Purification and characterization of norcoclaurine synthase. The first committed enzyme in benzylisoquinoline alkaloid biosynthesis in plants.
Norcoclaurine synthase (NCS; EC ) catalyzes the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in benzylisoquinoline alkaloid biosynthesis in plants. NCS was purified 1590-fold to homogeneity from cell suspension cultures of meadow rue (Thalictrum flavum ssp. glaucum). The purification procedure, which resulted in a 4.2% yield, involved hydrophobic interaction, anion exchange, hydroxyapatite, and gel filtration chromatography. Purified NCS displayed native and denatured molecular masses of approximately 28 and 15 kDa, respectively, suggesting that the enzyme is composed of two subunits. Two-dimensional polyacrylamide gel electrophoresis revealed two major and two minor isoforms with pI values between 5.5 and 6.2. NCS activity was maximal at pH 6.5 to 7.0 and temperatures between 42 and 55 degrees C and was not affected by divalent cations. The enzyme showed hyperbolic saturation kinetics for 4-HPAA (K(m) = 335 microm) but sigmoidal saturation kinetics for dopamine (Hill coefficient = 1.8) suggesting cooperativity between the dopamine binding sites on each subunit; thus, NCS might play a regulatory, or rate-limiting, role in controlling the rate of pathway flux in benzylisoquinoline alkaloid biosynthesis. Product inhibition kinetics performed at saturating levels of one substrate and with norlaudanosoline as the inhibitor showed that NCS follows an iso-ordered bi-uni mechanism with 4-HPAA binding before dopamine. NCS activity was highest in soluble protein extracts from roots followed by stems, leaves, and flower buds.
Samanani
Nailish
N
Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Facchini
Peter J
PJ
D000079:Acetaldehyde / Q000031:analogs & derivatives* / Q000378:metabolism; D000470:Alkaloids / Q000096:biosynthesis*; D019731:Carbon-Nitrogen Ligases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D019800:Phenol; D013696:Temperature; D044005:Tetrahydroisoquinolines; D031949:Thalictrum / Q000201:enzymology*
D000470:Alkaloids; D044005:Tetrahydroisoquinolines; D019800:Phenol; C097891:4-hydroxyphenylacetaldehyde; D019731:Carbon-Nitrogen Ligases; C450500:norcoclaurine synthase; D000079:Acetaldehyde; C012348:higenamine; D004298:Dopamine