title: Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.
abstract: Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer's disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4'-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4'-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.
authors: Donald Danforth Plant Science Center, St. Louis, Missouri, United States of America.
authors: Matthew B
authors: MB
authors: Kilgore
authors: Donald Danforth Plant Science Center, St. Louis, Missouri, United States of America.
authors: Megan M
authors: MM
authors: Augustin
authors: Sequoia Sciences, St Louis, Missouri, United States of America.
authors: Courtney M
authors: CM
authors: Starks
authors: Sequoia Sciences, St Louis, Missouri, United States of America.
authors: Mark
authors: M
authors: O'Neil-Johnson
authors: National Center for Genome Resources, Santa Fe, New Mexico, United States of America.
authors: Gregory D
authors: GD
authors: May
authors: National Center for Genome Resources, Santa Fe, New Mexico, United States of America.
authors: John A
authors: JA
authors: Crow
authors: Donald Danforth Plant Science Center, St. Louis, Missouri, United States of America.
authors: Toni M
authors: TM
authors: Kutchan
mesh_terms: D000470:Alkaloids / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D000544:Alzheimer Disease / Q000188:drug therapy / Q000473:pathology; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D005702:Galantamine / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D006801:Humans; D031427:Narcissus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011497:Protein O-Methyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000470:Alkaloids; D018076:DNA, Complementary; D005702:Galantamine; D011497:Protein O-Methyltransferase