Covalent Linkage of an R-ω-Transaminase to a d-Amino Acid Oxidase through Protein Splicing to Enhance Enzymatic Catalysis of Transamination.

R-ω-Transaminases (RTAs) catalyse the conversion of R-configured amines [e.g., (R)-1-phenylethylamine] into the corresponding ketones (e.g., acetophenone), by transferring an amino group from an amino donor [e.g., (R)-1-phenylethylamine] onto an amino acceptor (e.g., pyruvate), resulting in a co-product (e.g., d-alanine). d-Alanine can be deaminated back to pyruvate by d-amino acid oxidase (DAAOs). Here, through in vivo subunit splicing, the N terminus of an RTA subunit (RTA ) was specifically ligated to the C terminus of a DAAO subunit (DAAO ) through native peptide bonds (RTA&DAAO). RTA is in close proximity to DAAO , at a molecular-scale distance. Thus the transfer of pyruvate and d-alanine between RTA and DAAO can be directional and efficient. Pyruvate→d-alanine→pyruvate cycles are efficiently formed, thus promoting the forward transamination reaction. In a different, in vitro noncovalent approach, based on coiled-coil association, the RTA N terminus was specifically associated with the DAAO C terminus (RTA#DAAO). In addition, the two mixed individual enzymes (RTA+DAAO) were also studied. RTA&DAAO has a shorter distance between the paired subunits (RTA -DAAO ) than RTA#DAAO, and the number of the paired subunits is higher than in the case of RTA#DAAO, whereas RTA+DAAO cannot form the paired subunits. RTA&DAAO exhibited a transamination catalysis efficiency higher than that of RTA#DAAO and much higher than that of RTA+DAAO.