CHARACTERIZATION OF S-ADENOSYL-L-METHIONINE-(ISO)EUGENOL O-METHYLTRANSFERASE INVOLVED IN FLORAL SCENT PRODUCTION IN CLARKIA-BREWERI

Eugenol, isoeugenol, methyleugenol, and isomethyleugenol are volatiles found in the floral scent of Clarkia breweri. With their distinct aro mas, they are used in many perfumes and food seasonings. Here we repor t the purification and characterization of (iso)eugenol O-methyltransf erase (IEMT), the enzyme that methylates eugenol or isoeugenol to make methyleugenol or isomethyleugenol, respectively, using S-adenosyl-L-m ethionine as the methyl donor. C. breweri IEMT was copurified with caf feic acid O-methyltransferase (COMT) from petals and purified to homog eneity from a bacterial expression system. IEMT is active as a homodim er with a subunit molecular mass of 40 kDa. It is stable at temperatur es up to 35 degrees C. It shows optimum activity at pH 7.5, and it doe s not require any cofactors for enzymatic activity. Plant-purified IEM T has K-m values of 7 and 58 mu M for eugenol and isoeugenol, respecti vely, and 27 mu M for SAM (30, 74, and 19 mu M, respectively, for the plant IEMT expressed in Escherichia coli). By substituting coding regi ons from COMT into IEMT, it was determined that the regions in IEMT in volved in substrate specificity are located in the first half of the p rotein sequence and that a small segment of 82 amino acids (amino acid s 92-173) accounts for the main differences between IEMT and COMT in b oth substrate specificity and methylation regiospecificity. (C) 1998 A cademic Press.