Abstract
Cytochrome P-450CAM was shown to be the primary catalyst mediating reductive dehalogenation of polychlorinated ethanes byPseudomonas putida G786. Under anaerobic conditions, the enzyme catalyzed reductive elimination reactionsin vivo with the substrates hexachloroethane, pentachloroethane, and 1,1,1,2-tetrachloroethane; the products were tetrachloroethylene, trichloroethylene, and 1,1-dichloroethylene, respectively.In vivo reaction rates were determined. No reaction was observed with 1,1,2,2-tetrachloroethane or 1,1,1-trichloroethane. Purified cytochrome P-450CAM was used to measure dissociation constants of polychlorinated ethanes for the enzyme active site. Observed rates and dissociation constants were used to predict the course of a reaction with the three substrates simultaneously. Data obtained from experiments withP. putida G786 generally followed the simulated reaction curves. Oxygen suppressed the reductive dechlorination reactions and, in the case of 1,1,1,2-tetrachloroethane, 2,2,2-trichloroacetaldehyde was formed. Significant rates of reductive dechlorination were observed at 5% oxygen suggesting that these reactions could occur under partially aerobic conditions. These studies highlight the potential to use an aerobic bacterium,P. putida G786, under a range of oxygen tensions to reductively dehalogenate mixed wastes which are only degraded at very low rates by obligately anaerobic bacteria.
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Abbreviations
- GC/MS:
-
Gas chromatography/mass spectrometry
- P-450CAM :
-
Cytochrome m of the camphor oxidizing system ofP. putida
- pca:
-
Polychlorinated ethane
References
Ahr HJ, King LJ, Nastainczyk W & Ullrich V (1982) The mechanism of reductive dehalogenation of halothane by liver cytochrome P-450. Biochem. Pharmacol. 31:383–390
Bradshaw WH, Conrad HE, Corey EJ, Gunsalus IC & Lednicer D (1959) Microbiological degradation of (+)- Camphor. J. Am. Chem. Soc. 81:5507
Castro CE & Belser NO (1990) Biodehalogenation: Oxidative and reductive metabolism of 1,1,2-trichloroethane byPseudomonas putida — Biogeneration of vinyl chloride. Environ. Toxicol. Chem. 9:707–714
Castro CE, Wade RS & Belser NO (1985) Biodehalogenation: Reactions of cytochrome P-450 with polyhalomethanes. Biochemistry 24:204–210
Davies MD, Koga H, Horiuchi T & Sligar SG (1990) Site-directed mutagenesis of thePseudomonas cam Operon. In: Silver S, Chakrabarty AM, Iglewski B & Kaplan S (Eds)Pseudomonas: Biotransformations, Pathogenesis, and Evolving Biotechnology. Amer. Soc. Microbiol. Press, Washington, pp 101–110
DeWeerd KA & Suflita JM (1990) Anaerobic aryl reductive dehalogenation of halobenzoates by cell extracts ofDesulfomonile tiedjei. Appl. Environ. Microbiol. 56: 2999–3005
Dolfing J (1990) Reductive dechlorination of 3-chlorobenzoate is coupled to ATP production and growth in an anaerobic bacterium, strain DCB-1. Arch. Microbiol. 153: 264–266
Egli C, Scholtz R, Cook AM & Leisinger T (1987) Anaerobic dechlorination of tetrachloromethane and 1,2-dichloroethane to degradable products by pure cultures ofDesulfobacterium sp. andMethanobacterium sp. FEMS Microbiol. Lett. 43:257–261
Egli C, Tschan T, Scholtz R, Cook AM & Leisinger T (1988) Transformation of tetrachloromethane to dichloromethane and carbon dioxide byAcetobacterium woodii. Appl. Environ. Microbiol. 54:2819–2824
Fathepure BZ & Vogel TM (1991) Complete degradation of polychlorinated hydrocarbons by a two-stage biofilm reactor. Appl. Environ. Microbiol. 57: 3418–3422
Fersht A (1985) Enzyme Structure and Mechanism. WH Freeman & Co. New York.
Fisher MT & Sligar SG (1985) Control of heme protein potential and reduction rate: Linear free energy relation between potential and ferric spin state equilibrium. J. Am. Chem. Soc. 107: 5018–5019
Gunsalus IC & Wagner GC (1978) Bacterial P-450CAM Methylene Monooxygenase Components: Cytochromem, Putidaredoxin, and Putidaredoxin Reductase. In: Fleischer S & Packer L (Eds Methods in Enzymology, Vol LII, Academic Press, New York p166–188
Gunsalus IC, Meeks JR, Lipscomb JD, Debrunner P & Münck E (1974) Bacterial monooxygenases — The P-450 cytochrome system. In: Hayaishi O (Ed) Molecular Mechanisms of Oxygen Activation. Academic Press, New York. pp 559–613
Hedegaard J & Gunsalus IC (1965) Mixed function oxidation. IV. An induced methylene hydroxylase in camphor oxidation. J. Biol. Chem. 240: 4038–4043
Koga H, Aramaki H, Yamaguchi E, Takeuchi K, Horiuchi T & Gunsalus IC (1986)CAMR, a negative regulator locus of the cytochrome P-450CAM hydroxylase operon. J. Bacteriol. 166: 1089–1095
Luke BT & Loew GH (1986) A theoretical investigation of the first step in the metabolic reduction of halogenated methanes by cytochrome P-450. Internat. J. Quant. Chem.: Quant. Biol. Symp. 12: 99–112
Mikesell MD & Boyd SA (1990) Dechlorination of chloroform byMethanosarcina strains. Appl. Environ. Microbiol. 56: 1198–1201
Mohn WW & Tiedje JM (1990). Strain DCB-1 conserves energy for growth from reductive dechlorination coupled to formate oxidation. Arch. Microbiol.153: 267–271
Poulos TL, Finzel BC, Gunsalus IC, Wagner GC & Kraut J (1985) The 2.6Å crystal structure ofPseudomonas putida cytochrome P-450. J. Biol. Chem. 260: 16122–16130
Raag R & Poulos TL (1989) The structural basis for substrate-induced changes in redox potential and spin equilibrium in cytochrome P-450CAM. Biochemistry 28: 917–922
Raag R & Poulos TL (1991) Crystal structures of cytochrome P-450CAM complexed with camphane, thiochamphor, and adamantane: Factors controlling substrate hydroxylation. Biochemistry 30: 2674–2684
Schanke CA & Wackett LP (1992) Environmental reductive elimination reactions of polychlorinated ethanes mimicked by transition-metal coenzymes. Environ. Sci. Technol. 26: 830–833
Segel IH (1975) Enzyme Kinetics. John Wiley and Sons. New York
Sligar SG & Gunsalus IC (1976) A thermodynamic model of regulation: Modulation of redox equilibria in camphor monooxygenase. Proc. Natl. Acad. Sci. USA 73: 1078–1082
Stanier RY, Palleroni NJ & Doudoroff M (1966) The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43:159–271
Tiedje JM, Boyd SM & Fathepure BZ (1987) Anaerobic biodegradation of chlorinated hydrocarbons. Dev. Ind. Microbiol. 27: 117–127
Vogel TM, Criddle CS & McCarty, PL, (1987) Transformations of halogenated aliphatic compounds. Environ. Sci. Technol. 21: 722–736
Westerick JJ, Mello JW & Thomas RF (1984) The groundwater supply survey. J. Am. Water Works Assoc. 76: 52–59
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Logan, M.S.P., Newman, L.M., Schanke, C.A. et al. Cosubstrate effects in reductive dehalogenation byPseudomonas putida G786 expressing cytochrome P-450CAM . Biodegradation 4, 39–50 (1993). https://doi.org/10.1007/BF00701453
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DOI: https://doi.org/10.1007/BF00701453