Purification and Characterization of Norcoclaurine Synthase

Abstract

Norcoclaurine synthase (NCS; EC 4.2.1.78) 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 ϳ28 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°C and was not affected by divalent cations. The enzyme showed hyperbolic saturation kinetics for 4-HPAA (K m ‫؍‬ 335 M) 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.

Structural Basis of Enzymatic (S)-Norcoclaurine Biosynthesis

“…6) and the role of Tyr 108 on the electronic configuration of the dopamine substrate, still need to be fully clarified. Moreover, further studies will be necessary to understand the observed cooperativity in enzyme kinetics (7)(8)(9). At present, only minor ligand-linked tertiary structural changes have been detected at the active site (different rotameric arrangement of Phe 122 in subunit A versus subunit B), whereas the quaternary structures of the substratefree and substrate-bound proteins appear to be essentially superimposable.…”

“…So far, it is also unclear whether the enzyme is assembled in a quaternary structure that could explain the reported cooperativity. In fact, the association state in solution has been reported to be dimeric in gel filtration measurements, whereas NMR investigations indicated that the protein is mostly monomeric (7,11).…”

“…NCS from Thalictrum flavum (NCS; EC 4.2.1.78) has been identified recently (7) and has been fully characterized in terms of substrate specificity and enzymatic activity (8,9). The enzyme shares no sequence similarity to the functionally correlated indole alkaloid synthases, whereas it shows low (12-15% identity) but significant homology with proteins belonging to the vast family of Bet v1 and pathogen-related PR10 proteins, whose physiological functions are still poorly understood (9).…”

Functional Analysis of Norcoclaurine Synthase in Coptis japonica

“…tenuis cells also did not accept phenylpyruvate as a substrate as did Coptis enzyme. 5 Whereas the formation of norcoclaurine by the condensation of dopamine and 4-HPAA is now clearly established as the central pathway in isoquinoline alkaloid biosynthesis (4,24), the formation of norcoclaurine via the condensation of dopamine and 4-HPAA or via the condensation of dopamine and 4-hydroxyphenylpyruvate was also intensively investigated. The formation of norlaudanosoline through norlaudanosoline 1-carboxylic acid by the condensation of dopamine and 3,4-dihydroxyphenylpyruvate was once seriously considered to be a possible pathway using in vitro and in vivo tracer experiments (23,31).…”

“…Only recently has NCS been isolated and characterized from cell suspension cultures of meadow rue (Thalictrum flavum ssp. glaucum) (5,6), but the mechanism of the NCS reaction, including the stereospecificity of product, has not yet been determined.…”