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. 2022 Nov;24(11):5202-5216.
doi: 10.1111/1462-2920.16187. Epub 2022 Oct 17.

Fluoro-recognition: New in vivo fluorescent assay for toluene dioxygenase probing induction by and metabolism of polyfluorinated compounds

Kelly G Aukema  1 , Madison D Bygd  1 , Lambros J Tassoulas  1 , Jack E Richman  1 , Lawrence P Wackett  1
Affiliations

Fluoro-recognition: New in vivo fluorescent assay for toluene dioxygenase probing induction by and metabolism of polyfluorinated compounds

Kelly G Aukema et al. Environ Microbiol. 2022 Nov.

Abstract

The present study examined the regulatory and metabolic response of the aromatic degrader Pseudomonas putida F1 and its tod operon, controlling toluene degradation, to fluorinated aromatic and aliphatic compounds. The tod operon is upregulated by inducer binding to the TodS sensing domain of a two-component regulator. The induced enzymes include toluene dioxygenase that initiates catabolic assimilation of benzenoid hydrocarbons. Toluene dioxygenase was shown to oxidize 6-fluoroindole to a meta-stable fluorescent product, 6-fluoroindoxyl. The fluorescent output allowed monitoring relative levels of tod operon induction in whole cells using microtiter well plates. Mono- and polyfluorinated aromatic compounds were shown to induce toluene dioxygenase, in some cases to a greater extent than compounds serving as growth substrates. Compounds that are oxidized by toluene dioxygenase and undergoing defluorination were shown to induce their own metabolism. 1,2,4-Trifluorobenzene caused significant induction and computational modelling indicated productive binding to the TodS sensor domain of the TodST regulator. Toluene dioxygenase also showed preferential binding of 1,2,4-trifluorobenzene such that defluorination was favoured. Fluorinated aliphatic compounds were shown to induce toluene dioxygenase. An aliphatic ether with seven fluorine atoms, 1,1,1,2-tetrafluoro-2-trifluoromethoxy-4-iodobutane (TTIB), was an excellent inducer of toluene dioxygenase activity and shown to undergo transformation in cultures of P. putida F1.

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Conflict of interest statement

The authors declare that there are no competing interests associated with this manuscript.

Figures

FIGURE 1
FIGURE 1
Schematic of toluene dioxygenase (TDO) regulation and oxidation of indole to indoxyl and indigo. (A) The tod operon is controlled by todST gene products in which ligands are bound by the TodS with inducers causing a conformational change and phosphorylation of TodT that can bind to the promoter. (B) The operon induction expresses toluene dioxygenase that is known to dioxygenate indole compounds, followed by spontaneous dehydration to produce fluorescent indoxyl and further spontaneous oxidation produces indigo white and then indigo. Indole dihydrodiol and indoxyl formation occur on the time scale of seconds, subsequent reactions on the time scale of minutes and hours. Ascorbate helps maintain indoxyl that is fluorescent.
FIGURE 2
FIGURE 2
Fluorescence and stable structural products from the oxidation of 6‐fluoroindole by toluene dioxygenase. (A) Relative fluorescence for each substituted indole substrate of toluene dioxygenase compared to indole. (B) Characterization of the extracted product of toluene‐grown Pseudomonas putida F1 incubated with 6‐fluoroindole by UV–vis spectroscopy. (C) 19F‐NMR spectrum (at 400 MHz 1H) of extract dissolved in deuterochloroform (CDCl3) (dt, J 5 Hz, 10 Hz, F‐11 reference). The inset shows the material analysed by NMR and UV–vis after extraction from culture media.
FIGURE 3
FIGURE 3
Comparison of toluene dioxygenase (TDO) induction using the 6‐fluoroindole activity assay from this article (light bars) to previously published results using a β‐galactosidase reporter (dark bars). The percent induction for each chemical was determined by normalizing to TDO activity with toluene induction that was set at 100%.
FIGURE 4
FIGURE 4
Induction of toluene dioxygenase activity with four different classes of polyfluorinated benzene compounds as a percent of induction observed with toluene using a protocol described in the Experimental Procedures section. The four compounds named have been subjected to metabolic studies, including toluene, the archetypal substrate for the tod operon. The three fluorinated compounds highlighted in red have been shown to undergo toluene dioxygenase‐dependent defluorination: 2,2‐difluoro‐1,3‐benzodioxole (Bygd et al., 2021); 4‐fluorobenzotrifluoride (Bygd et al., 2022); and 1,2,4‐trifluorobenzene (this study, in a subsequent section).
FIGURE 5
FIGURE 5
1,2,4‐Trifluorobenzene induction by binding to TodS and metabolism by toluene dioxygenase and diol dehydrogenase with formation of 3,4,6 trifluorobenzene diol and 3,6‐difluorobenzene diol. (A) Docking and energy minimization with 1,2,4‐benzene, shown overlapped with toluene, bound in TodS as described in Experimental Procedures. (B) GC separation of catechols doubly derivatized with trimethylsilane and mass spectra of the respective peaks. (C) Pathways leading to a trifluorocatechol (top) and difluorocatechol and hydrogen fluoride (bottom). The latter derives from gem‐elimination of a dihydrodiol.
FIGURE 6
FIGURE 6
Induction of toluene dioxygenase activity by fluorinated aliphatic compounds. Induction controls were toluene, set at 100%, and no inducer that gave basal level of toluene dioxygenase. Compounds giving no significant induction were as follows: (I) heptafluoro‐2‐iodopropane; (II) 1,2‐dichloro‐3,3,3‐trifluoropropene; (III) hexafluoroacetylacetone; (IV) perfluoropropyl iodide. The compounds highlighted in red showed significant levels of induction.
FIGURE 7
FIGURE 7
Substrates and position of dioxygenation by toluene dioxygenase with known reactions and as proposed for 3,4,4,4‐tetrafluoro‐3‐trifluoromethoxy‐butene, the elimination product of 1,1,1,2‐tetrafluoro‐2‐trifluoromethoxy‐4‐iodobutane (TTIB). (A) Modelling of toluene (purple) and 1,2,4‐trifluorobenzene (green) in the active site of toluene dioxygenase showing the direction of dioxygen attack consistent with known absolute stereochemistry of the toluene dihydrodiol and of the major fluorinated product as demonstrated here. The red sphere represents the active site iron atom that binds and activates dioxygen for attack from the top face as shown by the blue arrow. (B) Space‐filling model for toluene showing attack from the top face of the ring with the methyl group oriented down. (C) Space‐filling model showing proposed direction of dioxygen attack catalyzed by TDO with 3,4,4,4‐tetrafluoro‐3‐trifluoromethoxy‐butene. (D) Space‐filling model showing known position of attack on styrene by TDO. (E) Space‐filling model showing known position of attack on indene by toluene dioxygenase.
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