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 S ) was specifically ligated to the C terminus of a DAAO subunit (DAAO S ) through native peptide bonds (RTA&DAAO). RTA S is in close proximity to DAAO S , 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 S N terminus was specifically associated with the DAAO S 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 S –DAAO S ) 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.