Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells
Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells
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DOI：
10.1016/j.phytochem.2012.02.013
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发表时间：
2013-07-01
期刊：
影响因子：
3.8
通讯作者：
Sato, Fumihiko
中科院分区：
文献类型：
--
作者：
Takemura, Tomoya;Ikezawa, Nobuhiro;Sato, Fumihiko
苯并菲啶生物碱，如血根碱，是由牛心果碱，一种常见的中间体，在苄基异喹啉生物碱生物合成，通过原阿片碱。四种细胞色素P450参与从牛心果碱生物合成血根碱;即cheilanthifoline合酶（CYP 719 A5; EC 1.14.21.2.），刺罂粟碱合酶（CYP 719 A2/A3; EC 1.14.21.1.），N-甲基刺罂粟碱羟化酶（MSH）和原阿片碱6-羟化酶（P6 H; EC 1.14.13.55.）。本研究通过对黄连Scoullium-9-O-methyltransferase转基因细胞的代谢产物和转录本表达谱的综合分析，从培养的黄连Scoullium-9-O-methyltransferase转基因细胞中分离到P6 H的cDNA。使用P6 H（CYP 82 N2 v2）的全长候选cDNA，在酿酒酵母中产生重组蛋白用于表征。含有重组CYP 82 N2 v2的微粒体部分显示出典型的P450的CO差异减少谱，以及分别从原阿片和别隐阿片产生二氢血根碱和二氢白屈菜红碱。对CYP 82 N2 v2底物特异性的进一步表征表明，6-羟基化在反应中起作用。(C)2012爱思唯尔有限公司保留所有权利。
Benzophenanthridine alkaloids, such as sanguinarine, are produced from reticuline, a common intermediate in benzylisoquinoline alkaloid biosynthesis, via protopine. Four cytochrome P450s are involved in the biosynthesis of sanguinarine from reticuline; i.e. cheilanthifoline synthase (CYP719A5; EC 1.14.21.2.), stylopine synthase (CYP719A2/A3; EC 1.14.21.1.), N-methylstylopine hydroxylase (MSH) and protopine 6-hydroxylase (P6H; EC 1.14.13.55.). In this study, a cDNA of P6H was isolated from cultured Eschscholzia californica cells, based on an integrated analysis of metabolites and transcript expression profiles of transgenic cells with Coptis japonica scoulerine-9-O-methyltransferase. Using the full-length candidate cDNA for P6H (CYP82N2v2), recombinant protein was produced in Saccharomyces cerevisiae for characterization. The microsomal fraction containing recombinant CYP82N2v2 showed typical reduced CO-difference spectra of P450, and production of dihydrosanguinarine and dihydrochelerythrine from protopine and allocryptopine, respectively. Further characterization of the substrate-specificity of CYP82N2v2 indicated that 6-hydroxylation played a role in the reaction. (C) 2012 Elsevier Ltd. All rights reserved.