Biocatalytic synthesis of phenyl benzoate esters using the amide ligase ClxA

TL;DR

Researchers found that the enzyme ClxA can efficiently create phenyl benzoate esters and can be modified to work with a wider range of substrates.

Contribution

The study demonstrates that ClxA, originally known for amide bond formation, can be repurposed and engineered for ester synthesis with expanded substrate tolerance.

Findings

ClxA catalyzes phenyl benzoate ester formation from benzoic acids and phenols in aqueous solution.

X-ray crystallography revealed the structure of ClxA with AMP and 3,4-aminohydroxybenzoic acid.

Engineered variants N226L and K140A of ClxA exhibit broader substrate specificity for ester synthesis.

Abstract

The synthesis of ester bonds using lipases is one of the most frequently performed reactions in biocatalysis, yet examples of the enzymatic synthesis of phenyl benzoate esters are comparatively rare. In this report we show that the ligase ClxA, from Clostridium cavendishii, initially reported to have roles in amide bond formation in the biosynthesis of benzoxazole antibiotics, is an effective catalyst for the formation of phenyl benzoate esters from acid and phenol substrates using ATP in an aqueous medium. The structure of ClxA in a complex with both AMP and 3,4-aminohydroxybenzoic acid was determined by X-ray crystallography to 2.15 Å resolution and used as a platform to engineer the enzyme to create variants N226L and K140A possessing broader substrate specificity for ester formation, and also the ability to enable the synthesis of native amide product oligomers.

The ligase ClxA catalyses the synthesis of phenyl benzoate esters from benzoic acids and phenols and can be engineered for broadened substrate specificity.

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