Of all NMR observable isotopes 19F is the one perhaps most convenient for studies on biodegradation of environmental pollutants. The reasons underlying this potential of 19F NMR are discussed and illustrated on the basis of a study on the biodegradation of fluorophenols by four Rhodococcus strains. The results indicate marked differences between the biodegradation pathways of fluorophenols among the various Rhodococcus species. This holds not only for the level and nature of the fluorinated biodegradation pathway intermediates that accumulate, but also for the regioselectivity of the initial hydroxylation step. Several of the Rhodococcus species contain a phenol hydroxylase that catalyses the oxidative defluorination of ortho-fluorinated di- and trifluorophenols. Furthermore, it is illustrated how the 19F NMR technique can be used as a tool in the process of identification of an accumulated unknown metabolite, in this case most likely 5-fluoromaleylacetate. Altogether, the 19F NMR technique proved valid to obtain detailed information on the microbial biodegradation pathways of fluorinated organics, but also to provide information on the specificity of enzymes generally considered unstable and, for this reason, not much studied so far.

Gram-positive bacteria of the genus Rhodococcus catabolize p-hydroxybenzoate (PHB) through the initial formation of 3,4-dihydroxybenzoate. High levels of p-hydroxybenzoate hydroxylase (PHBH) activity are induced in six different Rhodococcus species when these strains are grown on PHB as sole carbon source. The PHBH enzymes were purified to apparent homogeneity and appeared to be homodimers of about 95 kD with each subunit containing a relatively weakly bound FAD. In contrast to their counterparts from gram-negative microorganisms, the Rhodococcus PHBH enzymes prefer NADH to NADPH as external electron donor. All purified enzymes were inhibited by Cl- and for five of six enzymes more pronounced substrate inhibition was observed in the presence of chloride ions.

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 ...

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