Molecular cloning and characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica
Molecular cloning and characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica
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DOI：
10.1111/j.1742-4658.2007.05652.x
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发表时间：
2007-02-01
期刊：
影响因子：
5.4
通讯作者：
Sato, Fumihiko
中科院分区：
文献类型：
--
作者：
Ikezawa, Nobuhiro;Iwasa, Kinuko;Sato, Fumihiko
（S）-柱花草碱是合成血根碱等苯并菲啶类生物碱的重要中间体。柱花草碱的生物合成涉及两个亚甲二氧基桥的顺序形成。尽管已经从黄连中克隆了涉及小檗碱生物合成的亚甲基二氧基桥形成P450（CYP 719）[Ikezawa N，Tanaka M，Nagayoshi M，Shinkyo R，Sakaki T，Inouye K & Sato F（2003）J Biol Chem 278，38557-38565]，但是没有关于刺茉莉碱生物合成中亚甲基二氧基桥形成酶的基因的信息。用简并引物从C. japonica CYP719。在酿酒酵母中的异源表达表明，CYP 719 A2和CYP 719 A3都具有stylopine合成酶活性，催化cheilanthifoline到stylopine的亚甲基二氧基桥的形成，但没有cheilanthifoline合成酶活性，将scoulterine转化为cheilanthifoline。研究了CYP 719 A2和CYP 719 A3的功能差异和表达模式，以探讨它们在刺罂粟碱生物合成中的生理作用。酶促分析表明，CYP 719 A2仅对（R，S）-cheilanthifoline具有高底物亲和力，而CYP 719 A3对三种相似的底物（R，S）-cheilanthifoline、（S）-scoultamine和（S）-tetrahydrocolumbamine具有高亲和力。在E.结果表明，CYP 719 A2和CYP 719 A3在根中的表达模式与stylopine生物合成基因CYP 80 B1、小檗碱桥酶和S-腺苷-L-甲硫氨酸：3 '-羟基-N-甲基乌药碱4'-O-甲基转移酶的表达模式相似，而CYP 719 A3在根中的特异性表达显著。E.的治疗茉莉酸甲酯诱导的加州葡萄幼苗的CYP 719 A2和CYP 719 A3基因协同诱导。本文还讨论了CYP 719 A2和CYP 719 A3在刺罂粟碱生物合成中的生理作用。
(S)-Stylopine is an important intermediate in the biosynthesis of benzophenanthridine alkaloids, such as sanguinarine. Stylopine biosynthesis involves the sequential formation of two methylenedioxy bridges. Although the methylenedioxy bridge-forming P450 (CYP719) involved in berberine biosynthesis has been cloned from Coptis japonica[Ikezawa N, Tanaka M, Nagayoshi M, Shinkyo R, Sakaki T, Inouye K & Sato F (2003) J Biol Chem278, 38557-38565], no information is available regarding the genes for methylenedioxy bridge-forming enzymes in stylopine biosynthesis. Two cytochrome P450 cDNAs involved in stylopine biosynthesis were isolated using degenerate primers designed for C. japonica CYP719 from cultured Eschscholzia californica cells. Heterologous expression in Saccharomyces cerevisiae showed that both CYP719A2 and CYP719A3 had stylopine synthase activity to catalyze methylenedioxy bridge-formation from cheilanthifoline to stylopine, but not cheilanthifoline synthase activity to convert scoulerine to cheilanthifoline. Functional differences and expression patterns of CYP719A2 and CYP719A3 were examined to investigate their physiological roles in stylopine biosynthesis. Enzymatic analysis showed that CYP719A2 had high substrate affinity only toward (R,S)-cheilanthifoline, whereas CYP719A3 had high affinity toward three similar substrates (R,S)-cheilanthifoline, (S)-scoulerine, and (S)-tetrahydrocolumbamine. An expression analysis in E. californica plant tissues showed that CYP719A2 and CYP719A3 exhibited expression patterns similar to those of three stylopine biosynthetic genes (CYP80B1, berberine bridge enzyme, and S-adenosyl-L-methionine : 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase), whereas the specific expression of CYP719A3 in root was notable. Treatment of E. californica seedlings with methyl jasmonate resulted in the coordinated induction of CYP719A2 and CYP719A3 genes. The physiological roles of CYP719A2 and CYP719A3 in stylopine biosynthesis are discussed.