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

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%.

Transaminases for industrial biocatalysis: novel enzyme discovery

“…Discovery of R-selective wild-type TAms using activityguided approaches has resulted in limited success, with only a handful of such enzymes uncovered in this way. Using Ramines as the sole nitrogen source in enrichment media, Pavkov-Keller and co-workers discovered and characterized R-selective TAms from Curtobacterium pusillum and Microbacterium ginsengisoli (Pavkov-Keller et al 2016). These enzymes were not easily classified along with previously known TAms, given their structural similarity to D-amino acid TAms and branched-chain amino acid TAms, combined with their ability to accept amines as substrates.…”

Improvement in the Thermostability of a β‐Amino Acid Converting ω‐Transaminase by Using FoldX

“…The properties to accept non‐α‐keto acids make ω‐TAs desirable enzymes, as this enables the synthesis of a wide spectrum of chiral amines and amino acids from prochiral substrates with predefined stereochemistry depending on the use of either an R ‐ or S ‐selective enzyme . This difference in enantiopreference is accompanied by a different protein fold, known as fold type IV ( R ) and I ( S ) of the PLP‐dependent enzymes . The acceptor molecules can vary from classical α‐keto acids to β‐, γ‐, or ϵ‐keto acids, and even ketones and aldehydes are converted by this class of enzymes .…”

Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism

“…studied fold type I aminotransferases are L-aspartate ATs [17], branched L-amino acid ATs, b-amino acid ATs [18,19] and the x-aminotransferases (xATs) from Vibrio fluvialis, Chromobacterium violaceum, Paracoccus denitrificans, and Ochrobactrum anthropi [20][21][22][23][24]. Fold type IV enzymes include D-amino acid ATs and (R)-amine selective xATs [11][12][13][14]25,26]. Aminotransferases have also been classified in subgroups or classes, mainly based on substrate structural features, leading to a classification that partially agrees with grouping in fold types [13,15].…”