Asymmetric Synthesis of Optically Pure Aliphatic Amines with an Engineered Robust ω-Transaminase

Abstract

The production of chiral amines by transaminase-catalyzed amination of ketones is an important application of biocatalysis in synthetic chemistry. It requires transaminases that show high enantioselectivity in asymmetric conversion of the ketone precursors. A robust derivative of ω-transaminase from Pseudomonasjessenii (PjTA-R6) that naturally acts on aliphatic substrates was constructed previously by our group. Here, we explore the catalytic potential of this thermostable enzyme for the synthesis of optically pure aliphatic amines and compare it to the well-studied transaminases from Vibrio fluvialis (VfTA) and Chromobacterium violaceum (CvTA). The product yields indicated improved performance of PjTA-R6 over the other transaminases, and in most cases, the optical purity of the produced amine was above 99% enantiomeric excess (e.e.). Structural analysis revealed that the substrate binding poses were influenced and restricted by the switching arginine and that this accounted for differences in substrate specificities. Rosetta docking calculations with external aldimine structures showed a correlation between docking scores and synthetic yields. The results show that PjTA-R6 is a promising biocatalyst for the asymmetric synthesis of aliphatic amines with a product spectrum that can be explained by its structural features.

Computational Prediction of ω-Transaminase Specificity by a Combination of Docking and Molecular Dynamics Simulations

“…At least for the presented datasets, this correlation shows that modeling the amino donor (typically L-alanine or isopropylamine) is not necessary for the reactivity prediction task, in contrast to the approach of Seo et al, 49 where differential binding energies were considered for the substrates of both half-transamination reactions. We recently also reported the correlation between interface energies and experimental conversion in other Vf-TA and Pj-TA datasets 73 and used the Rosetta interface energy as the target function for the computational engineering of the Pj-TA substrate scope. 68 The protocol allowed the rapid design of Pj-TA variants accepting sterically hindered substrates.…”

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

“… 19 Thus, we hypothesized that mutations that increase the stability of the external aldimine complex would have an observable effect on product formation. 37 An added advantage of using a ligand covalently bound to a rigid cofactor is that the position of the reactive atom (C α ) is known beforehand, which reduces both the docking search space and the number of variants that need to be screened to find a good mutant. Although the methodology described in this paper shows promising results for engineering the ω-TA activity, special attention must be paid to the identity of the substrates.…”

“…We chose the external aldimine as the ligand for the docking calculations because its conversion to the geminal diamine intermediate in the amine synthesis half-reaction involves a high-energy transition state . Thus, we hypothesized that mutations that increase the stability of the external aldimine complex would have an observable effect on product formation . An added advantage of using a ligand covalently bound to a rigid cofactor is that the position of the reactive atom (C α ) is known beforehand, which reduces both the docking search space and the number of variants that need to be screened to find a good mutant.…”

“…The ability of the Rosetta-generated variants to accommodate the intended intermediates was evaluated by the Rosetta interface energy (docking score). 37 The results showed that there is a correlation between the interface energy and the experimental yield, which can aid in narrowing down the search in future mutagenesis efforts. Experimental verification showed that the majority (97%) of the mutants gave better reactivity than the initial scaffold, which was not able to produce any of the six targeted amines.…”

“…The search space included multiple residues around the active site (Table S1), and the resulting small mutant libraries selected for experimental verification contained mutations at eight positions of Pj TA-R6 (Scheme ). The ability of the Rosetta-generated variants to accommodate the intended intermediates was evaluated by the Rosetta interface energy (docking score) . The results showed that there is a correlation between the interface energy and the experimental yield, which can aid in narrowing down the search in future mutagenesis efforts.…”