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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Partial Purification, Kinetic Analysis, and Amino Acid Sequence Information of a Flavonol 3-O-Methyltransferase from Serratula tinctoria¹

Plant Biochemistry Laboratory and Centre for Structural and Functional Genomics, Concordia University, Montreal, Quebec, Canada H4B 1R6

Serratula tinctoria (Asteraceae) accumulates mainly 3,3'-dimethylquercetin and small amounts of 3-methylquercetin as an intermediate. The fact that 3-methylquercetin rarely accumulates in plants in significant amounts, and given its important role as an antiviral and antiinflammatory agent that accumulates in response to stress conditions, prompted us to purify and characterize the enzyme involved in its methylation. The flavonol 3-O-methyltransferase (3-OMT) was partially purified by ammonium sulfate precipitation and successive chromatography on Superose-12, Mono-Q, and adenosine-agarose affinity columns, resulting in a 194-fold increase of its specific activity. The enzyme protein exhibited an expressed specificity for the methylation of position 3 of the flavonol, quercetin, although it also utilized kaempferol, myricetin, and some monomethyl flavonols as substrates. It exhibited a pH optimum of 7.6, a pI of 6.0, and an apparent molecular mass of 31 kD. Its K_m values for quercetin as the substrate and S-adenosyl-L-Met (AdoMet) as the cosubstrate were 12 and 45 µM, respectively. The 3-OMT had no requirement for Mg²⁺, but was severely inhibited by p-chloromercuribenzoate, suggesting the requirement for SH groups for catalytic activity. Quercetin methylation was competitively inhibited by S-adenosyl-L-homo-Cys with respect to the cosubstrate AdoMet, and followed a sequential bi-bi reaction mechanism, where AdoMet was the first to bind and S-adenosyl-L-homo-Cys was released last. In-gel trypsin digestion of the purified protein yielded several peptides, two of which exhibited strong amino acid sequence homology, upon protein identification, to a number of previously identified Group II plant OMTs. The availability of peptide sequences will allow the design of specific nucleotide probes for future cloning of the gene encoding this novel enzyme for its use in metabolic engineering.

² Present address: Département de Biochimie, Université de Montréal, C.P. 6128, Succersale Centre-ville, Montréal, QC Canada H3C 3J7.

³ Present address: Université de Poitiers, UFR Sciences, UMR CNRS 6161, 86022 Poitiers cedex, France.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.036442.

^* Corresponding author; e-mail ragibra{at}vax2.concordia.ca; fax 1–514–848–2881.

Received November 25, 2003; returned for revision January 30, 2004; accepted February 1, 2004.