A Plurizyme with Transaminase and Hydrolase Activity Catalyzes Cascade Reactions

Engineering dual-function single polypeptide catalysts with two abiotic or biotic catalytic entities (or combinations of both) supporting cascade reactions is becoming an important area of enzyme engineering and catalysis. Herein we present the development of a PluriZyme, TR2E2, with efficient native transaminase (k(cat): 69.49 +/- 1.77 min(-1)) and artificial esterase (k(cat): 3908-0.41 min(-1)) activities integrated into a single scaffold, and evaluate its utility in a cascade reaction. TR2E2 (pH(opt): 8.0-9.5; T-opt: 60-65 degrees C) efficiently converts methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate into 3-(R)-amino-4-(2,4,5-trifluorophenyl)butanoic acid, a crucial intermediate for the synthesis of antidiabetic drugs. The reaction proceeds through the conversion of the beta-keto ester into the beta-keto acid at the hydrolytic site and subsequently into the beta-amino acid (e.e. >99 %) at the transaminase site. The catalytic power of the TR2E2 PluriZyme was proven with a set of beta-keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions.