Partial purification, biochemical characterization, kinetic analysis, and amino acid sequencing information of a flavonol 3-O-methyltransferase from the leaves of Serratula tinctoria

Serratula tinctoria L. (Dyer's savory, Asteraceae) accumulates small amounts of 3-methylquercetin as an intermediate. 3-Methylquercetin is known as a specific inhibitor of viral RNA replication (Castrillo et al ., 1986; Castrillo and Carrasco, 1987), an anti-inflammatory and antiviral agent (Middleton and Kandaswami, 1993), and a phytoanticipin in tobacco plants in response to wounding and insect herbivory (Roda et al ., 2003). The flavonol 3-O-methyltransferase (3-OMT) was partially purified from Serratula leaf tissues using ammonium sulfate precipitation, Superose 12, Mono Q, and adenosine-affinity columns. Result of the purification showed an increase of 194-folds of enzyme specific activity. It catalyzes the methylation reaction of quercetin at position 3, yielding 3-methylquercetin. This enzyme exhibited substrate specificity towards flavonols, which classifies it as a member of the Group II OMT. It has a pH optimum of 7.6, a pl of 6.0, and an apparent molecular mass of 31 kDa, which is an unusual small protein size in contrast to the 40 to 45 kDa reported for members of this group thus far. Its K m values for quercetin and AdoMet were 12 and 45 oM, respectively. It has no requirement for Mg 2+ , and was severely inhibited by p -chloromercuribenzoate ( p CMB), an indication of SH groups requirement for catalytic activity. Kinetic analyses indicated that the enzyme followed an ordered bi-bi reaction mechanism, with AdoMet as the first binding substrate and AdoHcy being released last. Results of LC-MS/MS of the protein yielded several peptides, which showed sequence homology to a number of Group II plant OMTs in the MASCOT database