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

Abstract

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.

Expanding the Toolbox of R‐Selective Amine Transaminases by Identification and Characterization of New Members

“…Instead, RxH is an important characteristic of d ‐ATAs. Together with Y36, these amino acids form the carboxylate trap and coordinate the α‐carboxyl group of a substrate in the O‐pocket thereby determining the pro‐ d ‐position of keto acids [8,13,21] . In ( R )‐ATAs, the O‐pocket is responsible for the dual substrate recognition.…”

“…Thus, the question arose if they still code for putative ( R )‐ATAs. In this context, a recent publication from the Bornscheuer group became interesting [21] . They enabled ( R )‐ATA activity in a d ‐ATA from Bacillus subtilis .…”

Direct 1,3-butadiene biosynthesis in Escherichia coli via a tailored ferulic acid decarboxylase mutant

“…With the evolution of computer simulation technology, many researchers have investigated enzyme developments based on the calculated parameter change of substrate-docked enzyme modeling before and after in silico mutation. 39,40,47,48,49,50,51,52 A good design in the substrate-binding site for improving enzymatic function can be applied to other enzymes as well. In this study, we focused on ccMA as a precursor compound of 1,3-butadiene.…”

Identification, Characterization and Site-Saturation Mutagenesis of a Thermostable ω-Transaminase From Chloroflexi Bacterium

“…The amino acid residues Y190, Q192, and G288 were selected for site-saturation mutagenesis on the basis of previous results and modeling analysis [19]. The site saturation mutagenesis was designed by NDT codon design.…”

“…In order to improve the performance of enzymes, protein engineering has usually been adopted to conduct enzyme design and optimization. A (R)-selective ω-TA has been created by bioinformatic analysis combined with computational redesign of the D-amino acid aminotransferase, exhibiting a specific activity close to those of natural (R)-selective ω-TAs [19].On the basis of a fluorescence-based screening system, a KnowVolution campaign has been carried out to optimize a (R)-selective ω-TA from Mycobacterium vanbaalenii and the best resulting mutant showed specific activity to acetonaphthone more than 100 times higher than parental enzyme [20]. In another study, a (R)-selective ω-TA from A. cumminsii ZJUT212 has been modified using a semi-rational protein design, and a mutant has been screened to produce sitagliptin intermediate on a kilogram scale with >99 % e.e.…”