J. Bacteriol., Jan 1997, 90-96, Vol 179, No. 1
Copyright © 1997, American Society for Microbiology

Trichloroethylene oxidation by purified toluene 2-monooxygenase: products, kinetics, and turnover-dependent inactivation

LM Newman and LP Wackett
Department of Biochemistry, the Biological Process Technology Institute, University of Minnesota, St. Paul 55108, USA.

Trichloroethylene is oxidized by several types of nonspecific bacterial oxygenases. Toluene 2-monooxygenase from Burkholderia cepacia G4 is implicated in trichloroethylene oxidation and is uniquely suggested to be resistant to turnover-dependent inactivation in vivo. In this work, the oxidation of trichloroethylene was studied with purified toluene 2- monooxygenase. All three purified toluene 2-monooxygenase protein components and NADH were required to reconstitute full trichloroethylene oxidation activity in vitro. The apparent Km and Vmax were 12 microM and 37 nmol per min per mg of hydroxylase component, respectively. Ten percent of the full activity was obtained when the small-molecular-weight enzyme component was omitted. The stable oxidation products, accounting for 84% of the trichloroethylene oxidized, were carbon monoxide, formic acid, glyoxylic acid, and covalently modified oxygenase proteins that constituted 12% of the reacted [14C]trichloroethylene. The stable oxidation products may all derive from the unstable intermediate trichloroethylene epoxide that was trapped by reaction with 4-(p-nitrobenzyl)pyridine. Chloral hydrate and dichloroacetic acid were not detected. This finding differs from that with soluble methane monooxygenase and cytochrome P-450 monooxygenase, which produce chloral hydrate. Trichloroethylene- dependent inactivation of toluene 2-monooxygenase activity was observed. All of the protein components were covalently modified during the oxidation of trichloroethylene. The addition of cysteine to reaction mixtures partially protected the enzyme system against inactivation, most notably protecting the NADH-oxidoreductase component. This suggested the participation of diffusible intermediates in the inactivation of the oxidoreductase.

This article has been cited by other articles:

• Holmes, V. F., He, J., Lee, P. K. H., Alvarez-Cohen, L. (2006). Discrimination of Multiple Dehalococcoides Strains in a Trichloroethene Enrichment by Quantification of Their Reductive Dehalogenase Genes. Appl. Environ. Microbiol. 72: 5877-5883 [Abstract] [Full Text]  
• Rui, L., Cao, L., Chen, W., Reardon, K. F., Wood, T. K. (2004). Active Site Engineering of the Epoxide Hydrolase from Agrobacterium radiobacter AD1 to Enhance Aerobic Mineralization of cis-1,2-Dichloroethylene in Cells Expressing an Evolved Toluene ortho-Monooxygenase. J. Biol. Chem. 279: 46810-46817 [Abstract] [Full Text]  
• Ayala-del-Rio, H. L., Callister, S. J., Criddle, C. S., Tiedje, J. M. (2004). Correspondence between Community Structure and Function during Succession in Phenol- and Phenol-plus-Trichloroethene-Fed Sequencing Batch Reactors. Appl. Environ. Microbiol. 70: 4950-4960 [Abstract] [Full Text]  
• Morono, Y., Unno, H., Tanji, Y., Hori, K. (2004). Addition of Aromatic Substrates Restores Trichloroethylene Degradation Activity in Pseudomonas putida F1. Appl. Environ. Microbiol. 70: 2830-2835 [Abstract] [Full Text]  
• Yu, E. W., Aires, J. R., Nikaido, H. (2003). AcrB Multidrug Efflux Pump of Escherichia coli: Composite Substrate-Binding Cavity of Exceptional Flexibility Generates Its Extremely Wide Substrate Specificity. J. Bacteriol. 185: 5657-5664 [Full Text]  
• Coleman, N. V., Mattes, T. E., Gossett, J. M., Spain, J. C. (2002). Phylogenetic and Kinetic Diversity of Aerobic Vinyl Chloride-Assimilating Bacteria from Contaminated Sites. Appl. Environ. Microbiol. 68: 6162-6171 [Abstract] [Full Text]  
• Park, J., Kukor, J. J., Abriola, L. M. (2002). Characterization of the Adaptive Response to Trichloroethylene-Mediated Stresses in Ralstonia pickettii PKO1. Appl. Environ. Microbiol. 68: 5231-5240 [Abstract] [Full Text]  
• Yeager, C. M., Bottomley, P. J., Arp, D. J. (2001). Requirement of DNA Repair Mechanisms for Survival of Burkholderia cepacia G4 upon Degradation of Trichloroethylene. Appl. Environ. Microbiol. 67: 5384-5391 [Abstract] [Full Text]  
• Hatzinger, P. B., McClay, K., Vainberg, S., Tugusheva, M., Condee, C. W., Steffan, R. J. (2001). Biodegradation of Methyl tert-Butyl Ether by a Pure Bacterial Culture. Appl. Environ. Microbiol. 67: 5601-5607 [Abstract] [Full Text]  
• Yeager, C. M., Bottomley, P. J., Arp, D. J. (2001). Cytotoxicity Associated with Trichloroethylene Oxidation in Burkholderia cepacia G4. Appl. Environ. Microbiol. 67: 2107-2115 [Abstract] [Full Text]  
• Magnuson, J. K., Romine, M. F., Burris, D. R., Kingsley, M. T. (2000). Trichloroethene Reductive Dehalogenase from Dehalococcoides ethenogenes: Sequence of tceA and Substrate Range Characterization. Appl. Environ. Microbiol. 66: 5141-5147 [Abstract] [Full Text]  
• Parales, R. E., Ditty, J. L., Harwood, C. S. (2000). Toluene-Degrading Bacteria Are Chemotactic towards the Environmental Pollutants Benzene, Toluene, and Trichloroethylene. Appl. Environ. Microbiol. 66: 4098-4104 [Abstract] [Full Text]  
• Lee, K. (1999). Benzene-Induced Uncoupling of Naphthalene Dioxygenase Activity and Enzyme Inactivation by Production of Hydrogen Peroxide. J. Bacteriol. 181: 2719-2725 [Abstract] [Full Text]  
• Yeager, C. M., Bottomley, P. J., Arp, D. J., Hyman, M. R. (1999). Inactivation of Toluene 2-Monooxygenase in Burkholderia cepacia G4 by Alkynes. Appl. Environ. Microbiol. 65: 632-639 [Abstract] [Full Text]  
• Ayoubi, P. J., Harker, A. R. (1998). Whole-Cell Kinetics of Trichloroethylene Degradation by Phenol Hydroxylase in a Ralstonia eutropha JMP134 Derivative. Appl. Environ. Microbiol. 64: 4353-4356 [Abstract] [Full Text]  
• Yee, D. C., Maynard, J. A., Wood, T. K. (1998). Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-Monooxygenase Constitutively. Appl. Environ. Microbiol. 64: 112-118 [Abstract] [Full Text]  
• Maeda, T., Takahashi, Y., Suenaga, H., Suyama, A., Goto, M., Furukawa, K. (2001). Functional Analyses of Bph-Tod Hybrid Dioxygenase, Which Exhibits High Degradation Activity toward Trichloroethylene. J. Biol. Chem. 276: 29833-29838 [Abstract] [Full Text]