Creation of (<i>R</i>)-Amine Transaminase Activity within an α-Amino Acid Transaminase Scaffold

Abstract

The enzymatic transamination of ketones into (<i>R</i>)-amines represents an important route for accessing a range of pharmaceuticals or building blocks. Although many publications have dealt with enzyme discovery, protein engineering, and the application of (<i>R</i>)-selective amine transaminases [(<i>R</i>)-ATA] in biocatalysis, little is known about the actual <i>in vivo</i> role and how these enzymes have evolved from the ubiquitous α-amino acid transaminases (α-AATs). Here, we show the successful introduction of an (<i>R</i>)-transaminase activity in an α-amino acid aminotransferase with one to six amino acid substitutions in the enzyme's active site. Bioinformatic analysis combined with computational redesign of the d-amino acid aminotransferase (DATA) led to the identification of a sextuple variant having a specific activity of 326 milliunits mg^-1 in the conversion of (<i>R</i>)-phenylethylamine and pyruvate to acetophenone and d-alanine. This value is similar to those of natural (<i>R</i>)-ATAs, which typically are in the range of 250 milliunits mg^-1. These results demonstrate that (<i>R</i>)-ATAs can evolve from α-AAT as shown here for the DATA scaffold.