Characterisation of a solvent-tolerant haloarchaeal (R)-selective transaminase isolated from a Triassic period salt mine

Abstract

Transaminase enzymes (TAms) are becoming increasingly valuable in the chemist's toolbox as a biocatalytic route to chiral amines. Despite high profile successes, the lack of (R)-selective TAms and robustness under harsh industrial conditions continue to prove problematic. Herein, we report the isolation of the first haloarchaeal TAm (BC61-TAm) to be characterised for the purposes of pharmaceutical biocatalysis. BC61-TAm is an (R)-selective enzyme, cloned from an extremely halophilic archaeon, isolated from a Triassic period salt mine. Produced using a Haloferax volcanii-based expression model, the resulting protein displays a classic halophilic activity profile, as well as thermotolerance (optimum 50 °C) and organic solvent tolerance. Molecular modelling predicts the putative active site residues of haloarchaeal TAms, with molecular dynamics simulations providing insights on the basis of BC61-TAm's organic solvent tolerance. These results represent an exciting advance in the study of transaminases from extremophiles, providing a possible scaffold for future discovery of biocatalytic enzymes with robust properties.

Keywords: Archaea; Biocatalysis; Halophile; Organic solvent; Transaminase.

Fig. 1

SDS-PAGE of pre-purified clarified cell-free extract (lane 2) and BC61-TAm fraction, purified by IMAC (lane 4), alongside SeeBlue® Plus2 Protein standard (lane 1) and buffer used for protein elution (lane 3)

Fig. 2

Multiple alignment of amino acid sequences of BC61-TAm with a number of previously reported TAm sequences, including (S)-selective TAms from mesophilic bacteria (Cv-TAm, Vf-TAm, and Pp-TAm) and halophilic bacteria (Hs-TAm and Ad2-TAm), (R)-selective ω-TAms from bacteria and fungi (Arth-TAm and At-TAm respectively), and BCATs from bacteria (Ec-TAm) and haloarchaea (Hp-TAm and BC61-TAm)

Fig. 3

The effect of changing various parameters on the amination of α-KG using purified BC61-TAm and (R)-MBA as amino donor, with acetophenone formation measured at 240 nm. Plotted values are the mean of triplicate measurements, with error bars representing ± standard deviation. In each graph, conversion is reported relative to the highest value observed for each parameter. (Abbreviations shown in panel B: DMSO, dimethyl sulfoxide; THF, tetrahydrofuran; DMF, dimethylformamide; MeOH, methanol; iPrOH, isopropanol)

Fig. 4

a Molecular model of BC61-TAm constructed using I-TASSER server followed by energy minimization using Yasara, showing docked α-KG substrate (AKG) and cofactor (PMP). Models and docked complexes were viewed and manipulated using PyMol. b 2D ligand map showing docking and interacting residues between α-KG substrate (Akg1302), cofactor PMP (Pmp310) and BC61-TAm. Construction of residue interaction maps was carried out using LigPlus with subsequent visualisation in PyMol

Fig. 5

a Simulation showing Root Mean Square Fluctuation across individual residues of BC61-TAm in both water and 30% DMF. b Simulation predicting number of hydrogen bonds formed between water molecules and BC61-TAm protein in water (black line) and 30% DMF (red line). Number of H bonds between DMF and BC61-TAm in 30% DMF medium is also shown (green line). Solvation shell models simulating BC61-TAm in water (c) and 30% DMF (d). Water molecules were coloured according to a blue/white/red scheme corresponding to increasing density