A Plurizyme with Transaminase and Hydrolase Activity Catalyzes Cascade Reactions

Abstract

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, TR₂ E₂ , 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. TR₂ E₂ (pH_opt : 8.0-9.5; T_opt : 60-65 °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 β-keto ester into the β-keto acid at the hydrolytic site and subsequently into the β-amino acid (e.e. >99 %) at the transaminase site. The catalytic power of the TR₂ E₂ PluriZyme was proven with a set of β-keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions.

Keywords: Cascade Reactions; Computational Engineering; Esterase; Plurizyme; Transaminase.