Title
Covalent Linkage of an R‐ω‐Transaminase to a d‐Amino Acid Oxidase through Protein Splicing to Enhance Enzymatic Catalysis of Transamination
Author
Kun Du, Rong Li, Dongrui Zhang, Wei Feng
Year
2018
Journal
ChemBioChem
Abstract
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 (RTAS) was specifically ligated to the C terminus of a DAAO subunit (DAAOS) through native peptide bonds (RTA&DAAO). RTAS is in close proximity to DAAOS, 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 RTAS N terminus was specifically associated with the DAAOS 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 (RTAS–DAAOS) 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.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Biochemistry