Protein Engineering for Enhanced Enantioselectivity of Carboxylesterase Est924 Toward Ethyl 2-Arylpropionates

Abstract

BackgroundNon-steroidal anti-inflammatory drugs (NSAIDs) are the world's most used drugs with anti-inflammatory, analgesic and antipyretic effects. 2-arylpropionate drugs are a very important class of chiral drugs in NSAIDs. In the pharmaceutical industry, there is an increasing demand for esterase-mediated chiral resolution of racemic 2-arylpropionate esters due to the high activity and low side effects of the (S)-enantiomers. Esterases are important biocatalysts for chemical synthesis owing to their enantioselectivity, regioselectivity, broad substrate specificity and no need for cofactors. Several bHSL family esterases have been used to resolve racemic 2-arylpropionates. However, each reported enzyme was able to synthesize only one NSAIDs drug, which does not meet the industry requirement for catalysts and limits their application.ResultsThrough careful analysis of the structural information and molecular docking, we present the discovery of key residues that controlled the enantioselectivity of bHSL carboxylesterases to ethyl 2-arylpropionates. Est924 was identified as a bHSL family esterase and a promising catalyst for kinetic resolution of racemic ethyl 2-arylpropionates with sight (R)-stereopreference. Using Est924 as the starting enzyme, protein engineering at these key residues was conducted, and the substitution of A203 was shown to affect the enantioselectivity. The stereopreference of the mutant M1(A203W) was inverted to ethyl 2-(S)-arylpropionates, and this stereopreference was further improved in variant M3(I202F/A203W/G208F). In addition, the optimal variant, M3, was also suitable for the resolution of ibuprofen ethyl ester and ketoprofen ethyl ester, and their efficient (S)-isomers were synthesized.ConclusionsOur results suggested that Est924 variants could kinetically resolve economically important racemates for industrial production and further offer the opportunity for the rational design of enzyme enantioselectivity.