Pentachlorophenol by a Flavobacterium Pentachlorophenol ...

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Agriculture Canada, Central Experimental Farmn, Ottawa, Canada KJA OC62 ... The purified enzyme incorporated 180 from 1802 but not from H2180 into the ...
Vol. 174, No. 17

JOURNAL OF BACTERIOLOGY, Sept. 1992, p. 5745-5747

0021-9193/92/175745-03$02.00/0 Copyright © 1992, American Society for Microbiology

Confirmation of Oxidative Dehalogenation of Pentachlorophenol by a Flavobacterium Pentachlorophenol Hydroxylaset LUYING XUN,l EDWARD TOPP,2 AND CINDY S. ORSER'* Department of Bacteriology and Biochemisty and Center for Hazardous Waste Remediation Research, University of Idaho, Moscow, Idaho 83843, and Centre for Land and Biological Resources Research, Agriculture Canada, Central Experimental Farmn, Ottawa, Canada KJA OC62 Received 10 March 1992/Accepted 22 June 1992

Pentachlorophenol (PCP) hydroxylase purified from Flavobacterium sp. strain ATCC 39723 converted PCP or 2,3,5,6-tetrachlorophenol to tetrachloro-p-hydroquinone (TeCH) with the coconsumption Of 02 and NADPH. The purified enzyme incorporated 180 from 1802 but not from H2180 into the reaction end product TeCH. The results clearly demonstrate that PCP is oxidatively converted to TeCH by a monooxygenase-type

enzyme from Flavobacterium sp. strain ATCC 39723.

Pentachlorophenol (PCP) is an important pollutant because of its widespread use as a wood preservative, fungicide, insecticide, algicide, and bactericide (4). PCP degradation by several microorganisms has been reported (1, 3, 5, 8, 10, 12), but there is some disagreement over the reaction mechanisms involved in PCP degradation. Although all reports propose tetrachloro-p-hydroquinone (TeCH) as the first intermediate during PCP degradation, they differ on whether the reaction is hydrolytic or oxidative (2, 6, 9). The previous "O experiments with either whole cells or cell extract fractions demonstrated that an 180 from H2180 but not from 1802 was incorporated into TeCH during PCP degradation (2, 6, 9). As pointed out by Schenk et al. (6), 180 from H2180 could exchange with 160 in TeCH when incubated with their partially purified enzyme. Therefore, they could not conclude whether the first step in PCP degradation was an oxidative or a hydrolytic reaction. We recently reported the purification and characterization of a Flavobacterium PCP hydroxylase which converts PCP to TeCH in the presence of NADPH and 02, indicating an oxidative reaction (14). We report here data from 180 labelling experiments confirming that the mechanism of PCP hydroxylation by purified PCP hydroxylase is indeed oxidative. Labelling experiments with H2180 or 1802 were performed by using purified PCP hydroxylase from Flavobacterium sp. strain ATCC 39723 (14). H2 80 (81% 180) was purchased from Cambridge Isotope Laboratories (Woburn, Mass.), and 1802 (98% 180) was purchased from Merck Sharp and Dohme Isotopes (Rahway, N.J.). The 1802 labelling experiment was performed with a 20-ml serum bottle. The reaction mixture (250-pl total volume), which contained 20 ,umol of potassium phosphate (pH 7.5), 0.4% Tween 20, 75 nmol of PCP, and 150 ,ug of purified PCP hydroxylase, was placed in an anaerobic chamber (85% N2, 5% CO2, and 10% H2) and equilibrated for 10 min before the serum bottle was sealed with a butyl rubber stopper. Five milliliters of «as was removed from the reaction bottle, and 5 ml of 1 02 was injected into the bottle. The reaction was started by injecting *

Corresponding author.

t Idaho Agricultural Experiment Station Journal article no. 92506.

Centre for Land and Biological Research control no. 92-45.

15 RI of freshly prepared 20 mM NADPH in H20, which had been equilibrated in the anaerobic chamber for 10 min, into the reaction bottle. After 30 min of incubation at 23°C, PCP was completely converted to TeCH. TeCH was extracted by ether and analyzed by gas chromatography-mass spectrometry (11). The H2180 labelling experiment was similarly conducted with a total volume of 250 ,l and a final concentration of 61% H218O. The final concentrations of potassium phosphate, Tween 20, PCP, and purified PCP hydroxylase were the same as for the 1802 experiment. The reaction was also started by the addition of 15 p1 of 20 mM NADPH followed by incubation with atmospheric 02 at 23°C for 30 min. TeCH was similarly extracted and analyzed. Authentic 2,3,5,6-TeCH (C6Cl4H21602), purchased from Sigma Chemical Co. (St. Louis, Mo.), was used as a control for gas chromatography-mass spectrometry analysis. It had a mass spectrum (retention time, 8.3 min) with peaks at mlz 252 (M + 6), 250 (M + 4), 248 (M + 2, base peak), and 246 (M+), whose masses and relative intensities are characteristic of a molecule containing four chlorine atoms (Fig. 1). The relative intensities are consistent with a natural abundance of 76% for 35Cl and 24% for 37CI. Major fragments were observed at mlz 210, 212, and 214 (loss of H35Cl from 246, 248, 250; loss of H37CI from 248, 250, 252); m/z 182 and 184 (loss of CO from 210 or 212); mlz 175 and 177 (loss of 35Cl from 210 or 212; loss of 37CI from 212), mlz 154 and 156 (loss of CO from 182 or 184); and mlz 149 and 147 (loss of 35CI from 182 or 184; loss of 37CI from 184). Because PCP hydroxylase hydroxylates both PCP and 2,3,5,6-tetrachlorophenol (TeCP) to a common end product, TeCH (15), we used both PCP and TeCP in our 180 experiments. Incubation of PCP hydroxylase with H218O and PCP or TeCP under air yielded a product whose retention time and mass spectrum were identical to those of TeCH (C6CI4H21602). The mass spectrum of the end product from PCP is presented in Fig. 2. However, when the enzyme was incubated with PCP under an atmosphere enriched with 1802, the product had the same retention time but a mass spectrum with all major peaks larger by 2 mass units (Fig. 3), consistent with a molecular formula of C6Cl4H216"802. Likewise, incubation of PCP hydroxylase with TeCP and 1802 yielded the same highermolecular-weight product (data not shown). In both 1802 5745

J. BACTERIOL.

NOTES

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FIG. 1. Mass spectrum of authentic 2,3,5,6-TeCH.

experiments, the peak at mlz 246 indicated that there was some residual 1602 in the reaction vessel. Nevertheless, the results clearly demonstrate that the hydroxyl group incorporated into thepara position originated from dioxygen but not from water. We have previously reported the stoichiometries of representative reactions catalyzed by PCP hydroxylase (15). Two moles of NADPH and 1 mol of 02 are consumed for 1 mol of PCP hydroxylation, while only 1 mol of NADPH and 1 mol of 02 are consumed for 1 mol of TeCP hydroxylation (15). The stoichiometry of TeCP hydroxylation catalyzed by PCP hydroxylase indicates that the enzyme is a monooxygenase. The stoichiometric data and our 18Q labelling results confirm that PCP hydroxylase is a monooxygenase. Therefore, "PCP 4-monooxygenase" should be the official name for PCP hydroxylase (13). Steiert and Crawford reported that 2,6-dichlorohydroquinone was labelled with 180 when incubated with H2180 but not when incubated with 1802 during PCP degradation by a mutant of Flavobacterium sp. strain ATCC 39723 (9). They proposed that the first reaction was by a hydrolytic reaction 248

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(9). Similarly, Apajalahti and Salkinoja-Salonen (2) reported that TeCH was transitionally accumulated during PCP degradation in the culture supematant of Rhodococcus chlorophenolicus and that TeCH was labelled with 18Q when incubated with H2180 but not when incubated with 1802 They also proposed a hydrolytic reaction for the conversion of PCP to TeCH (2). Recently, Schenk et al. (7) reported enzymatic conversion of PCP to TeCH in the presence of NADPH and 02 by a fraction of cell extract from Arthrobacter sp. strain ATCC 33790. Subsequently, they found that a protein fraction which was further purified (by ammonium sulfate fractionation and gel filtration) incorporated 180 into PCP to produce TeCH from H2180 but not from 1802 (6). However, they incubated authentic TeCH with H2180 in the presence of the protein fraction and found that 180 from H2180 was exchanged into TeCH. They suggested that it was also possible that the first dechlorinating step was by oxidative dehalogenation (6).

We report here the results of our 180 labelling experiments with the purified PCP hydroxylase. Our results show that the enzyme incorporated '80 from 802 but not from H2180. PCP is oxidatively converted to TeCH by a monooxygenasetype enzyme. Results from the previous studies on the incorporation of 180 from H2180 into TeCH, done with cell extracts or whole cells, were probably due to other proteins present which can enzymatically catalyze the exchange, as demonstrated by Schenk et al. (6). We did not observe such an exchange with the purified PCP hydroxylase. We thank Pierre Lafontaine of the Plant Research Centre for

performing the

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REFERENCES

83

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and R. Crawford and M. Morra for reviewing the manuscript. This research was supported in part by grant 91-34214-6051 from the USDA.

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1. Apajalahti, J. H. A., P. Kiirpinoja, and M. S. Salkinoja-Salonen. 1986. Rhodococcus chlorophenolicus sp. nov., a chlorophenolmineralizing actinomycete. Int. J. Syst. Bacteriol. 36:246-251. 2. Apajalahti, J. H. A., and M. S. Salkinoja-Salonen. 1987. Dechlorination and para-hydroxylation of polychlorinated phenols by Rhodococcus chlorophenolicus. J. Bacteriol. 169:675-681.

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3. Chu, J. P., and E. J. Kirsch. 1972. Metabolism of pentachlorophenol by an axenic bacterial culture. Appl. Microbiol. 23: 1033-1035. 4. Crosby, D. G. 1981. Environmental chemistry of pentachlorophenol. Pure Appl. Chem. 53:1051-1080. 5. Saber, D. L., and R. L. Crawford. 1985. Isolation and characterization of Flavobacterium strains that degrade pentachlorophenol. Appl. Environ. Microbiol. 50:1512-1518. 6. Schenk, T., R. Muller, and F. Lingens. 1990. Mechanism of enzymatic dehalogenation of pentachlorophenol by Arthrobacter sp. strain ATCC 33790. J. Bacteriol. 172:7272-7274. 7. Schenk, T., R. Muller, F. Morsberger, M. K. Otto, and F. Lingens. 1989. Enzymatic dehalogenation of pentachlorophenol by extracts from Arthrobacter sp. strain ATCC 33790. J. Bacteriol. 171:5487-5491. 8. Stanlake, G. J., and R. K. Finn. 1982. Isolation and characterization of a pentachlorophenol-degrading bacterium. Appl. Environ. Microbiol. 44:1421-1427. 9. Steiert, J. G., and R. L. Crawford. 1986. Catabolism of pen-

NOTES

10. 11. 12. 13.

14. 15.

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tachlorophenol by a Flavobacterium sp. Biochem. Biophys. Res. Commun. 141:825-830. Suzuki, T. 1977. Metabolism of pentachlorophenol by a soil microbe. J. Environ. Sci. Health Part B 12:113-127. Topp, E., and M. H. Akhtar. 1990. Mineralization of 3-phenoxybenzoate by a two-membered bacterial co-culture. Can. J. Microbiol. 36:495-499. Watanabe, I. 1973. Isolation of pentachlorophenol-decomposing bacteria from soil. Soil Sci. Plant Nutr. 19:109-116. Webb, E. C. 1984. Recommendations of the nomenclature committee of the International Union of Biochemistry on the nomenclature and classification of enzyme-catalyzed reactions. Academic Press, Inc., Orlando, Fla. Xun, L., and C. S. Orser. 1991. Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723. J. Bacteriol. 173:4447-4453. Xun, L., E. Topp, and C. S. Orser. 1992. Diverse substrate range of a Flavobacterium pentachlorophenol hydroxylase and reaction stoichiometries. J. Bacteriol. 174:2898-2902.