title: Cloning and characterization of a Nicotiana tabacum methylputrescine oxidase transcript.
abstract: The oxidative deamination of N-methylputrescine is an essential step in both pyridine and tropane alkaloid biosynthesis. Reverse genetic approaches have not resulted in the cloning of a methylputrescine oxidase gene (MPO). However, we have used a homology-based approach to clone a full-length tobacco MPO1 cDNA. The MPO1 gene is part of a small multigene family comprised of approximately six members. MPO1-like transcript levels increased in roots that were either deprived of auxin or treated with methyl jasmonic acid. Similar to other known nicotine biosynthetic genes in domesticated tobacco, MPO1-like mRNA levels were lower in roots with the mutant a and b alleles. The MPO1 protein was expressed in bacteria as a recombinant Thioredoxin-His(6)-MPO1 fusion protein. The recombinant MPO1 protein utilized N-methylputrescine more efficiently than other diamines. Therefore, the kinetic properties of the MPO1 enzyme may play an important role in determining the pyridine alkaloid profiles observed in tobacco roots.
authors.affiliation: Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, 540 Latham Hall, Blacksburg, VA 24061-0390, USA.
authors.forename: William G
authors.initials: WG
authors.lastname: Heim
authors.forename: Katie A
authors.initials: KA
authors.lastname: Sykes
authors.forename: Sherry B
authors.initials: SB
authors.lastname: Hildreth
authors.forename: Jian
authors.initials: J
authors.lastname: Sun
authors.forename: Rong-He
authors.initials: RH
authors.lastname: Lu
authors.forename: John G
authors.initials: JG
authors.lastname: Jelesko
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D018744:DNA, Plant / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D016415:Sequence Alignment; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D018744:DNA, Plant; D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D010088:Oxidoreductases