Isolation and partial characterization of norcoclaurine synthase, the first committed step in benzylisoquinoline alkaloid biosynthesis, from opium poppy

Norcoclaurine synthase (NCS) catalyzes the condensation of dopamine and 4-h ydroxyphenylacetaldehyde (4-HPAA) to yield norcoclaurine, the common precur sor to all benzylisoquinoline alkaloids produced in plants. In opium poppy (Papaver somniferum L.), NCS activity was detected in germinating seeds, yo ung seedlings, and all mature plant organs.. especially stems and roots. Ho wever, the highest levels of activity were found in cell-suspension culture s treated with a fungal elicitor. NCS activity was induced more than 20-fol d over an 80-h period in response to elicitor treatment. Compared to opium poppy, basal NCS activity was 3-and 5-fold higher in benzylisoquinoline alk aloid-producing cell cultures of Eschscholzia californica and Thalictrum fl avum ssp. glaucum, respectively. In contrast, NCS activity was not detected in cultured cells of Nicotiana tabacum and Catharanthus roseus, which do n ot produce benzylisoquinoline alkaloids. NCS displayed maximum activity bet ween pH 6.5 and 7.0, and a broad temperature optimum between 42 and 55 degr eesC. Enzyme activity was not affected by Ca2+ or Mg2+, and was not inhibit ed by a variety of benzylisoquinoline alkaloids. NCS showed hyperbolic satu ration kinetics for 4-HPAA, with an apparent K-m of 1.0 mM. However, the en zyme exhibited sigmoidal saturation kinetics for dopamine with a Hill coeff icient of 1.84. NCS enzymes from E. californica and T. flavum displayed sim ilar properties. These data indicate that NCS exhibits positive cooperativi ty between substrate-binding sites. Enzymes of this type catalyze regulator y, or rate-limiting, steps in metabolism, suggesting that NCS plays a role in controlling the rate of pathway flux in benzylisoquinoline alkaloid bios ynthesis.