Structure-function relationships in plant terpene synthases

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dc.contributor.advisor Professor Ted Baker en
dc.contributor.advisor Dr William Laing en
dc.contributor.author Green, Sol Alexander en
dc.date.accessioned 2009-11-19T20:58:11Z en
dc.date.available 2009-11-19T20:58:11Z en
dc.date.issued 2009 en
dc.identifier.citation Thesis (PhD--Biological Sciences)--University of Auckland, 2009. en
dc.identifier.uri http://hdl.handle.net/2292/5560 en
dc.description.abstract Terpene synthases (TPS) are a family of enzymes responsible for synthesising the vast array of terpenoid compounds known to exist in nature. In plants these compounds serve to increase ecological fitness, while commercially they represent one of the key compound classes in determining the quality of food products, including flavour and aroma in fruit crops. Preliminary functional characterisation of an apple (Malus x domestica) TPS responsible for the production of α-farnesene (MdAFS1), which constitutes the major terpene volatile in apple, showed novel features not previously reported for a TPS enzyme. These included the apparent ability of MdAFS1 to generate α-farnesene by a prenyltransferase reaction and being the first angiosperm TPS with a dependence on potassium for activity. The focus of this PhD study was to extend the preliminary functional analysis of MdAFS1 and provide definitive proof for this prenyltransferase activity and also the location and nature of the MdAFS1 potassium binding region. In the research presented here, mutagenesis of the MdAFS1 aspartate-rich divalent metal ion binding motif removed TPS and prenyltransferase activities, showing not only that MdAFS1 possessed intrinsic prenyltransferase function but that both TPS and prenyltransferase catalysis were occurring through the same active site. Protein modelling also revealed a surface-exposed loop (H-αl loop) in MdAFS1 that fulfilled the necessary requirements for a potassium binding region. Site-directed mutagenesis analysis of specific residues within this loop then revealed their crucial importance to this potassium response and strongly implicated specific residues in direct potassium binding. The role of the H-αl loop in terpene synthase potassium coordination was confirmed in a conifer pinene synthase also using site-directed mutagenesis. These findings provide the first direct evidence for a specific potassium binding region in two functionally and phylogenetically divergent terpene synthases. They also provide a basis for understanding potassium activation in other TPS enzymes and establish a new role for the H-αl loop region in TPS catalysis. Structural insights were also sought for how MdAFS1 might have evolved from an ancestral mono-TPS enzyme. A combination of contact mapping and mutagenesis analysis identified a single non-synonymous nucleotide substitution that could explain one route for the requisite shift in substrate specificity necessary for α-farnesene synthesis. Abstract II Taken together, the work presented in this thesis provides the foundation for future investigation into the catalytic mechanisms and evolution of plant terpene synthases. Broadening the scope of this and related studies so as to begin unravelling the complex interplay between plant ecological fitness and TPS catalytic specificity still remains an enormous future challenge. en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.relation.isreferencedby UoA1937132 en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.title Structure-function relationships in plant terpene synthases en
dc.type Thesis en
thesis.degree.discipline Forensic Science en
thesis.degree.grantor The University of Auckland en
thesis.degree.level Doctoral en
thesis.degree.name PhD en
dc.date.updated 2009-11-19T20:58:12Z en
dc.rights.holder Copyright: The author en
pubs.local.anzsrc 06 - Biological Sciences en
pubs.org-id Faculty of Science en


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