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Degree Name

MS (Master of Science)

Program

Biology

Date of Award

8-2015

Committee Chair or Co-Chairs

Cecilia A. McIntosh

Committee Members

Aruna Kilaru, Shivakumar P. Devaiah

Abstract

A flavonol specific glucosyltransferase cloned from Citrus paradisi has strict substrate and regio-specificity (Cp3OGT). The amino acid sequence of Cp3OGT was aligned with sequences of an anthocyanidin UDP- dependant glucosyltransferase (UGT) from Clitorea ternatea and a UGT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling to identify candidate regions followed by site directed mutagenesis, three double mutations were constructed and biochemically characterized. S20G+T21S mutant protein retained activity with flavonols similar to the wildtype Cp3OGT but the mutant had optimum activity at 60°C and broadened substrate acceptance to include the flavanone naringenin. S290C+S319A mutant protein retained 40% activity with quercetin relative to WT, and had an optimum pH shift. H154Y+Q87I mutant protein was only 10% active with quercetin relative to WT. Docking analysis revealed that H154, Q87 and S20 could be involved in orienting the acceptor molecules within the acceptor binding site whereas S319 and S290 residues are involved in maintaining the active site conformation.

Document Type

Thesis - Campus Only

Copyright

Copyright by the authors.

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