Computational-Guided Hinge-Region Engineering Unlocks QtPheDH for Synthesizing Bulky Naphthyl Amino Acids

Abstract

Asymmetric reductive amination of α-keto acids catalyzed by amino acid dehydrogenases (AADHs) provides an atom-efficient and sustainable strategy for synthesizing optically pure non-natural amino acids. However, the narrow substrate spectrum of AADHs often restricts their broader application in green synthesis. Herein, a functional hinge region composed of three helices was identified as a key structural determinant governing substrate recognition and entry in both phenylalanine and leucine dehydrogenases. By engineering the hinge region, the catalytic performance of QtPheDH, a phenylalanine dehydrogenase from Quasibacillus thermotolerans, was significantly improved. Specifically, variant M2(V311G/S158G) alleviated the steric barrier and facilitated the entry of bulky substrates, enabling the initial amination activity of 1-naphthylglyoxylic acid (5a). Further optimization through binding energy reconfiguration led to the development of M4.2(V311G/S158G/L308M/T126I), which exhibited a 10-fold increase in catalytic efficiency and achieved complete conversion of 100 mM 5a with >99% ee. MD simulations revealed that the enhanced conformational flexibility in the active pocket and widened substrate tunnel were indispensable for accommodating bulkier substrates. These findings highlight the potential of hinge-region engineering in evolving AADHs toward powerful biocatalysts for sustainable and scalable synthesis of bulky non-natural amino acids.