Discovery and structural characterisation of new fold type IV-transaminases exemplify the diversity of this enzyme fold.

Tea Pavkov-Keller,Gernot A Strohmeier,Martin Schürmann,Wilco Peeters,Kerstin Steiner,Helmut Schwab,Karl Gruber,Natascha Smeets,Matthias Diepold

doi:10.1038/srep38183

Abstract

Transaminases are useful biocatalysts for the production of amino acids and chiral amines as intermediates for a broad range of drugs and fine chemicals. Here, we describe the discovery and characterisation of new transaminases from microorganisms which were enriched in selective media containing (R)-amines as sole nitrogen source. While most of the candidate proteins were clearly assigned to known subgroups of the fold IV family of PLP-dependent enzymes by sequence analysis and characterisation of their substrate specificity, some of them did not fit to any of these groups. The structure of one of these enzymes from Curtobacterium pusillum, which can convert d-amino acids and various (R)-amines with high enantioselectivity, was solved at a resolution of 2.4 Å. It shows significant differences especially in the active site compared to other transaminases of the fold IV family and thus indicates the existence of a new subgroup within this family. Although the discovered transaminases were not able to convert ketones in a reasonable time frame, overall, the enrichment-based approach was successful, as we identified two amine transaminases, which convert (R)-amines with high enantioselectivity, and can be used for a kinetic resolution of 1-phenylethylamine and analogues to obtain the (S)-amines with e.e.s >99%.

Highlights

Biocatalytic processes have become increasingly important in chemical industry[1,2,3]
Comparison of the sequences to those of fold IV transaminases with known structures (Fig. 1) revealed that AspTA1 and RaqTA1 are most similar to D-amine transaminases (ATAs), CpuTA2, MgiTA2 and RaqTA2 to branched chain aminotransferases (BCATs) and RaqTA3 to amino-4-deoxychorismate lyases (ADCL)
In addition to α-transaminases, which catalyse the conversion of α-amino acids to α-keto acids and vice versa, and βand ω-transaminases, which transfer amino groups which are more distant from the carboxylic group, the group of amine transaminases was discovered and defined

Summary

Introduction

Biocatalytic processes have become increasingly important in chemical industry[1,2,3]. Transaminases catalyse the transfer of an amino group from an amino donor to a carbonyl acceptor with pyridoxal 5′-phosphate (PLP) as cofactor They belong to fold types I and IV of PLP-dependent enzymes. All (R)-amine transaminases described in the literature so far are members of the fold type IV PLP-dependent enzyme family[13,14,15,16,17,18]. This family includes d-amino acid aminotransferases, L-branched chain aminotransferases and 4-amino-4-deoxychorismate lyases (ADCL)[19]. We report the discovery of a new class of fold IV-transaminases, which was discovered during our quest for novel (R)-selective transaminases using an enrichment-based approach

Methods

Results

Conclusion