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dc.creatorHayes, Robert Patrick
dc.date.accessioned2014-08-27T18:25:37Z
dc.date.available2014-08-27T18:25:37Z
dc.date.issued2013
dc.identifier.urihttp://hdl.handle.net/2376/5066
dc.descriptionThesis (Ph.D.), Washington State Universityen_US
dc.description.abstractPolychlorophenols are toxic compounds that exist as persistent environmental pollutants. Several bacteria have been discovered which are capable of the complete metabolism of various polychlorophenols and thus have been proposed as potential bioremediation solutions. The bacteria contain operons encoding enzymes that work together to dechlorinate the aromatic ring, cleave the ring to its constituent carbon skeleton and ultimately produce a compound that enters the tricarboxylic acid cycle. We studied polychlorophenol degradation in Cupriavidus necator JMP134, Burkholderia phenoliruptrix AC1100 and Sphingobium chlorophenolicum, which degrade 2,4,6-trichlorophenol, 2,4,5-trichlorophenol and pentachlorophenol respectively. This dissertation is composed of three studies that investigate enzymes, one from each organism mentioned above, that conduct unique chemistry and thus were selected for structure determination, biochemical characterization and mechanistic analysis. The first study was a structural and catalytic comparison between 2,4,6-trichlorophenol-4-monooxygenase (TcpA) from C. necator JMP134 and 2,4,5-trichlorophenol-4-monooxygenase (TftD) from B. phenoliruptrix AC1100. We determined the crystal structure of TcpA and conducted docking experiments that suggest Alanine 293 in TcpA and Isoleucine 292 in TftD are the direct cause for differences in substrate specificity between the two enzymes. We also propose a mechanism for hydrolytic dechlorination in TcpA. The second study investigated a non-heme Fe (II) dioxygenase, 2,6-dichloro-p-hydroquinone 1,2-dioxygenase (PcpA), from S. chlorophenolicum. The crystal structure, catalytic residues and enzyme kinetics for PcpA were determined. We discovered the substrate p-hydroxyl group was essential for binding and thus phenols and catechols did not bind to PcpA, distancing it from other known intradiol and extradiol catechol dioxygenases. We propose a general reaction mechanism for the novel p-hydroquinone 1,2-dioxygenases. The third study characterized 5-chlorohydroxyhydroquinone dehydrochlorinase (TftG) from B. phenoliruptrix AC1100. We determined the crystal structure of TftG in both apo-form and complex-form with the product analog 2,5-dihydroxybenzoquinone. Site-directed mutagenesis was conducted to confirm the role of several active site residues. Our data support the presence of a histidine-aspartic acid catalytic dyad and an oxyanion hole in the active site. The residues implicated for catalysis in TftG were conserved across the YCII-superfamily proteins of largely unknown function. We determined the signature sequence for the YCII-superfamily proteins and propose they are likely a family of aromatic ring lyases.en_US
dc.description.sponsorshipDepartment of Chemistry, Washington State Universityen_US
dc.language.isoEnglish
dc.rightsIn copyright
dc.rightsNot publicly accessible
dc.rightsclosedAccess
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/
dc.rights.urihttp://www.ndltd.org/standards/metadata
dc.rights.urihttp://purl.org/eprint/accessRights/ClosedAccess
dc.subjectBiochemistryen_US
dc.titleEnzymatic degradation of polychlorophenols
dc.typeElectronic Thesis or Dissertation


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