| This dissertation describes the biochemistry and molecular characterization of the (iso)eugenol O-methyltransferase (IEMT), an enzyme that catalyzes the formation of two floral scent compounds, methyleugenol and isomethyleugenol. IEMT enzyme was purified from C. breweri petals and characterized in respect to molecular weight, kinetic parameters Km and K cat, pH optimum, and other biochemical parameters. A cDNA encoding IEMT was isolated, and its sequence was determined. The regulation of the IEMT gene in C. breweri was further studied by northern blots, in situ hybridization and western blots. A cDNA clone encoding caffeic acid O-methyltransferase (COMT) was also isolated from C. breweri . The sequence of IEMT was found to be very similar to that of COMT. The substrate specificity of IEMT was studied using recombinant protein and domain swapping with COMT. To test the hypothesis that IEMT evolved from COMT in Clarkia and in other plant species, COMT and other O-methyltransferases capable of methylating eugenol and chavicol were purified and partly characterized from basil (Ocimum basilicum). This study provides a better understanding of floral scent biogenesis and the evolution of floral scent, as well as contributing to the general understanding of methyltransferases involved in other plant secondary metabolic pathways. |
