Functional analysis of norcoclaurine synthase in Coptis japonica
Functional analysis of norcoclaurine synthase in Coptis japonica
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DOI：
10.1074/jbc.m608933200
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发表时间：
2007-03-02
影响因子：
4.8
通讯作者：
Sato, Fumihiko
中科院分区：
文献类型：
--
作者：
Minami, Hiromichi;Dubouzet, Emilyn;Sato, Fumihiko
（S）-去甲乌药碱是苄基异喹啉生物碱生物合成中的入门化合物，通过去甲乌药碱合酶（NCS）（EC 4.2.1.78）使多巴胺和4-羟基苯乙醛（4-HPAA）缩合而产生。尽管从Thalumtrumflavum中分离出了病程相关（PR）10家族的cDNA（其翻译产物催化NCS反应），但其详细的酶学性质尚未得到表征。本文报道了一个分离自日本黄连的cDNA（CjNCS 1）也催化NCS反应，它与黄连的PR 10同源。粳稻（CjPR 10A）。两种重组蛋白通过多巴胺和4-HPAA的缩合立体特异性地产生（S）去甲乌药碱。由于CjNCS 1 cDNA编码352个氨基酸的序列相似性，2-酮戊二酸依赖的双加氧酶的植物来源，我们的特点是本地酶的特性。序列分析表明，CjNCS 1只含有一个Fe ~（2+）结合位点，缺乏2-酮戊二酸结合结构域。事实上，从培养的日本跳虫细胞中分离的天然NCS的NCS反应不依赖于2-酮戊二酸或氧，但确实需要亚铁离子。邻菲咯啉的加入抑制了天然NCS和重组CjNCS 1的NCS反应，但对CjPR 10A的NCS反应没有影响。此外，天然NCS和重组CjNCS 1接受苯乙醛和3，4-二羟基苯乙醛以及4-HPAA，用于与多巴胺缩合，而重组CjPR 10A除了上述醛之外还可以使用4-羟基苯丙酮酸和丙酮酸。这些结果表明，CjNCS 1是C japonica的主要NCS，而从培养的C.与重组CjNCS 1相比，Japonica细胞活性更高，形成的复合物更大。
(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 Thalictrumflavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isotated from Coptisjaponica (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 Fe2+-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.