Carboxylic acid reductases (CARs) play crucial roles in the biosynthesis of optically pure aldehydes with no side products. It has inspired synthetic organic chemists and biotechnologists to exploit them as catalysts in practical applications. However, levels of activity and substrate specificity are not routinely sufficient. Recent developments in protein engineering have produced numerous biocatalysts with new catalytic properties, whereas such efforts in CARs are limited. In this study, we show that the exploitation of information derived from catalytic mechanism analysis and molecular dynamics simulations assisted the semi-rational engineering of a CAR from Segniliparus rugosus (SrCAR) with the aim of increasing activity. Guided by protein-ligand interaction fingerprinting analysis, 17 residues at the substrate binding pockets were first identified. We then performed single site saturation mutagenesis and successfully obtained variants that gave high activities using benzoic acid as the model substrate. As a result, the best mutant K524W enabled 99% conversion and 17.28 s-1 mM-1kcat/Km, with 7- and 2-fold improvement compared to the wild-type, respectively. The engineered catalyst K524W as well as a second variant K524Q proved to be effective in the reduction of other benzoic acid derivatives. Insight into the source of enhanced activity was gained by molecular dynamics simulations.
Keywords: Biocatalysis; Carboxylic acid reductase; Enzyme activity; Rational design; Saturation mutagenesis.
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