title: A flavonol O-methyltransferase from Catharanthus roseus performing two sequential methylations.
abstract: Protein extracts from dark-grown cell suspension cultures of Catharanthus roseus (Madagascar periwinkle) contained several O-methyltransferase (OMT) activities, including the 16-hydroxytabersonine O-methyltransferase (16HT-OMT) in indole alkaloid biosynthesis. This enzyme was enriched through several purification steps, including affinity chromatography on adenosine agarose. SDS-PAGE of the purified protein preparation revealed a protein band at the size expected for plant OMTs (38-43 kDa). Mass spectrometry indicated two dominant protein species of similar mass in this band, and sequences of tryptic peptides showed similarities to known OMTs. Homology-based RT-PCR identified cDNAs for four new OMTs. Two of these cDNAs (CrOMT2 and CrOMT4) encoded the proteins dominant in the preparation enriched for 16HT-OMT. The proteins were closely related (73% identity), but both shared only 48-53% identity with the closest relatives found in the public databases. The enzyme functions were investigated with purified recombinant proteins after cDNA expression in Escherichia coli. Unexpectedly, both proteins had no detectable 16HT-OMT activity, and CrOMT4 was inactive with all substrates investigated. CrOMT2 was identified as a flavonoid OMT that was expressed in dark-grown cell cultures and copurified with 16HT-OMT. It represented a new type of OMT that performs two sequential methylations at the 3'- and 5'-positions of the B-ring in myricetin (flavonol) and dihydromyricetin (dihydroflavonol). The resulting methylation pattern is characteristic for C. roseus flavonol glycosides and anthocyanins, and it is proposed that CrOMT2 is involved in their biosynthesis.
authors.affiliation: Institut für Biologie II, Universität Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Cacace
authors.forename: Gudrun
authors.initials: G
authors.lastname: Schröder
authors.forename: Elke
authors.initials: E
authors.lastname: Wehinger
authors.forename: Dieter
authors.initials: D
authors.lastname: Strack
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schmidt
authors.forename: Joachim
authors.initials: J
authors.lastname: Schröder
mesh_terms: D000595:Amino Acid Sequence; D030008:Catharanthus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D018506:Gene Expression Regulation, Plant; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015394:Molecular Structure; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D008780:Methyltransferases