Identification and Characterization of D-Amino Acid Aminotransferase Mutants Using Oxidase-coupled Assays

Abstract

D-amino acid aminotransferase (DAAT) is a pyridoxal-dependent enzyme that catalyzes the formation of a variety of D-amino acids, making it an attractive biocatalyst for the production of enantiopure D-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity towards non-native substrates are desired. In this work, we first developed a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of D-amino acid oxidase (DAAO). In this assay, the D-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and o-dianisidine. Using this assay, we identified three DAAT mutants (V33D, V33G and V33Y) displaying altered substrate specificity via the screening of cell lysates in 96-well plates. More specifically, the V33G and V33Y mutants display a ≈3-fold increase in kcat/KM for the non-native acceptor phenylpyruvate over wild type. Furthermore, we developed a coupled enzyme assay for the activity of DAAT with almost any D-amino acid using D-aspartate oxidase (DDO) as a coupling enzyme. Using this assay, we measured the specific activity of wild-type DAAT and the V33D, V33G, and V33Y mutants with 16 D-amino acid substrates. We demonstrate that the V33D, V33G, and V33Y mutants are more active than wild type with the aromatic substrates D-phenylalanine and 3-methyl-D-phenylalanine, while V33D and V33G are more active than wild type with D-valine, a branched chain amino acid. We expect the assays developed herein and subsequent mutants discovered to serve as valuable tools in the further development of biocatalysts for the production of enantiopure D-amino acids.