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Multifaceted in vivo and in vitro Characterization of cis-Prenyltransferase 5 from Solanum lycopersicum

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dc.contributor.advisor Akhtar, Tariq Rea, Kevin 2017-09-22T16:02:38Z 2017-09-22T16:02:38Z 2017-09 2017-09-15 2017-09-22
dc.description.abstract The widespread occurrence of polyprenols throughout the plant kingdom is well documented. These compounds are believed to be assembled by a class of enzymes designated as cis-prenyltransferases (CPTs), which are encoded by small, yet largely uncharacterized, CPT gene families in plants. This research aimed to identify and characterize the CPT from Solanum lycopersicum (tomato) responsible for polyprenol synthesis. RNAi-mediated knockdown of one member of the tomato CPT family (SlCPT5) reduced polyprenols in leaves by ~70%. Assays with recombinant SlCPT5 determined that the enzyme synthesizes polyprenols of approximately C50-C55 in length and accommodates a variety of trans-prenyl diphosphate precursors as substrates. Introduction of SlCPT5 into the polyprenol-deficient yeast rer2∆ mutant resulted in the accumulation of C55 polyprenols in yeast cells, restored proper protein N-glycosylation, and rescued the temperature sensitive growth phenotype associated with its polyprenol deficiency. Finally, site-directed mutagenesis of SlCPT5 identified a suite of amino acids that are catalytically essential. en_US
dc.description.sponsorship Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA). Natural Sciences and Engineering Research Council of Canada (NSERC). Canada Foundation for Innovation (CFI). en_US
dc.language.iso en en_US
dc.subject isoprenoids en_US
dc.subject polyisoprenoids en_US
dc.subject cis-prenyltransferase en_US
dc.subject CPT en_US
dc.subject polyprenol en_US
dc.subject dolichol en_US
dc.subject solanum lycopersicum en_US
dc.subject tomato en_US
dc.subject prenyltransferase en_US
dc.subject functional complementation en_US
dc.subject serial dilution en_US
dc.subject CPT5 en_US
dc.subject rer2 en_US
dc.subject substrate specificity en_US
dc.subject preferred substrate en_US
dc.subject enzyme characterization en_US
dc.subject carotenoids en_US
dc.title Multifaceted in vivo and in vitro Characterization of cis-Prenyltransferase 5 from Solanum lycopersicum en_US
dc.type Thesis en_US Molecular and Cellular Biology en_US Master of Science en_US Department of Molecular and Cellular Biology en_US
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