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

Bygd, Madison D., Aukema, Kelly G., Richman, Jack E., Wackett, Lawrence P.·2021·mBio
DOI 10.1128/mbio.03001-21PMC PMC8593668
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Abstract

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.

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Figures

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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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).CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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.
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.CC BY · PubMed Central

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Metadata

Authors
Bygd, M. D., Aukema, K. G., Richman, J. E., & Wackett, L. P.
Affiliations
  • Microbial Engineering, University of Minnesota, St. Paul, Minnesota, USA
  • Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minnesota, USA
ORCID
Venue
mBio
Publisher
American Society for Microbiology
Published
2021-12-21
Volume
12
Issue
6
ISSN
2150-7511
DOI
10.1128/mbio.03001-21
PMC
PMC8593668
Cited by
28
References
1
Language
en
Licence
Funders
MnDRIVE Industry and the Environment

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