S-Adenosyl-L-methionine:3-hydroxy-N-methylcoclaurine 4-O-methyltransferase (4-OMT) catalyzes the conversion of 3-hydroxy-N-methylcoclaurine to reticuline, an important intermediate in synthesizing isoquinoline alkaloids. In an earlier step in the biosynthetic pathway to reticuline, another O-methyltransferase, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase (6-OMT), catalyzes methylation of the 6-hydroxyl group of norcoclaurine. We isolated two kinds of cDNA clones that correspond to the internal amino acid sequences of a 6-OMT/4-OMT preparation from cultured Coptis japonica cells. Heterologously expressed proteins had 6-OMT or 4-OMT activities, indicative that each cDNA encodes a different enzyme. 4-OMT was purified using recombinant protein, and its enzymological properties were characterized. It had enzymological characteristics similar to those of 6-OMT; the active enzyme was the dimer of the subunit, no divalent cations were required for activity, and there was inhibition by Fe 2؉ , Cu 2؉ , Co 2؉ , Zn 2؉ , or Ni 2؉ , but none by the SH reagent. 4-OMT clearly had different substrate specificity. It methylated (R,S)-6-O-methylnorlaudanosoline, as well as (R,S)-laudanosoline and (R,S)-norlaudanosoline. Laudanosoline, an N-methylated substrate, was a much better substrate for 4-OMT than norlaudanosoline. 6-OMT methylated norlaudanosoline and laudanosoline equally. Further characterization of the substrate saturation and product inhibition kinetics indicated that 4-OMT follows an ordered Bi Bi mechanism, whereas 6-OMT follows a Ping-Pong Bi Bi mechanism. The molecular evolution of these two related O-methyltransferases is discussed.

Plant alkaloids, one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the biosynthetic pathways for scopolamine, nicotine, and berberine have been cloned, making the metabolic engineering of these alkaloids possible. Expression of two branching-point enzymes was engineered: putrescine N-methyltransferase (PMT) in transgenic plants of Atropa belladonna and Nicotiana sylvestris and (S)-scoulerine 9-Omethyltransferase (SMT) in cultured cells of Coptis japonica and Eschscholzia californica. Overexpression of PMT increased the nicotine content in N. sylvestris, whereas suppression of endogenous PMT activity severely decreased the nicotine content and induced abnormal morphologies. Ectopic expression of SMT caused the accumulation of benzylisoquinoline alkaloids in E. californica. The prospects and limitations of engineering plant alkaloid metabolism are discussed.berberine ͉ nicotine ͉ polyamine ͉ sanguinarine ͉ scopolamine H igher plants constitute one of our most important natural resources. They provide not only foodstuffs, fibers, and woods, but many chemicals, such as oils, flavorings, dyes, and pharmaceuticals. Although plants are renewable resources, some species are becoming more difficult to obtain in sufficient amounts to meet increasing demands. Destruction of natural habitats and technical difficulties in cultivation also are driving the drastic reductions in plant availability. For example, it is claimed that the demand for paclitaxel, a potent anticancer compound, could endanger forests of Taxus brevifolia (Pacific yew) because of the low paclitaxel content (40-100 mg͞kg of bark) in and slow growth of the trees (1).For many natural chemicals it is possible to synthesize alternatives from petroleum, coal, or both. The economic limitations of chemical synthesis and the pollution that accompanies this type of chemical synthesis, however, have led to the development of cell culture and molecular engineering of plants for the production of important and commodity chemicals. Plant cell and organ culture offer promising alternatives for the production of chemicals because totipotency enables plant cells and organs to produce useful secondary metabolites in vitro (2). Cell culture is also advantageous in that useful metabolites are obtained under a controlled environment, independent of climatic changes and soil conditions. In addition, the products are free of microbe and insect contamination. Fermentation technology also can be used to produce desired metabolites and can be optimized to maintain high and stable yields of known quality by cellular and molecular breeding techniques to further improve productivity and quality. After extensive empirical trials, some metabolites are now being produced by large-scale cell culture (e.g., shikonin and berberine; ref. 2), but the numbers of compounds that are producible commercially by cell culture technology are still very few. The main limitations are low productivity and the necessity of the down-stream pr...

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Benzylisoquinoline alkaloids are one of the most important secondary metabolite groups, and include the economically important analgesic morphine and the antimicrobial agent berberine. To improve the production of these alkaloids, we investigated the effect of the overexpression of putative rate-limiting step enzymes in benzylisoquinoline alkaloid biosynthesis. We introduced two O-methyltransferase [Coptis japonica norcoclaurine 6-O-methyltransferase (6OMT) and 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT)] expression vectors into cultured California poppy cells to avoid the gene silencing effect of endogenous genes. We established 20 independent lines for 6OMT transformants and 15 independent lines for 4'OMT transformants. HPLC/liquid chromatography-mass spectrometry (LC-MS) analysis revealed that the overexpression of C. japonica 6OMT was associated with an average alkaloid content 7.5 times greater than that in the wild type, whereas the overexpression of C. japonica 4'OMT had only a marginal effect. Further characterization of 6OMT in California poppy cells indicated that a 6OMT-specific gene is missing and 4OMT catalyzes the 6OMT reaction with low activity in California poppy, which supports the notion that the 6OMT reaction is important for alkaloid biosynthesis in this plant species. We discuss the importance of 6OMT in benzylisoquinoline alkaloid biosynthesis and the potential for using a rate-limiting step gene to improve alkaloid production.