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Investigations of the Z-selective sesquiterpene cyclase; 7-epizingiberene synthase

Jones, Christopher 2018. Investigations of the Z-selective sesquiterpene cyclase; 7-epizingiberene synthase. PhD Thesis, Cardiff University.
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Sesquiterpene cyclases convert linear isoprenyl diphosphates into complex hydrocarbon structures that include multiple ring systems, chiral centres and various stereochemistry. The wild tomato plant (Solanum habrochaites) has shown resistance against herbivore attack that is not observed in the domesticated moneymaker tomato species. Wild tomatoes release sesquiterpenes; 7-epizingiberene and its dehydrogenated form (R)-curcumene to repel adult whiteflies prior to landing, whilst moneymaker tomatoes do not exhibit this ability. 7-Epizingiberene has been reported as toxic to whitefly and a phytoalexin that causes birth defects in offspring. As a result, the use of 7-epizingiberene to combat whitefly infestation gives a new possibility for crop protection, circumventing the use of potentially dangerous and environmentally damaging pesticides. This thesis focuses on 7-epizingiberene synthase (EZS), a sesquiterpene cyclase that defies convention by using (2Z,6Z) farnesyl diphosphate as its substrate and not the universal substrate (2E,6E) farnesyl diphosphate. The project is divided into three sections. The first section involved the synthesis of (2Z,6Z) farnesyl diphosphate followed by the expression and purification of 7-epizingiberene synthase. Due to cost implications of purchasing (2Z,6Z) farnesol, multiple synthetic routes were investigated for the synthesis (2Z,6Z) farnesyl diphosphate. A Horner-Wadsworth-Emmons olefination using a Still-Gennari modified reagent gave the greatest yield of the Z isomer which was used for further reactions. Various cell lines and expression tags were used to optimise EZS expression and purification, whilst incubations of synthesised (2Z,6Z) farnesyl diphosphate with EZS gave catalytic products consistent with literature. The second portion of the project was the synthesis of (2Z,6Z)-[1-3H]-farnesyl diphosphate for the measurement of steady state parameters of EZS and EZS mutants. Multiple oxidations were attempted during the synthesis of radiolabelled substrate, with an oxoammonium-catalysed oxidation shown to retain the stereochemistry of the C2,C3 double bond. EZS mutants were used to investigate the metal binding motifs responsible for class I activity whilst numerous aromatic residues were subjected to single point mutation to investigate their role within the active site pocket. The third section involved the synthesis of substrate analogues to investigate the catalytic mechanism and substrate specificity of EZS. The synthesis of deuterated and fluorinated analogues were used to investigate prospective hydride shifts and carbocation intermediates respectively. The stereochemistry required for substrate turnover was probed by the synthesis of all isomers of farnesyl diphosphate whilst oxygenated and methylated substrates were synthesised for the potential production of novel sesquiterpene products.

Item Type: Thesis (PhD)
Date Type: Publication
Status: Unpublished
Schools: Chemistry
Subjects: Q Science > QD Chemistry
Date of First Compliant Deposit: 2 April 2019
Last Modified: 04 Aug 2022 02:05

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