Cloning and characterization of a Nicotiana tabacum methylputrescine oxidase transcript.
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.
Heim
William G
WG
Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, 540 Latham Hall, Blacksburg, VA 24061-0390, USA.
Sykes
Katie A
KA
Hildreth
Sherry B
SB
Sun
Jian
J
Lu
Rong-He
RH
Jelesko
John G
JG
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
D018744:DNA, Plant; D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D010088:Oxidoreductases