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-hydroxyphenylacetaldehyde (4-HPAA) to yield norcoclaurine, the common precursor to all benzylisoquinoline alkaloids produced in plants. In opium poppy (Papaver somniferum L.), NCS activity was detected in germinating seeds, young seedlings, and all mature plant organs, especially stems and roots. However, the highest levels of activity were found in cell-suspension cultures treated with a fungal elicitor. NCS activity was induced more than 20-fold 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 alkaloid-producing cell cultures of Eschscholzia californica and Thalictrum flavum ssp. glaucum, respectively. In contrast, NCS activity was not detected in cultured cells of Nicotiana tabacum and Catharanthus roseus, which do not produce benzylisoquinoline alkaloids. NCS displayed maximum activity between pH 6.5 and 7.0, and a broad temperature optimum between 42 and 55 degrees C. Enzyme activity was not affected by Ca2+ or Mg2+, and was not inhibited by a variety of benzylisoquinoline alkaloids. NCS showed hyperbolic saturation kinetics for 4-HPAA, with an apparent Km of 1.0 mM. However, the enzyme exhibited sigmoidal saturation kinetics for dopamine with a Hill coefficient of 1.84. NCS enzymes from E. californica and T. flavum displayed similar properties. These data indicate that NCS exhibits positive cooperativity between substrate-binding sites. Enzymes of this type catalyze regulatory, or rate-limiting, steps in metabolism, suggesting that NCS plays a role in controlling the rate of pathway flux in benzylisoquinoline alkaloid biosynthesis.
Samanani
N
N
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Facchini
P J
PJ
D000079:Acetaldehyde / Q000031:analogs & derivatives* / Q000378:metabolism; D000470:Alkaloids / Q000096:biosynthesis* / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D004298:Dopamine / Q000378:metabolism; D018525:Germination; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010207:Papaver / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D019800:Phenol; D018515:Plant Leaves / Q000201:enzymology; D018517:Plant Roots / Q000201:enzymology; D018547:Plant Stems / Q000201:enzymology; D012639:Seeds / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism; D013379:Substrate Specificity; D044005:Tetrahydroisoquinolines
D000470:Alkaloids; D044005:Tetrahydroisoquinolines; D019800:Phenol; C097891:4-hydroxyphenylacetaldehyde; D019731:Carbon-Nitrogen Ligases; C450500:norcoclaurine synthase; D000079:Acetaldehyde; C012348:higenamine; D004298:Dopamine