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

Abstract

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 ε-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.

Reductive Enzyme Cascades for Valorization of PET Deconstruction Products

“…S6C). 67,68 However, we did not see a correlation of our experimental results to the obtained d1 values with our docked AMP-acyl TPA. Interestingly, we observed binding of MHET in the predicted srCAR structure, indicating that the bulky ethylene glycol sidechain could be accommodated at position A315 in the binding pocket (Fig.…”

“…S6C). 67,68 However, we did not see a correlation of our experimental results to the obtained d1 values with our (which was not certified by peer review) is the author/funder. All rights reserved.…”

Biocatalytic Carboxylate Reduction – Recent Advances and New Enzymes

“…Indeed, a double mutant of a M. smegmatis CAR with 8-fold increased k cat was identified, at unchanged K m for vanillic acid. [96] Extensive protein engineering was employed to engineer the MmCAR towards selective activity on shorter chain fatty acids. A combination of directed evolution of the fulllength MmCAR and its A-domain, rational design of the terminal domains, and structure-guided semi-rational design of the Rdomain, was used to tailor substrate specificity and catalytic activity, leading to a variant that was able to generate 2.8-fold more medium chain fatty-alcohols in Saccharomyces cerevisiae.…”

Reductive Enzyme Cascades for Valorization of Polyethylene Terephthalate Deconstruction Products

“…Prior studies have suggested that the ε-nitrogen atom of a conserved lysine binding pocket residue can form hydrogen bonds with the acid acyl−AMP complex, particularly with the ribose-ring oxygen atom, and that the distance (d1) between the nitrogen atom and the oxygen atom of the Lys residue and ribose ring, respectively, could be a nimble indicator of CAR activity (Figure S6C). 65,66 However, we did not see a correlation of our experimental results with the obtained d1 values with our docked AMP-acyl TPA. Interestingly, we observed binding of MHET in the predicted srCAR structure, indicating that the bulky ethylene glycol sidechain could be accommodated near position A315 in the binding pocket (Figure S7C).…”