Rational Engineering of <i>Mesorhizobium</i> Imine Reductase for Improved Synthesis of <i>N-</i>Benzyl Cyclo-tertiary Amines

Abstract

The effective synthesis of N-benzyl cyclo-tertiary amines using imine reductase, key components in natural products and pharmaceutical synthesis, is a green approach. Traditional methods faced challenges with enzyme activity and selectivity. This study focused on enhancing Mesorhizobium imine reductase (MesIRED) for better N-benzyl cyclo-tertiary amine production. Through alanine scanning and consensus mutation, 12 single-site MesIRED mutants were identified from 23 candidates, showing improved conversion of N-benzylpyrrolidine and N-benzylpiperidine. Notably, mutants from I177, V212, I213, and A241 significantly boosted conversions. The best-performing mutant for N-benzylpyrrolidine, MesIREDV212A/I213V (M1), increased conversion from 23.7% to 74.3%. For N-benzylpiperidine, MesIREDV212A/I177A/A241I (M2) enhanced conversion from 22.8% to 66.8%. Tunnel analysis revealed M1 and M2 have more efficient tunnels for larger product movement compared to wild-type MesIRED. Using recombinant E. coli coexpressing MesIRED and glucose dehydrogenase (GDH), high conversions were achieved: 75.1% for N-benzylpyrrolidine (M1) and 88.8% for N-benzylpiperidine (M2). A preparative experiment resulted in 86.2% conversion and 60.2% yield for N-benzylpiperidine. This research offers an efficient method for engineering IRED, significantly improving conversion and selectivity for N-benzyl cyclo-tertiary amines, aiding drug synthesis and providing insights into rational design of other enzymes.

Keywords

• imine reductase
• <i>N-</i>benzyl cyclo-tertiary amines
• alanine scanning
• consensus mutation
• whole-cell biocatalysis
• coexpression