Characterization of Two cDNA Clones Which EncodeO-Methyltransferases for the Methylation of both Flavonoid and Phenylpropanoid Compounds

Abstract

EnzymaticO-methylation of phenylpropanoid and flavonoid compounds is believed to be catalyzed by distinct classes ofO-methyltransferases [EC 2.1.1.6x]. The O-methylated derivatives of phenylpropanoids and flavonoids play an important role in lignification and as antimicrobial compounds, respectively. Two cDNA clones,OMT1andOMT2,which differ in three amino acid residues were isolated and characterized from the semiaquatic freshwater weedChrysosplenium americanum(Saxifragaceae). These two novel cDNA clones encode enzymes which catalyze the 3′-O-methylation of the flavonoid aglycones luteolin and quercetin, although they also catalyze the efficient 3/5-O-methylation of the phenylpropanoids caffeic and 5-hydroxyferulic acids, respectively. Both recombinant proteins were partially purified from anEscherichia coliexpression system and their kinetic parameters were compared using two flavonoids and two phenylpropanoids as substrates. Although both gene products methylate caffeic acid and 5-hydroxyferulic acid to a similar extent, they exhibit a threefold higher affinity for and a four- to sixfold increase in turnover of flavonoid compounds. The gene product ofOMT1accepts the flavonoid substrates luteolin and quercetin for methylation at a higher rate than that ofOMT2,as indicated by a two- to threefold increase in itsVmaxvalues and turnover ratios. The fact thatC. americanumaccumulates a variety of highly methylated flavonols and exhibits little lignification suggests that these two flavonoid OMT clones have retained their ability toO-methylate phenylpropanoids as well. These results are discussed in relation to differences in the amino acid sequences of these two clones, as well as with otherO-methyltransferases, and the evolutionary divergence of these genes in plants.