Characterization ofS-Adenosyl-l-Methionine:(Iso)eugenolO-Methyltransferase Involved in Floral Scent Production inClarkia breweri

Abstract

Eugenol, isoeugenol, methyleugenol, and isomethyleugenol are volatiles found in the floral scent ofClarkia breweri.With their distinct aromas, they are used in many perfumes and food seasonings. Here we report the purification and characterization of (iso)eugenolO-methyltransferase (IEMT), the enzyme that methylates eugenol or isoeugenol to make methyleugenol or isomethyleugenol, respectively, usingS-adenosyl-l-methionine as the methyl donor.C. breweriIEMT was copurified with caffeic acidO-methyltransferase (COMT) from petals and purified to homogeneity from a bacterial expression system. IEMT is active as a homodimer with a subunit molecular mass of 40 kDa. It is stable at temperatures up to 35°C. It shows optimum activity at pH 7.5, and it does not require any cofactors for enzymatic activity. Plant-purified IEMT hasKmvalues of 7 and 58 μM for eugenol and isoeugenol, respectively, and 27 μM for SAM (30, 74, and 19 μM, respectively, for the plant IEMT expressed inEscherichia coli). By substituting coding regions from COMT into IEMT, it was determined that the regions in IEMT involved in substrate specificity are located in the first half of the protein sequence and that a small segment of 82 amino acids (amino acids 92–173) accounts for the main differences between IEMT and COMT in both substrate specificity and methylation regiospecificity.