Fluorochemicals are persistent, bioaccumulative, and toxic compounds that are widely tributed in the environment. Developing efficient biodegradation strategies to decompose the fluorochemicals via breaking the inert C−F bonds presents a holistic challenge. As a promising biodegradation enzyme candidate, fluoroacetate dehalogenase (FAcD) has been reported as the only non-metallic enzyme to catalyze the cleavage of the strong C−F bond. Here, we systematically investigated the catalytic actions of FAcD toward its natural substrate fluoroacetate using molecular dynamics simulations and quantum mechanism/molecular mechanism calculations. We propose that the enzymatic transformation involves four elementary steps, (I) C−F bond activation, (II) nucleophilic attack, (III) C−O bond cleavage, and (IV) proton transfer. Our results show that nucleophilic attack is the rate-determining step. However, for difluoroacetate and trifluoroacetate, C−F bond activation, instead of nucleophilic attack, becomes the rate-determining step. We show that FAcD, originally recognized as α-fluorocarboxylic acid degradation enzyme, can catalyze the defluorination of difluoroacetate to glyoxylate, which is captured by our high-resolution mass spectrometry experiments. In addition, we employed amino acid electrostatic analysis method to screen potential mutation hotspots for tuning FAcD's electrostatic environment to favor substrate conversion. The comprehensive understanding of catalytic mechanism will inform a rational enzyme engineering strategy to degrade fluorochemicals for benefits of environmental sustainability.

Glycosyltransferases have attracted increasing interest for the ability to construct glycosylated molecules in a facile way. However, promiscuous chemoselectivity and poor regioselectivity hinder their widespread application in the synthetic field, especially in the pharmaceutical area. Here, a plant glycosyltransferase, MiCGT, was engineered by directed evolution to catalyze the glycosylation of flavonoids, which opens the door to pharmaceutical applications. Combining an alanine scan and iterative saturation mutagenesis, mutants with enhanced chemo- and regioselectivity and significantly improved activities toward seven different flavonoids were evolved, and two glycosylated products were prepared on a large scale. The best quadruple mutant VFAH enables strict 3-O glycosylation selectivity and a 120-fold activity enhancement toward the model substrate quercetin relative to the wild type (WT). Moreover, the crystal structures of the WT and mutant VFAH were obtained, a breakthrough of its kind in plant glycosyltransferase research. The origin of substrate specificity and regioselectivity was elucidated by combining the experimental data with the unique structure information. We anticipate that this work will aid future protein engineering of this type of enzyme.