Identification of Fluoropyrogallols as New Intermediates in Biotransformation of Monofluorophenols in Rhodococcus opacus 1cp

Abstract

Halophenols and their derivatives are priority pollutants of mainly anthropogenic origin. Over several decades, these compounds have been widely used as building blocks in chemical and pharmaceutical syntheses and as herbicides and pesticides, and they have caused serious local contamination of the environment. Soil microorganisms have developed the capacity of utilizing halophenols for their growth by a diverse set of biodegradation pathways (8). Aerobic soil microorganisms generally degrade mono-and dihalophenols through the initial action of (chloro)phenol ortho-hydroxylases, leading to the formation of halocatechols (1,7,9,10,12). In the framework of a project devoted to the biodegradation of halophenols by gram-positive bacteria, we investigated the formation of hydroxylated intermediates formed upon the conversion of halophenols by various Rhodococcus species and previously demonstrated the formation of (halo)catechols as initial intermediates in the biodegradation pathways (3). However, identification of the subsequent biodegradation pathways of the chlorocatechols appeared hampered by the fact that unequivocal identification of the site of introduction of a third hydroxyl group is difficult because 1 H nuclear magnetic resonance (NMR) splitting patterns combined with 1 H chemical shift data of the protons present in these metabolites can be compatible with more than one substitution pattern (13). Therefore, in this paper, we have studied the possible formation of trihydroxyfluorobenzene metabolites from fluorophenols by whole cells of Rhodococcus opacus 1cp in detail. The fluorine substituent provides the possibility to detect and quantify the possible hydroxyfluorobenzene intermediates by 19 F NMR, allowing the identification of the exact substitution pattern. Using this technique we unambiguously demonstrate the formation of fluoropyrogallols (1,2,3-trihydroxyfluorobenzenes) as new intermediates in the biotransformation of monofluorophenols by R. opacus 1cp. MATERIALS AND METHODS Chemicals.Phenol was purchased from Merck (Darmstadt, Germany). 2-Fluorophenol, 3-fluorophenol, and 4-fluorophenol were purchased from Janssen Chimica (Beerse, Belgium). Fluorocatechols were prepared from the corresponding fluorophenols using purified phenol hydroxylase from Trichosporon cutaneum (14). Fluoromuconates were prepared and identified as described previously (2) by incubating the fluorocatechols with catechol 1,2-dioxygenase from Pseudomonas arvilla C-1.Growth of R. opacus 1cp. The strain R. opacus 1cp was isolated and maintained as described previously (6). The strain can grow on phenol as the sole source of carbon. For cultivation, a mineral synthetic medium containing, per liter, 1 g of NH 4 NO 3 , 1 g of K 2 HPO 4 , 1 g of KH 2 PO 4 , 0.2 g of MgSO 4 ⅐ 7H 2 O, 0.02 g of CaCl 2 , and 2 drops of a saturated solution of FeCl 3 (pH 7.2) was used. Phenol was used as the source of carbon and was initially added at a 200-mg/liter final concentration. R. opacus 1cp did not grow on either of the three monofluorophenols ...

The biodegradation vs. biotransformation of fluorosubstituted aromatics

“…strain FP1 (Duque et al 2012) as well as in Burkholderia fungorum strain FLU100 (Strunk and Engesser 2013). Moreover, further hydroxylated by-products like fluorohydroquinone or fluoropyrogallols may be formed (Finkelstein et al 2000;Kim et al 2010).…”

“…In Pseu do noc ardia benzenivorans, 2-fluorohydroquinone was detected as an additional product (Kim et al 2010), and in R. opacus 1cp, 4-fluorocatechol was hydroxylated to form 5-fluoropyrogallol (Finkelstein et al 2000). Tyrosinase from Streptomyces antibioticus, an enzyme known to catalyze the ortho-hydroxylation of monophenols and the subsequent oxidation of diphenols to quinones, converted 3-fluorophenol into 4-fluorinated 1,2-benzoquinone which then underwent nonenzymatic polymerization reactions, thereby releasing about 50 % of the fluorine contained.…”

Isolation and characterization of a Rhodococcus strain able to degrade 2-fluorophenol

“…Aerobic biotransformation of 2-FP by several Rhodococcus strains (Bondar et al 1999;Finkelstein et al 2000), acclimated activated sludge (Chaojie et al 2007), Gloeophyllum striatum (Kramer et al 2004) and Penicillium strains (Hofrichter et al 1994;Marr et al 1996), has been reported. In all cases, 2-FP was degraded by cometabolism in the presence of other carbon sources.…”

Biotransformation of monofluorophenols by cultured cells of Eucalyptus perriniana

“…Therefore, soil microorganisms have developed the capacity of utilizing halophenols for their growth by a diverse set of biodegradation pathways (Haggblom 1992). Furthermore, the microbial transformation of monofluorophenols by the whole cells of Rhodococcus opacus 1cp was investigated (Finkelstein et al 2000). However, up to now there are no reports concerning the biotransformation of monofluorophenols by cultured plant cells.…”