Examination of 2-Oxoglutarate Dependant Dioxygenases Leading to the Production of Flavonols in Arabidopsis thaliana

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dc.contributor.authorOwens, Daniel Kennethen
dc.contributor.committeechairWinkel, Brenda S. J.en
dc.contributor.committeememberSible, Jill C.en
dc.contributor.committeememberWalker, Richard A.en
dc.contributor.committeememberRutherford, Charles L.en
dc.contributor.committeememberMcDowell, John M.en
dc.contributor.departmentBiologyen
dc.date.accessioned2014-03-14T20:16:55Zen
dc.date.adate2005-10-21en
dc.date.available2014-03-14T20:16:55Zen
dc.date.issued2005-09-22en
dc.date.rdate2007-10-21en
dc.date.sdate2005-09-30en
dc.description.abstractThe flavonols are a varied and abundant sub-class of flavonoids that are associated with a number of essential physiological functions in plants and pharmacological activities in animals. The 2-oxoglutarate-dependant dioxygenases(2-ODDs), flavonol synthase (FLS) and flavanone 3-hydroxylase (F3H), are essential for flavonol synthesis. The primary goal of this study has been to gain a deeper understanding of the biochemistry of these enzymes in Arabidopsis. To accomplish this goal, an activity assay employing recombinant protein expression and HPLC as a detection system was developed for F3H and adapted for use with FLS. The assay was employed to establish the biochemical parameters of F3H from Arabidopsis, and to further characterize the F3H mutant allele, <i>tt6</i>(87). Enzymatic activity was demonstrated for F3H enzymes from <i>Ipomoea alba</i> (moonflower), <i>Ipomoea purpurea</i> (common morning glory), <i>Citrus sinensis</i> (sweet orange), and <i>Malus X domestica</i> (newton apple), each of which had previously been identified solely based on sequence homology. Arabidopsis contains six genes with high similarity to <i>FLS</i> from other plant species; however, all other central flavonoid pathway enzymes in Arabidopsis are encoded by single genes. The hypothesis that differential expression of FLS isozymes with varying substrate specificities is responsible for observed tissue-specific differences in flavonol accumulation was tested. Sequence analysis revealed that <i>AtFLS2, 4</i> and <i>6</i> contain premature stop codons that eliminate residues essential for enzyme activity. AtFLS1 was found to have a strong preference for dihydrokaempferol as a substrate. However, no enzyme activity was observed for AtFLS3 or AtFLS5 with a number of different substrates under a variety of reaction conditions. To identify structural elements that may contribute to the observed differences in biochemical activity, homology models for each of the isoforms were generated utilizing Arabidopsis anthocyanin synthase (ANS) as a template. A domain at the N-terminus of AtFLS1 that is missing in the other isozymes was insufficient to convey activity to an AtFLS1/5 chimera. These findings suggest a single catalytically-active form of FLS exists in Arabidopsis. The possibility that the apparently expressed but non-catalytic proteins, AtFLS2, 3, and 5, serve noncatalytic roles in flavonol production were explored by yeast 2-hybrid analysis.en
dc.description.degreePh. D.en
dc.identifier.otheretd-09302005-204631en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-09302005-204631/en
dc.identifier.urihttp://hdl.handle.net/10919/29144en
dc.publisherVirginia Techen
dc.relation.haspartdowens_final_update.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subject2-oxoglutarate dependant dioxygenasesen
dc.subjectMichaelis-Menten kineticsen
dc.subjectflavanone 3-hydroxylaseen
dc.subjectflavonoid biosynthesisen
dc.subjectflavonol synthaseen
dc.titleExamination of 2-Oxoglutarate Dependant Dioxygenases Leading to the Production of Flavonols in <i>Arabidopsis thaliana</i>en
dc.typeDissertationen
thesis.degree.disciplineBiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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