A Trifunctional Imine Reductase Enables a Three-Step Biocatalytic Cascade

Area of Science:

• Biocatalysis
• Organic Synthesis
• Enzyme Engineering

Background:

• Traditional biocatalytic cascades rely on multiple discrete enzymes for sequential synthetic steps.
• Complex synthetic pathways often require intermediate isolation, increasing process time and cost.

Purpose of the Study:

• To develop a single enzyme capable of catalyzing multiple sequential transformations.
• To streamline the synthesis of enantiomerically pure 2-aryl pyrrolidines.
• To elucidate the mechanism of step-selective catalysis in multifunctional imine reductases.

Main Methods:

• Engineering of trifunctional imine reductases (IREDs) with alkene reduction, intramolecular reductive amination, and imine reduction activities.
• Density functional theory (DFT) calculations to investigate catalytic mechanisms.
• Mechanistic studies to understand enzyme step-selectivity.

Main Results:

• Demonstration of unprecedented trifunctional IREDs catalyzing three sequential reactions in a single catalytic cycle.
• Direct transformation of linear substrates into enantiomerically pure 2-aryl pyrrolidines without intermediate isolation.
• Elucidation of the mechanism enabling step-selective catalysis within the IRED.

Conclusions:

• Multifunctional enzymes can significantly simplify complex biocatalytic cascades.
• This work provides a proof-of-concept for designing single-enzyme systems for streamlined synthesis.
• The developed IREDs offer a powerful strategy for efficient production of chiral pyrrolidines.