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dc.contributor.advisorKang, ChulHee
dc.creatorSattler, Steven
dc.date.accessioned2019-08-21T17:57:05Z
dc.date.available2019-08-21T17:57:05Z
dc.date.issued2018
dc.identifier.urihttp://hdl.handle.net/2376/16368
dc.descriptionThesis (Ph.D.), Molecular Biosciences, Washington State Universityen_US
dc.description.abstractWe conducted investigations of the structural and biochemical properties of two distinct types of enzyme from different pathways: cinnamoyl-CoA reductases (CCRs) and the persulfide dioxygenases (PDOs). CCR is an enzyme of the monolignol biosynthesis pathway in plants that uses NADPH to reduce any of three major hydroxycinnamoyl-CoA thioesters, thus catalyzing the formation of hydroxycinnamaldehydes. These aldehydes are further reduced by cinnamyl alcohol dehydrogenase (CAD) to form the alcohol substrates required for lignin biosynthesis. One CCR, SbCCR1, was crystallized in the presence of NADPH and the structure was determined at 2.9 Å resolution. It was determined—through site-directed mutagenesis, ITC, molecular docking, and kinetics assays—that not only is feruloyl-CoA the preferred substrate for SbCCR1, but residues Thr154 and Tyr310 are essential to binding functional groups about the aromatic ring of the substrate. Production of a T154Y mutant of SbCCR1 resulted in significant reduction in substrate preference for feruloyl-CoA over p-coumaroyl-CoA, providing support for the hypothesis that substitutions can be made at this position leading to favorable reductions in or softening of lignin content in S. bicolor. Furthermore, confirmed the existence of an additional CCR in S. bicolor through multiple-sequence alignment and kinetics analyses. PDOs catalyze the oxidation of a sulfane sulfur on glutathione persulfide (GSSH) or higher glutathione polysulfanes (GSS¬nH, where n > 1) during the process of detoxifying dihydrogen sulfide (H2S). In this study, the structures of PDOs from M. xanthus (MxPDO1) and P. putida (PpPDO2) were determined in the presence of a catalytic iron, as well as in the presence of both iron and product molecule glutathione for the PDO from P. putida. Structure alignments with other PDOs, in addition to the 1.46 Å PpPDO2/GSH complex structure, revealed key substrate binding residues and provided a basis for activity differences between PDOs and a closely related group of enzymes, the glyoxalases II. Static light scattering further showed that, despite high identity to the glyoxalases II, PDOs are likely dimeric. In summary, we described interactions between PDOs and GSH, as well as provided information as to how two highly related groups of enzymes could differ in form and function.en_US
dc.description.sponsorshipWashington State University, Molecular Biosciencesen_US
dc.languageEnglish
dc.rightsIn copyright
dc.rightsPublicly accessible
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/
dc.subjectBiochemistry
dc.subjectBiophysics
dc.subjectChemistry
dc.titleSTRUCTURAL AND MECHANISTIC CHARACTERIZATION OF ENZYMES IN PERSULFIDE OXIDATION AND MONOLIGNOL BIOSYNTHESIS PATHWAYS
dc.typeElectronic Thesis or Dissertation


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