Comprehensive Understanding of Fluoroacetate Dehalogenase-Catalyzed Degradation of Fluorocarboxylic Acids: A QM/MM Approach

Abstract

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.

EnzyHTP: A High-Throughput Computational Platform for Enzyme Modeling

“…This cleavage of the strong C−F bond contributes to the ratedetermining step. 45 The process of breaking the C−F bond involves charge separation, which can be stabilized by the interior electrostatic environment of FAcD. Upon mutation, the electrostatic environment is likely perturbed, which can influence the catalytic efficiency.…”

“…In the first step of the catalyzed reaction, upon the binding of the substrate, the Asp 110 attacks the C–F bond in an S N 2 manner and forms the covalent intermediate. This cleavage of the strong C–F bond contributes to the rate-determining step . The process of breaking the C–F bond involves charge separation, which can be stabilized by the interior electrostatic environment of FAcD.…”

Challenges and opportunities in bringing nonbiological atoms to life with synthetic metabolism

“…Although it appears plausible that F uptake and assimilation into metabolites also serves as a defense mechanism in bacteria, the underlying evolutionary reason has not yet been identified [82]. Several detoxification mechanisms for fluorinated molecules have been identified in bacteria, such as through dehalogenation [83] or deacylation of tRNAs loaded with fluorinated amino acid moieties that would otherwise end up in proteins [84]. As discussed in the preceding text, most biochemical mechanisms involving F incorporation are probably based on misrecognition, due to the small atomic size of this halogen.…”

Nothing lasts forever: understanding microbial biodegradation of polyfluorinated compounds and perfluorinated alkyl substances

“…Difluoroacetate has been found to be much more recalcitrant to biodegradation (Alexandrino et al ., 2018 ), while dichloroacetate is readily biodegraded (Slater et al ., 1985 ; Busto et al ., 1992 ; Thomas et al ., 1992 ; Blackburn et al ., 2000 ; Dixon et al ., 2000 ; Pandey et al ., 2017 ; Chen et al ., 2021 ). Recently, the enzymatic defluorination of difluoracetate and 2,3,3,3‐tetrafluoropropionic acid has been reported (Li et al ., 2019 ; Yue et al ., 2021 ). While the lesser fluorinated analogs are not major pollution problems, trifluoroacetate is used in larger scale industrially, and bioremediation systems have been sought, such as in peptide synthesis companies where this strong acid is used in deblocking chemistry (Sakakibara and Inukai, 1965 ; Pearson et al ., 1989 ).…”