Rational Design of a (S)-Selective-Transaminase for Asymmetric Synthesis of (1S)-1-(1,1′-biphenyl-2-yl)ethanamine

Abstract

Amine transaminases offer an environmentally sustainable synthesis route for the production of pure chiral amines. However, their catalytic efficiency toward bulky ketone substrates is greatly limited by steric hindrance and therefore presents a great challenge for industrial synthetic applications. We hereby report an example of rational transaminase enzyme design to help alleviate these challenges. Starting from the Vibrio fluvialis amine transaminase that has no detectable catalytic activity toward the bulky aromatic ketone 2-acetylbiphenyl, we employed a rational design strategy combining in silico and in vitro studies to engineer the transaminase enzyme with a minimal number of mutations, achieving an high catalytic activity and high enantioselectivity. We found that, by introducing two mutations W57G/R415A, detectable enzyme activity was achieved. The rationally designed variant, W57F/R88H/V153S/K163F/I259M/R415A/V422A, showed an improvement in reaction rate by more than 1716-fold toward the bulky ketone under study, producing the corresponding enantiomeric pure (S)-amine (enantiomeric excess (ee) value of >99%).

Isopropylamine as Amine Donor in Transaminase‐Catalyzed Reactions: Better Acceptance through Reaction and Enzyme Engineering

“…In contrast to ω‐TA, which accept carbonylic substrates with a distal carboxylate group, ATA tolerate substrates without a carboxyl moiety and therefore a wide range of ketones and aldehydes. Hence, in the last decade ATA became very attractive targets for enzyme engineering representing an environmentally benign alternative for the chemical transition metal‐catalyzed amine synthesis in pharmaceutical and agrochemical industries . For instance, the production of imagabaline, ( S )‐rivastigmine, or ( S )‐ivabradine has been realized on larger scale using ATA.…”

“…Additionally, in terms of shifting the equilibrium, acetone can be easily removed from the reaction solution . In general, it was reported in many cases that an excess of IPA needs to be applied to drive the transamination of various substrates to the desired product side,,,, when enzyme engineering did not lead to a better IPA acceptance, as mentioned above. In particular, different reaction conditions were reported such as varying donor‐acceptor‐ratios from 1.5‐fold over 40‐fold to 200‐fold and a pH range from 7.3 to 9.5 …”

Transaminases for industrial biocatalysis: novel enzyme discovery

“…The number of studies involving protein engineering of TAms has continued to increase in recent years. Using an in silico design approach, seven mutations to the wellcharacterized Vf-TAm resulted in a 1716-fold increase in reaction rate towards the bulky ketone substrate, 2-acetylbiphenyl (Dourado et al 2016). This focused mainly on enlarging the large binding pocket and increasing hydrophobicity at key residues in order to improve specificity and binding.…”

Amine Transaminase Engineering for Spatially Bulky Substrate Acceptance

“…[5] In another recent example VF-ATA was engineered to catalyze the synthesis of (S)-1-(1,1'-biphenyl-2-yl)ethanamine. [6] The ATAf rom Ruegeria sp. TM1040 [7] (PDB ID:3FCR;Rsp-ATA) differs significantly from VF-ATA (~33 %s equence identity).…”