Creation of (R)‑Amine Transaminase Activity within an α‑Amino Acid Transaminase Scaffold

• Published in: ACS chemical biology Vol. 15; no. 2; pp. 416 - 424
• Main Authors: Voss, Moritz, Xiang, Chao, Esque, Jérémy, Nobili, Alberto, Menke, Marian J, André, Isabelle, Höhne, Matthias, Bornscheuer, Uwe T
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.02.2020
• Subjects: Bacillus subtilis - enzymology; Biochemistry, Molecular Biology; Escherichia coli - enzymology; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Life Sciences; Mutagenesis, Site-Directed; Mutation; Phenethylamines - chemistry; Phenethylamines - metabolism; Protein Binding; Stereoisomerism; Substrate Specificity; Transaminases - genetics; Transaminases - metabolism
• ISSN: 1554-8929; 1554-8937; 1554-8937
• DOI: 10.1021/acschembio.9b00888

The enzymatic transamination of ketones into (R)-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 (R)-selective amine transaminases [(R)-ATA] in biocatalysis, little is known about the actual in vivo role and how these enzymes have evolved from the ubiquitous α-amino acid transaminases (α-AATs). Here, we show the successful introduction of an (R)-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 (R)-phenylethylamine and pyruvate to acetophenone and d-alanine. This value is similar to those of natural (R)-ATAs, which typically are in the range of 250 milliunits mg–1. These results demonstrate that (R)-ATAs can evolve from α-AAT as shown here for the DATA scaffold.