Semirational Design of Fluoroacetate Dehalogenase RPA1163 for Kinetic Resolution of alpha-Fluorocarboxylic Acids on a Gram Scale

摘要

Here the synthetic utility of fluoroacetate dchalogenase RPA1163 is explored for the production of enantiomerically pure (R)-alpha-fluorocarboxylic acids and (R)-alpha-hydroxylcarboxylic acids via kinetic resolution of racemic alpha-fluorocarboxylic acids. While wild-type (WT) RPA1163 shows high thermostability and fairly wide substrate scope, many interesting yet poorly or moderately accepted substrates exist. In order to solve this problem and to develop upscaled production, in silico calculations and semirational mutagenesis were employed. Residue W185 was engineered to alanine, serine, threonine, or asparagine. The two best mutants, W185N and W185T, showed significantly improved performance in the reactions of these substrates, while in silico calculations shed light on the origin of these improvements. Finally, 10 alpha-fluorocarboxylic acids and 10 alpha-hydroxycarboxylic acids were prepared on a gram scale via kinetic resolution enabled by WT, W185T, or WI 85N. This work expands the biocatalytic toolbox and allows a deep insight into the fluoroacetate dehalogenase catalyzed C-F cleavage mechanism.