Rational hinge engineering of carboxylic acid reductase from Mycobacterium smegmatis enhances its catalytic efficiency in biocatalysis

Background: Carboxylic acid reductases (CARs) represent useful tools for the production of aldehydes from ubiquitous organic carboxylic acids. However, the low catalytic efficiency of these enzymes hampers their application. Methods: Herein, a CAR originating from Mycobacterium smegmatis was redesigned through rational hinge engineering to enhance the catalytic efficiency. Results: Based on the unique domain architecture of CARs and their superfamily, a mutagenesis library of the hinge region was designed. The best mutant R505I/N506K showed a 6.57-fold improved catalytic efficiency. Molecular dynamics simulations showed the increased catalytic efficiency was due to the strong binding of the acyl-AMP complex with it. Meanwhile, the epsilon-nitrogen atom of Lys610 frequently interacted with the ribose-ring oxygen atom of the complex, the distance (d1) between them represents a great indicator for that. The d1 value was used as a nimble indicator to evaluate unexplored mutants of that region for enhanced activity by in silico mutational experiments. Overall, eight mutants were identified to show higher enhanced activity compared with wild-type enzyme and R505F/N506G showed the highest catalytic efficiency. Conclusion: Altogether, the two-step strategy used here provided useful references for the engineering of CARs and other similar multiple-domain enzymes.

Automated summary

A carboxylic acid reductase (CAR) originating from Mycobacterium smegmatis was redesigned through rational hinge engineering to improve catalytic efficiency. Mutagenesis of the hinge region resulted in a mutant showing a 6.57-fold increase in catalytic efficiency. Molecular dynamics simulations revealed the key interactions leading to enhanced activity were due to strong binding of the acyl-AMP complex. This two-step strategy provided useful references for the engineering of CARs and other similar enzymes with multiple domains.