Characterization of a Vitis vinifera cv. Cabernet Sauvignon 3',5'-O-methyltransferase showing strong preference for anthocyanins and glycosylated flavonols.
At ripening initiation in red grapevine (Vitis vinifera) berries, the exocarp turns color from green to red and then to purple due to the accumulation and extent of methylation of anthocyanins. The accumulation of transcripts encoding an O-methyltransferase was recently shown to be closely correlated with the onset of ripening and the degree of blue/purple pigmentation in grapevine berries; however, the biochemical function of this gene has remained uncharacterized. In this study, an O-methyltransferase cDNA that showed a distinct expression pattern when compared to closely related sequences was expressed in Escherichia coli and enzyme assays were carried out with a broad array of anthocyanin and other flavonoid substrates. We demonstrate that this enzyme carries out 3',5'-O-methylation of anthocyanins and flavonol compounds in vitro, which are known to be present in grape berries, with a preference for glycosylated substrates. The highest relative specific activity for the enzyme was found with delphinidin 3-O-glucoside as substrate. The enzyme is not able to methylate flavan type skeletons with chiral centers, such as either catechins or dihydroquercetin. The enzyme showed negligible specific activity for caffeoyl-CoA, compared to flavonol and anthocyanin substrates. Phylogenetic analysis of the O-methyltransferase suggests that it may be a member of a distinct subclass of Type 2 bivalent metal-dependent S-adenosyl-methionine O-methyltransferases.
Lücker
Joost
J
Faculty of Land and Food Systems, Wine Research Centre, University of British Columbia, Vancouver, B.C., Canada. jlucker@gmail.com
Martens
Stefan
S
Lund
Steven T
ST
D000214:Acyl Coenzyme A / Q000378:metabolism; D000872:Anthocyanins / Q000737:chemistry / Q000378:metabolism*; D044948:Flavonols / Q000737:chemistry / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D006027:Glycosides / Q000378:metabolism; D006031:Glycosylation; D008274:Magnesium / Q000378:metabolism; D008780:Methyltransferases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012436:S-Adenosylmethionine / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D027843:Vitis / Q000201:enzymology* / Q000235:genetics
D000214:Acyl Coenzyme A; D000872:Anthocyanins; D044948:Flavonols; D006027:Glycosides; C058645:caffeoyl-coenzyme A; D012436:S-Adenosylmethionine; D008780:Methyltransferases; D008274:Magnesium