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J. Biol. Chem., Vol. 282, Issue 20, 14741-14751, May 18, 2007

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Molecular Cloning and Characterization of Tetrahydroprotoberberine cis-N-Methyltransferase, an Enzyme Involved in Alkaloid Biosynthesis in Opium Poppy^*

From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada

S-Adenosyl-L-methionine:tetrahydroprotoberberine cis-N-methyltransferase (EC 2.1.1.122 [EC] ) catalyzes the conversion of (S)-stylopine to the quaternary ammonium alkaloid, (S)-cis-N-methylstylopine, as a key step in the biosynthesis of protopine and benzophenanthridine alkaloids in plants. A full-length cDNA encoding a protein exhibiting 45 and 48% amino acid identity with coclaurine N-methyltransferase from Papaver somniferum (opium poppy) and Coptis japonica, respectively, was identified in an elicitor-treated opium poppy cell culture expressed sequence tag data base. Phylogenetic analysis showed that the protein belongs to a unique clade of enzymes that includes coclaurine N-methyltransferase, the predicated translation products of the Arabidopsis thaliana genes, At4g33110 and At4g33120, and bacterial S-adenosyl-L-methionine-dependent cyclopropane fatty acid synthases. Expression of the cDNA in Escherichia coli produced a recombinant enzyme able to convert the protoberberine alkaloids stylopine, canadine, and tetrahydropalmatine to their corresponding N-methylated derivatives. However, the protoberberine alkaloids tetrahydroxyberbine and scoulerine, and simple isoquinoline, benzylisoquinoline, and pavine alkaloids were not accepted as substrates, demonstrating the strict specificity of the enzyme. The apparent K_m values for (R,S)-stylopine and S-adenosyl-L-methionine were 0.6 and 11.5 µM, respectively. TNMT gene transcripts and enzyme activity were detected in opium poppy seedlings and all mature plant organs and were induced in cultured opium poppy cells after treatment with a fungal elicitor. The enzyme was detected in cell cultures of other members of the Papaveraceae but not in species of related plant families that do not accumulate protopine and benzophenanthridine alkaloids.

Received for publication, December 29, 2006 , and in revised form, March 23, 2007.

^* This work was funded by Natural Sciences and Engineering Research Council of Canada (NSERC) Strategic and Discovery grants (to P. J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-Bank^TM/EBI Data Bank with accession number(s) DQ028579 [GenBank] .

The on-line version of this article (available at http://www.jbc.org) contains two supplemental figures and supplemental references.

¹ The recipient of an NSERC postgraduate scholarship.

² Holds the Canada Research Chair in Plant Metabolic Processes Biotechnology. To whom correspondence should be addressed. Tel.: 403-220-7651; Fax: 403-289-9311; E-mail: pfacchin{at}ucalgary.ca.

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