Evidence for modified mechanisms of chloroethene oxidation in Pseudomonas butanovora mutants containing single amino acid substitutions in the hydroxylase -subunit of butane monooxygenase

Molecular and Cellular Biology Program, Department of Microbiology, Department of Botany and Plant Pathology, Oregon State University, Cordley 2082, Corvallis, OR 97331-2902

^* To whom correspondence should be addressed. Email: arpd{at}science.oregonstate.edu.

	Abstract

The properties of dichloroethene (DCE) and trichloroethylene (TCE) oxidation by three mutant strains of P. butanovora containing single amino acid substitutions in the -subunit of butane monooxygenase hydroxylase (BMOH-) were compared to the wild-type strain (Rev WT). The rates of oxidation of the three chloroethenes (CEs) were reduced in mutant strain G113N and corresponded with slower 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 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 chlorine during 1,2 cis-DCE and TCE oxidation, and strain G113N released between 14 and 25%, and 56% of available chlorine during oxidation of DCE and TCE, respectively. 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 for 1,2 cis-DCE epoxide being a substrate for 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 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 not with strains L279F and G113N. Lactate-dependent O₂ uptake rates were differentially affected by DCE degradation among the mutant strains providing evidence that some products released by the altered BMOs have reduced the impact CE on cellular toxicity. The use of CEs as substrates in combination with P. butanovora BMOH- mutants might allow for insights into the catalytic mechanism of BMO.