Isoprenoid metabolism in lepidopteran insects and plants

• Main Author: Denton, Ryan Edward
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2009
• Subjects: Biochemistry; Organic chemistry
• ISBN: 9781124171579; 1124171576

Isoprenoids are important biological molecules required for a variety of metabolic processes, including electron transport (ubiquinones), glycoprotein biosynthesis (dolichols), and hydrophobic interactions (protein prenylation). This research focuses on two enzymes, one in insects and one in plants, that are essential for isoprenoid metabolism. Isopentenyl diphosphate isomerase (IPPI or IDI) is essential for the construction of isoprenyl diphosphates that are precursors to essentially all insect isoprenoid natural products. In the insect order Lepidoptera, IDI must also catalyze its respective chemistry with “homologous” substrates, as this provides various carbon skeletons that are precursors to juvenile hormones, an essential class of hormones found only in Lepidoptera. If the unique characteristics of IDI were better understood, organism-specific insecticides could be investigated that negatively affect the production of juvenile hormone. We have recently cloned the enzyme from two different lepidopteran insect species and have developed a computational 3D model for this insect IDI. Interestingly, the active site of the lepidopteran enzyme is not only larger but contains additional acidic and basic residues, which may be responsible for its unique specificity. Specific single point mutations within the active site of C. fumiferana IDI have been performed. Enzyme kinetics, substrate specificity, and the effect of these mutations with respect to catalytic efficiency and isomerization specificity will be presented. The second enzyme under investigation involves protein prenylation, which is vital to many biological processes (i.e. actin organization and calcium-mediated signal transduction). The fate of these prenylated proteins in plants and of the farnesylcysteine residue was recently linked to a plant farnesylcysteine lyase and a subsequent isoprenoid recycling and salvage pathway. The lyase enzyme is responsible for the oxidative metabolism of farnesylcysteine to farnesal and cysteine. Various isoprenyl cysteines were synthesized for enzymatic assays. Also in this report, farnesylcysteine lyase was investigated with respect to the enzyme’s relation to abscisic acid (ABA) signaling. To this end, an extraction/derivatization protocol allowed quantitation of farnesylcysteine by gas chromatography in Arabidopsis seedlings.