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Journal of Bacteriology, July 2007, p. 5068-5074, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.00189-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Evidence for Modified Mechanisms of Chloroethene Oxidation in Pseudomonas butanovora Mutants Containing Single Amino Acid Substitutions in the Hydroxylase {alpha}-Subunit of Butane Monooxygenase{triangledown}

Kimberly H. Halsey,1 David M. Doughty,2 Luis A. Sayavedra-Soto,3 Peter J. Bottomley,2 and Daniel J. Arp3*

Molecular and Cellular Biology Program,1 Department of Microbiology,2 Department of Botany and Plant Pathology, Oregon State University, Cordley 2082, Corvallis, Oregon 97331-29023

Received 5 February 2007/ Accepted 29 April 2007

The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of Pseudomonas butanovora containing single amino acid substitutions in the {alpha}-subunit of butane monooxygenase hydroxylase (BMOH-{alpha}) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes (CEs) were reduced in mutant strain G113N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-cis-DCE and TCE oxidation, and strain G113N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-cis-DCE epoxide during oxidation of 1,2-cis-DCE, only about 50% of the 1,2-cis-DCE oxidized by strain G113N was detected as the epoxide. Evidence was obtained that 1,2-cis-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-cis-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain G113N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent O2 uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with P. butanovora BMOH-{alpha} mutants might allow insights into the catalytic mechanism of BMO to be obtained.

* Corresponding author. Mailing address: Department of Botany and Plant Pathology, Cordley 2082, Oregon State University, Corvallis, OR 97331. Phone: (541) 737-1294. Fax: (541) 737-5310. E-mail: arpd{at}science.oregonstate.edu

{triangledown} Published ahead of print on 11 May 2007.

Journal of Bacteriology, July 2007, p. 5068-5074, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.00189-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Cooley, R. B., Dubbels, B. L., Sayavedra-Soto, L. A., Bottomley, P. J., Arp, D. J. (2009). Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly 'Pseudomonas butanovora'. Microbiology 155: 2086-2096 [Abstract] [Full Text]  


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