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

Madison D. Bygd, Kelly G. Aukema, Jack E Richman, Lawrence P. Wackett

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

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.

Original languageEnglish (US)
Article numbere03001-21
JournalmBio
Volume12
Issue number6
DOIs
StatePublished - Dec 1 2021

Bibliographical note

Funding Information:
We thank Becky Parales for providing the recombinant E. coli strains: E. coli pDTG601a and E. coli pDTG602. We acknowledge Thomas Niehaus for making available the Cary spectrophotometer. We acknowledge Mikael Elias and Amir Shimon for helpful discussion about this project. This project was funded by MnDRIVE Environment.

Publisher Copyright:
© 2021 American Society for Microbiology. All rights reserved.

Keywords

  • Bacteria
  • Defluorination
  • Dioxygenase
  • Fluoride
  • Organofluorine
  • Oxygenase
  • PFAS
  • Pesticides
  • Pseudomonas putida F1
  • Pyrogallol
  • Rapid rate

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