Graduate Thesis Or Dissertation
 

Investigating the basis of substrate specificity in butane monooxygenase and chlorinated ethene toxicity in Pseudomonas butanovora

Public Deposited

Downloadable Content

Download PDF
https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/02870z43h

Descriptions

Attribute NameValues
Creator
Abstract
  • Pseudomonas butanovora, Mycobacterium vaccae, and Nocardioides sp. CF8 utilize distinctly different butane monooxygenases (BMOs) to initiate degradation of recalcitrant chlorinated ethenes (CEs) that pollute aquifers and soils. BMO-dependent degradation of CEs such as trichloroethylene (TCE) can lead to cellular toxicities. The type and severity of TCE transformation-dependent damage can have different impacts on the bacterial host and community, potentially allowing for tolerance to TCE transformation. The physiological consequences of TCE transformation by the three butane-oxidizers were examined. Although the primary toxic event resulting from TCE cometabolism by these three strains was loss of BMO activity, species differences were observed. BMO of P. butanovora is the only member of the soluble methane monooxygenase (sMMO) subfamily of soluble diiron monooxygenases in which methane oxidation had not been measured. To investigate the fundamental differences in substrate specificity between BMO and MMO, single amino acid substitutions were made to the hydroxylase α-subunit of BMO (BMOH-α). Striking differences in specific activities and regiospecificity were observed for mutant strains G113N and L279F. The predominantly sub-terminal oxidation of propane and butane by strain G113N suggests the single amino acid substitution caused a significant alteration of BMOH-α active site geometry. The sensitivity of methane oxidation by BMO to methanol may have significant implications associated with product release. The differences in regiospecificity of P. butanovora mutant strains relative to wild-type extend to CEs. Although the wild-type strain released nearly all available chlorine during CE exposures, strain G113N released less than 25% of available DCE chlorine and only 56% of available TCE chlorine. Half the amount of CE epoxide was formed by strain G113N during CE degradation as compared to the wild-type strain. Furthermore, differences in CE epoxide degradation suggest the mutant strains have altered activity towards epoxides. Lactate-dependent O2 uptake rates were differentially affected by DCE degradation, providing evidence that different products or product ratios are released by the altered BMOs that have remarkable impacts on cellular toxicity. The use of CEs as mechanistic probes in combination with P. butanovora BMOH-α mutants provided unexpected insights to the catalytic mechanism of BMO.
License
Resource Type
Date Available
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Committee Member
Non-Academic Affiliation
Subject
Rights Statement
Publisher
Peer Reviewed
Language
File Format
File Extent
  • 3056915 bytes
Replaces

Relationships

Parents:

This work has no parents.

In Collection:

Items

Thumbnail Title Date Uploaded Visibility Actions
file details KHH_Dissertation.pdf 2017-08-07 Public Download