19F nuclear magnetic resonance as a tool to investigate microbial degradation of fluorophenols to fluorocatechols and fluoromuconates

Abstract

A method was developed to study the biodegradation and oxidative biodehalogenation of fluorinated phenols by 19F nuclear magnetic resonance (NMR). Characterization of the 19F NMR spectra of metabolite profiles of a series of fluorophenols, converted by purified phenol hydroxylase, catechol 1,2-dioxygenase, and/or by the yeast-like fungus Exophiala jeanselmei, provided possibilities for identification of the 19F NMR chemical shift values of fluorinated catechol and muconate metabolites. As an example, the 19F NMR method thus defined was used to characterize the time-dependent metabolite profiles of various halophenols in either cell extracts or in incubations with whole cells of E. jeanselmei. The results obtained for these two systems are similar, except for the level of muconates observed. Altogether, the results of the present study describe a 19F NMR method which provides an efficient tool for elucidating the metabolic pathways for conversion of fluorine-containing phenols by microorganisms, with special emphasis on possibilities for biodehalogenation and detection of the type of fluorocatechols and fluoromuconates involved. In addition, the method provides possibilities for studying metabolic pathways in vivo in whole cells.

FIG. 1

¹⁹F NMR spectra of culture medium after 1 h of incubation of whole cells of E. jeanselmei with 2-fluorophenol at 30°C (A), cell extracts from E. jeanselmei incubated for 1 h with 2-fluorophenol at 30°C (B), incubation of purified phenol hydroxylase with 2-fluorophenol (C), and incubation of purified phenol hydroxylase plus purified catechol 1,2-dioxygenase with 2-fluorophenol (D). ¹⁹F NMR chemical shift values were identified as described previously (19) and on the basis of results from the present study (see text and Table 1). The resonance marked “is” is from the internal standard, 4-fluorobenzoate.

FIG. 2

Time-dependent changes in the metabolic profile of incubations with 4-fluorophenol (A and D), 2,4-difluorophenol (B and E), and 2,3,4-trifluorophenol (C and F), where panels A to C show the profiles of incubations with cell extracts and panels D to F show the profiles of incubations with whole cells of E. jeanselmei. Metabolites were identified and quantified by ¹⁹F NMR. 4FOH, 4-fluorophenol; 4Fcat, 4-fluorocatechol; 3Fmuc, 3-fluoromuconate; F−, fluorine.

FIG. 3

Pathways for the biodegradation and oxidative biodehalogenation of phenol and its halogenated analogs. Numbers in parentheses refer to literature references. The enzymes involved are indicated as follows: PH, phenol hydroxylase; C12O, catechol 1,2-dioxygenase; (C)MCl, (chloro)muconate cycloisomerase; DLH, dienelactone hydrolase; ELH, enol-lactone hydrolase; MAR, maleylacetate reductase. Pathways identified so far for fluorophenols, either in the literature or in the present study for E. jeanselmei, are indicated in boldface.