Partial Purification, Kinetic Analysis, and Amino Acid Sequence Information of a Flavonol 3-<em>O</em>-Methyltransferase from <em>Serratula tinctoria</em><sup><a href="#fn1" rid="fn1" class=" fn">1</a></sup>
Abstract
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 Km 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 Mg, 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.
Flavonoid compounds constitute one of the most ubiquitous groups of natural plant products. They exhibit a wide range of functions and play important roles in the biochemistry, physiology, and ecology of plants. These include their contribution to flower color, protection against UV radiation and pathogenic organisms, promotion of pollen germination and pollen fertility, and activation of Rhizobium nodulation genes. They also act as growth regulators, enzyme inhibitors, insect antifeedants, and antioxidants, and are of potential benefit to human health (Bohm, 1998, and references therein). Flavonoids owe their structural biodiversity to a number of enzyme-catalyzed substitution reactions (Ibrahim and Anzellotti, 2003). Of these, enzymatic O-methylation, which is catalyzed by a family of S-adenosyl-l-Met (AdoMet)-dependent O-methyltransferases (OMTs; Ibrahim and Muzac, 2000), involves the transfer of the methyl group of AdoMet to the hydroxyl groups of an acceptor molecule, with the concomitant formation of the corresponding methyl ether derivative and S-adenosyl-l-homo-Cys (AdoHcy) as products. O-Methylation of flavonoids neutralizes the reactivity of their hydroxyl groups and alters their solubility and, hence, their intracellular compartmentation.
Flavonoid OMTs are substrate-specific, position-oriented enzymes, as was shown with a number of distinct enzymes catalyzing the stepwise O-methylation in Chrysosplenium americanum of the pentahydroxyflavone, quercetin (Q) → 3-methylquercetin (3-MeQ) → 3,7-diMeQ → 3,7,4′-triMeQ. After hydroxylation of the latter intermediate at positions 6 and/or 2′, 3,7,4′-triMeQ is further methylated to 3,7,4′,5′-tetraMeQ or to 3,6,7,2′,4′-pentaMe quercetagetin (Ibrahim et al., 1987), both of which are among the major flavonoid metabolites that accumulate in this plant (Collins et al., 1981). Stepwise O-methylation of Q by distinct OMTs has also been reported in apple (Malus domestica) cell cultures (Macheix and Ibrahim, 1984) and in spinach (Spinacia oleracea) leaves (Thresh and Ibrahim, 1985). In all these examples, O-methylation at position 3 constitutes the first committed step of the methylation sequence. This may explain the reason why 3-O-methyl flavonols rarely accumulate in plants, since they serve as intermediates in the biosynthetic pathway of partially/highly methylated flavonoids. However, 3-MeQ has been detected in small amounts in a number of plant species including Begonia, Centauria, Greyia, Nicotiana, and Serratula (for review, see Gottlieb, 1975).
3-O-Methylation of Q confers some distinct properties to this compound. In addition to being an antiinflammatory and antiviral agent (Malhotra et al., 1996; Middleton and Kandaswami, 1993), 3-MeQ selectively inhibits poliovirus RNA replication (Castrillo and Carrasco, 1987) and more selectively, phosphodiesterase Subtype 3 (Ko et al., 2003). Furthermore, 3-MeQ has recently been reported to accumulate in tobacco (Nicotiana tabacum) leaf trichomes, together with other methylated flavonols, in response to wounding stress and herbivory (Roda et al., 2003). With the exception of the cDNA encoding the 3-O-methylation of flavonols, several flavonoid OMT cDNA clones have been isolated and characterized, including two for chalcones, one for a flavone, two for isoflavones, and three for flavonols (Ibrahim and Muzac, 2000, and references therein).
Serratula tinctoria accumulates mainly 3,3′-dimethylquercetin (3,3′-diMeQ) and small amounts of 3-MeQ as an intermediate (Fig. 1), suggesting the existence in this plant of Q 3-OMT and 3-MeQ 3′-OMT enzyme proteins. Besides its high content of ecdysteroids, these methylated flavonols contribute to the yellow color of the root sap that is used as a folkloric dye, for which it became known as Dyer's savory (Corio-Costet et al., 1991).
Chemical structures of the substrates used in this study.
We describe in this paper the characterization of the methylated flavonols of S. tinctoria, the partial purification of the flavonol 3-OMT and its physico-chemical properties, as well as the acquisition of internal amino acid sequence information for future cloning of its gene. In spite of the ubiquitous occurrence of flavonols in plants, especially Q (Wollenweber and Dietz, 1981), 3-MeQ rarely accumulates in significant amounts. However, given its role as an antiviral and antiinflammatory agent, as well as being a phytoanticipin (Roda et al., 2003), it would be desirable to bioengineer its constitutive expression in transgenic plants.
The choice of Serratula as the experimental plant material was dictated by the availability of its seeds and the fact that it contains only two flavonol OMTs, in addition to the lignin monomer OMT, as compared with C. americanum, a semi-aquatic weed that contains at least five flavonol OMTs with similar physico-chemical properties (Ibrahim et al., 1987) rendering the purification of any of them an extremely difficult task. Because of their abundance in Serratula roots, the methylated flavonols were isolated and identified from these tissues. On the other hand, the low growth rate of root relative to leaf tissues, and the fact that both purification and characterization of the flavonol 3-OMT require significant amounts of tissue, prompted us to use the leaves for enzyme work, after verifying the similarity of the flavonoid methylation pattern in both organs.
Leaves (approximately 20 g) were extracted with Pi buffer, the homogenate filtered and centrifuged, and the protein that precipitated between 30% and 70% ammonium sulfate saturation was desalted on PD-10 column before successive chromatography on Superose-12, Mono-Q, and adenosine-agarose columns.
The partially purified protein was applied to a Mono-Q column and eluted with a linear (50–500 mm) NaCl gradient in buffer C, and 2-mL fractions were collected and assayed for 3-OMT, 3-MeQ 3′-OMT, and 5-HFA OMT activities using Q, 3-MeQ, and 5-HFA as substrates, respectively, as described in the “Materials and Methods” section.
The enzyme protein fraction II eluted from the Mono-Q column (Fig. 3A) was assayed against the indicated substrates at a concentration of 50 μm as described in “Materials and Methods”.
Acknowledgments
We wish to thank Aka Meyers (Göttingen Botanical Gardens, Germany) for the generous gifts of Serratula seeds, Prof. E. Wollenweber (TU Darmastadt, Germany) for the 3,3′-diMeQ and 3,4′-diMeQ, Dr. M. Abou-Zaid (Natural Resources Canada, Sault Ste Marie, Canada) for caffeoyl CoA, Dr. Y. Fukuski (Hokkaido University, Japan) for the 3-MeQ, Dr. M. Di Falco (Genome Québec, Montreal) for the MS/MS peptide analysis, and Dr. L. Davin (Washington State University, Pullman, WA) for MS analysis of the enzyme reaction product and 3-MeQ reference.
Notes
This work was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada, and by Formation des chercheurs et l'aide á la recherche (FCAR), Department of Higher Education, Québec. D.A. was the recipient of both NSERC and FCAR postgraduate scholarships.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.036442.