title: Isolation and Characterization of O-methyltransferases Involved in the Biosynthesis of Glaucine in Glaucium flavum.
abstract: Transcriptome resources for the medicinal plant Glaucium flavum were searched for orthologs showing identity with characterized O-methyltransferases (OMTs) involved in benzylisoquinoline alkaloid biosynthesis. Seven recombinant proteins were functionally tested using the signature alkaloid substrates for six OMTs: norlaudanosoline 6-OMT, 6-O-methyllaudanosoline 4'-OMT, reticuline 7-OMT, norreticuline 7-OMT, scoulerine 9-OMT, and tetrahydrocolumbamine OMT. A notable alkaloid in yellow horned poppy (G. flavum [GFL]) is the aporphine alkaloid glaucine, which displays C8-C6' coupling and four O-methyl groups at C6, C7, C3', and C4' as numbered on the 1-benzylisoquinoline scaffold. Three recombinant enzymes accepted 1-benzylisoquinolines with differential substrate and regiospecificity. GFLOMT2 displayed the highest amino acid sequence identity with norlaudanosoline 6-OMT, showed a preference for the 6-O-methylation of norlaudanosoline, and O-methylated the 3' and 4' hydroxyl groups of certain alkaloids. GFLOMT1 showed the highest sequence identity with 6-O-methyllaudanosoline 4'OMT and catalyzed the 6-O-methylation of norlaudanosoline, but more efficiently 4'-O-methylated the GFLOMT2 reaction product 6-O-methylnorlaudanosoline and its N-methylated derivative 6-O-methyllaudanosoline. GFLOMT1 also effectively 3'-O-methylated both reticuline and norreticuline. GFLOMT6 was most similar to scoulerine 9-OMT and efficiently catalyzed both 3'- and 7'-O-methylations of several 1-benzylisoquinolines, with a preference for N-methylated substrates. All active enzymes accepted scoulerine and tetrahydrocolumbamine. Exogenous norlaudanosoline was converted to tetra-O-methylated laudanosine using combinations of Escherichia coli producing (1) GFLOMT1, (2) either GFLOMT2 or GFLOMT6, and (3) coclaurine N-methyltransferase from Coptis japonica. Expression profiles of GFLOMT1, GFLOMT2, and GFLOMT6 in different plant organs were in agreement with the O-methylation patterns of alkaloids in G. flavum determined by high-resolution, Fourier-transform mass spectrometry.
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors: Limei
authors: L
authors: Chang
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors: Jillian M
authors: JM
authors: Hagel
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada pfacchin@ucalgary.ca.
authors: Peter J
authors: PJ
authors: Facchini
mesh_terms: D001060:Aporphines / Q000378:metabolism*; D044182:Benzylisoquinolines / Q000378:metabolism; D001600:Berberine Alkaloids / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D007546:Isoquinolines / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D028101:Papaveraceae / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018517:Plant Roots / Q000378:metabolism; D010946:Plants, Medicinal / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013765:Tetrahydropapaveroline / Q000378:metabolism
chemical_list: D001060:Aporphines; D044182:Benzylisoquinolines; D001600:Berberine Alkaloids; D007546:Isoquinolines; D010940:Plant Proteins; D011994:Recombinant Proteins; D013765:Tetrahydropapaveroline; C010941:discretamine; C001522:laudanosine; D008780:Methyltransferases; C005004:glaucine; C003298:reticuline