Functional analysis of norcoclaurine synthase in Coptis japonica.
(S)-Norcoclaurine is the entry compound in benzylisoquinoline alkaloid biosynthesis and is produced by the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS) (EC 4.2.1.78). Although cDNA of the pathogenesis-related (PR) 10 family, the translation product of which catalyzes NCS reaction, has been isolated from Thalictrum flavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isolated from Coptis japonica (CjNCS1) also catalyzed NCS reaction as well as a PR10 homologue of C. japonica (CjPR10A). Both recombinant proteins stereo-specifically produced (S)-norcoclaurine by the condensation of dopamine and 4-HPAA. Because a CjNCS1 cDNA that encoded 352 amino acids showed sequence similarity to 2-oxoglutarate-dependent dioxygenases of plant origin, we characterized the properties of the native enzyme. Sequence analysis indicated that CjNCS1 only contained a Fe(2+)-binding site and lacked the 2-oxoglutarate-binding domain. In fact, NCS reaction of native NCS isolated from cultured C. japonica cells did not depend on 2-oxoglutarate or oxygen, but did require ferrous ion. On the other hand, CjPR10A showed no specific motif. The addition of o-phenanthroline inhibited NCS reaction of both native NCS and recombinant CjNCS1, but not that of CjPR10A. In addition, native NCS and recombinant CjNCS1 accepted phenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, as well as 4-HPAA, for condensation with dopamine, whereas recombinant CjPR10A could use 4-hydroxyphenylpyruvate and pyruvate in addition to the above aldehydes. These results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C. japonica cells was more active and formed a larger complex compared with recombinant CjNCS1.
Minami
Hiromichi
H
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
Dubouzet
Emilyn
E
Iwasa
Kinuko
K
Sato
Fumihiko
F
D000470:Alkaloids / Q000096:biosynthesis; D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D039323:Coptis / Q000166:cytology* / Q000201:enzymology; D018076:DNA, Complementary; D018744:DNA, Plant; D004298:Dopamine / Q000378:metabolism; D007501:Iron / Q000737:chemistry; D010940:Plant Proteins; D017422:Sequence Analysis, DNA; D044005:Tetrahydroisoquinolines
D000470:Alkaloids; D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D044005:Tetrahydroisoquinolines; D007501:Iron; D019731:Carbon-Nitrogen Ligases; C450500:norcoclaurine synthase; C012348:higenamine; D004298:Dopamine