Characterization of the interactions of a lignostilbene alpha-beta dioxygenase with both stilbene and carotenoid substrates
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Authors
Zara, Anthony
Date
Type
thesis
Language
eng
Keyword
molecular biology , structural biology , enzymes , carotenoids , stilbenes , dioxygenase
Alternative Title
Abstract
As one of many evolutionary compensations for immobility, plants synthesize an array of natural products and metabolites which afford advantage against environmental stresses. The utility of these products is exemplified by stilbene and carotenoid-derived metabolites which are important in plant processes such as immune response and protection against UV-light damage. Not limited to plants, these metabolites even play roles in the maintenance of human health as antioxidants and in vision. Stilbene and carotenoid derivatives are generated by a family of non-heme, iron-coordinating enzymes, divided into two functionally distinct sub-families, including lignostilbene cleavage dioxygenases (LCDs) and carotenoid cleavage oxygenases (CCOs). While these enzymes have highly similar structure and identical reaction mechanisms, LCDs and CCOs have widely different substrate specificities, mediated by factors that currently remain enigmatic. To gain insight into these factors, a previously characterized bacterial LCD from the bio-control agent Pseudomonas brassicacearum (PbLSD) was compared to an array of CCOs by amino acid sequence and structural alignments, as well as in silico docking. This yielded several target residues predicted to contribute to substrate selectivity within the binding pocket. These predictions were tested using an in vivo carotenoid-cleavage assay system combined with site-directed mutagenesis. Current results emphasize that the structure of the binding pocket plays a key role in mediating substrate specificities, with potential applications for the commercial production of desirable carotenoid derivatives. Additional work characterizing specificities and reaction products of purified PbLSD-mutants in vitro, by liquid chromatography and mass spectroscopic methods is ongoing.
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Attribution 3.0 United States
Queen's University's Thesis/Dissertation Non-Exclusive License for Deposit to QSpace and Library and Archives Canada
ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
Queen's University's Thesis/Dissertation Non-Exclusive License for Deposit to QSpace and Library and Archives Canada
ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.