Unexpected Mechanism of Biodegradation and Defluorination of 2,2-Difluoro-1,3-Benzodioxole by Pseudomonas putida F1

Abstract

Perfluorinated carbon atoms in a diether linkage are common in commercial anesthetics, drugs, fungicides, and insecticides. An important chemical group comprising perfluorodiethers is the 2,2-fluoro-1,3-benzodioxole (DFBD) moiety. The fluorine atoms stabilize the molecule by mitigating against metabolism by humans and microbes, as used in drugs and pesticides, respectively. Pseudomonas putida F1 catalyzed defluorination of DFBD at an initial rate of 2,100 nmol/h per mg cellular protein. This is orders of magnitude higher than previously reported microbial defluorination rates with multiply fluorinated carbon atoms. Defluorination rates declined after several hours, and the medium darkened. Significant defluorination activity was observed with cells grown on toluene but not l-arginine. Defluorination required only toluene dioxygenase. Pseudomonas and recombinant Escherichia coli cells expressing toluene dioxygenase oxidized DFBD to DFBD-4,5-dihydrodiol. The dihydrodiol could be oxidized to 4,5-dihydroxy-DFBD via the dihydrodiol dehydrogenase from P. putida F1. The dihydrodiol dehydrated with acid to yield a mixture of 4-hydroxy-DFBD and 5-hydroxy-DFBD. All those metabolites retained the difluoromethylene group; no fluoride or dark color was observed. The major route of DFBD-4,5-dihydrodiol decomposition produced fluoride and 1,2,3-trihydroxybenzene, or pyrogallol, and that was shown to be the source of the dark colors in the medium. A mechanism for DFBD-4,5-dihydrodiol transformation to two fluoride ions and pyrogallol is proposed. The Pseudomonas genome database and other databases revealed hundreds of bacteria with enzymes sharing high amino acid sequence identity to toluene dioxygenase from P. putida F1, suggesting the mechanism revealed here may apply to the defluorination of DFBD-containing compounds in the environment. IMPORTANCE There are more than 9,000 polyfluorinated compounds developed for commercial use, some negatively impacting human health, and they are generally considered to be resistant to biodegradation. Only a limited number of studies have identified microbes with enzymes sufficiently reactive to defluorinate difluoromethylene carbon groups. The present study examined one important group of commercial fluorinated chemicals and showed its rapid defluorination by a bacterium and its key enzyme, a Rieske dioxygenase. Rieske dioxygenases are common in environmental bacteria, and those closely resembling toluene dioxygenase from Pseudomonas putida F1 are candidates for biodegradative defluorination of the common 2,2-fluoro-1,3-benzodioxole (DFBD) moiety.

Keywords: PFAS; Pseudomonas putida F1; bacteria; defluorination; dioxygenase; fluoride; organofluorine; oxygenase; pesticides; pyrogallol; rapid rate.

FIG 1

Examples of commercially relevant polyfluorinated compounds. The center compound in bold is 2,2-difluro-1,3-benzodioxole (DFBD). Compounds containing the DFBD moiety shown are the experimental drug DiFMDA (difluoromethylenedioxyamphetamine), the fungicide fludioxonil, the anticancer agent AS-604850, and the reagent for enantioselective synthesis (R)-difluorophos.

FIG 2

Fluoride release (A) and color change (B) in medium of a culture of Pseudomonas putida F1 incubated with DFBD with shaking over 48 h. P. putida F1 was grown with toluene as the carbon source, which induces the toluene catabolic pathway, and then the culture was switched to DFBD. For fluoride measurements, cells were removed, and the supernatant liquid was analyzed for fluoride, using an ion-specific electrode, as shown in panel A. Aliquots of the culture were taken at the times indicated and stored frozen, and then all were directly photographed, as shown in panel B.

FIG 3

Toluene dioxygenase oxidation of DFBD to a cis-dihydrodiol. Analytical demonstration of 4,5-cis-dihydroxy-dihydro-2,2-difluoro-1,3-benzodioxole (4,5-DD-DFBD) by GC, MS, and NMR. The structure of the compound is shown in the upper right of panel A. The material analyzed by GC and MS was derivatized as detailed in Materials and Methods. While the data are consistent with a cis-dihydrodiol, the absolute stereochemistry has not been determined, and that shown is consistent with dihydrodiols produced by toluene dioxygenase. (A) Gas chromatograph of the extracted product derivatized with trimethylsilane. (B) Mass spectrum of the compound represented by the 12.64-min peak. The parent compound has an m/z of 336, and the m/z = 337,338 envelope is consistent with 1.1% natural abundance of ¹³C and a compound containing 13 carbon atoms. The disilyl fragment with an m/z of 147 is characteristic of compounds with two adjacent derivatized hydroxyl groups. (C) ¹⁹F-NMR spectrum at 400 MHz of 4,5-DD-DFBD. The two coupled doublets are consistent with two fluorines on differentiated faces, in this case arising from the cis configuration of the hydroxyl groups. The 3-dimensional depiction of 4,5-DD-DFBD illustrates the asymmetry imposed by the cis-dihydroxylation of DFBD.

FIG 4

Comparison of dihydrodiol dehydration products from E. coli pDTG601a with synthetic standards following derivatization and mass spectrometry. (A) Mass spectra of trimethylsilane (TMS)-derivatized metabolite from cell cultures incubated with DFBD (top) and standard DFBD-4-ol (bottom). (B) Mass spectra of TMS-derivatized metabolite from cell cultures incubated with DFBD (top) and standard DFBD-5-ol (bottom).

FIG 5

Extracted product derived from E. coli (pDTG602) expressing toluene dioxygenase and toluene dihydrodiol dehydrogenase. (A) Gas chromatogram of the compound identified as TMS-derivatized 4,5-dihydroxy-DFBD. (B) Mass spectrum of TMS-derivatized 4,5-dihydroxy-DFBD. The parent compound shows an m/z of 334.

FIG 6

Evidence for the defluorinated aromatic product, pyrogallol, or 1,2,3-benzenetriol. (A) Mass spectrometry fragmentation pattern of derivatized 1,2,3-benzenetriol found in cell cultures from E. coli pDTG601a (top) compared to standard derivatized 1,2,3-benzenetriol (bottom). (B) UV-vis spectrum of E. coli pDTG601a cell culture supernatant after incubation with DFBD for 72 h. (C) UV-vis spectrum of standard 1,2,3-benzenetriol in MSB medium after 72 h. Time course data are shown in Fig. S10.

FIG 7

Scheme showing 2,2-difluoro-1,3-benzodioxole (DFBD) oxidation by toluene dioxygenase to 4,5-dihyro-dihydroxy-DFBD (4,5-DD-DFBD) and subsequent reactions producing fluoride ion and dark medium. The major path is highlighted by darker arrows. The three intermediates between 4,5-DD-DFBD and fluoride plus pyrogallol are expected to have a very short lifetime, cannot be demonstrated directly, and represent a proposed mechanism for fluoride and medium darkening. The minor products on the upper right are stable and were demonstrated in this work, as was pyrogallol and the pathway for color formation.