Site-directed mutagenesis and stability of the carboxylic acid reductase MAB4714 from Mycobacterium abscessus.

Journal of Biotechnology
Tatiana P FedorchukAlexander F Yakunin

Abstract

Carboxylic acid reductases (CARs) catalyze ATP- and NADPH-dependent reduction of carboxylic acids to corresponding aldehydes. Although successful applications of these enzymes for the bioconversion of monocarboxylic acids have already been reported, their applicability for the reduction of dicarboxylic acids is not well understood. Here, we explored the possibility of engineering CARs for enhanced activity toward succinic acid for potential applications in 1,4-butanediol production. Structural models of the carboxylate-binding pocket of the CAR enzyme MAB4714 from Mycobacterium abscessus suggested that its reactivity toward succinic acid could be enhanced by reducing the pocket volume. Using site-directed mutagenesis, we introduced larger side chains into the MAB4714 carboxylate binding pocket and compared the activity of 16 mutant proteins against cinnamic and succinic acids. These experiments revealed that, although the reaction rates remain low, the replacement of Leu284 or Thr285 with Trp increased activity toward succinic acid more than two times. The T285E mutant protein also showed increased activity toward succinic acid, but it was lower than that of T285W. The mutated residues of MAB4714 are located on the flexible loo...Continue Reading

Citations

Sep 3, 2020·Proceedings of the National Academy of Sciences of the United States of America·Yating HuVerena Siewers
Oct 20, 2019·Journal of Biotechnology·Neil Butler, Aditya M Kunjapur

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