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 conserved sequences with other acyl-CoA ligases/nonribosomal peptide synthetases/luciferases superfamily members, a mutagenesis library of the hinge region was designed. The best mutant identified (R505I/N506K) showed a 6.57-fold improved catalytic efficiency toward vanillic acid, of which the corresponding aldehyde is one of the most widely used flavors. Molecular dynamics simulations showed that the observed increased catalytic efficiency was due to the strong binding of the acyl-AMP complex with R505I/N506K. Meanwhile, the ε-nitrogen atom of Lys610 frequently interacted with the ribose-ring oxygen atom of the acyl-AMP complex in R505I/N506K, thereby distance (d1) between them represents a great indicator of increased catalytic activity. The d1 value was used as a nimble indicator to evaluate previously unexplored mutants of that region for enhanced activity by in silico mutational experiments. Overall, eight mutants with shorter d1 were identified to show higher enhanced activity compared with WT among which R505F/N506G showed the shortest d1 and the highest catalytic efficiency of 8.26-fold higher compared with the wild-type enzyme.

Conclusion: Altogether, our two-step strategy used here provided useful references for the engineering of CARs and other similar multiple-domain enzymes.

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