified as potential targets for mutagenesis.…”

Biocatalytic routes to anti-viral agents and their synthetic intermediates

“…Although yet a pipe dream, recent protein engineering initiatives have already shown that enzymatic transamination of similarly bulky substrates is possible. 296,462 Coupling of biocatalytically generated amino acid (analogues) through enzymatic amide bond formation would mean yet another advancement in the sustainable nature of biocatalytic anti-viral drug production. Currently, coupling is generally Chart 13 Structures of cobicistat with its morpholine-based amino acid highlighted in blue (A) and (1R,2S)-1-aminocyclopropanecarboxylic acid (ACCA) derivatives (B).…”