Semirational Engineering of a Distal Loop Region to Enhance the Catalytic Activity and Stability of Leucine...

Enzymatic asymmetric synthesis of l-phenylglycine by amino acid dehydrogenases has potential for industrial applications; however, this is hindered by their low catalytic efficiency toward high-concentration substrates. We identified and characterized a novel leucine dehydrogenase (MsLeuDH) with a high catalytic efficiency for benzoylformic acid via directed metagenomic approaches. Further, we obtained a triple-point mutant MsLeuDH-EER (D332E/G333E/L334R) with improved stability and catalytic efficiency through the rational design of distal loop 13. A coexpression system of MsLeuDH-EER and formate dehydrogenase completely converted a 300 mM substrate within 4 h with >99.9% enantiomeric excess. Molecular dynamics simulations revealed that mutations on loop 13 enhanced the overall structural rigidity of the protein to improve its stability but also stabilized the "closed" conformation through rigidifying the hinge region loop by distant modulation. Our results show that distal loop 13 can serve as a new hotspot region for enhancing the catalytic performance of leucine dehydrogenases.