Pauli H H, Kutchan T M
Laboratorium für Molekulare Biologie, Universität München, Germany.
Plant J. 1998 Mar;13(6):793-801. doi: 10.1046/j.1365-313x.1998.00085.x.
Alkaloids derived from the tetrahydrobenzylisoquinoline alkaloid (S)-N-methylcoclaurine represent a vast and varied structural array of physiologically active molecules. These compounds range from the dimeric bisbenzylisoquinolines, such as the muscle relaxant (+)-tubocurarine, to the powerful anaesthetic opiate morphine, the antimicrobial berberine and the anti-microbial benzo[c]-phenanthridine sanguinarine. The 3'-hydroxylation of (S)-N-methylcoclaurine is a branch point that is the penultimate step in the biosynthesis of the central alkaloidal intermediate (S)-reticuline. This study identified this enzyme as a cytochrome P-450-dependent mono-oxygenase that has until now eluded attempts at identification using in vitro enzyme assays. Two alleles encoding this new enzyme (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B1) were isolated from a cDNA library prepared from poly(A)+ RNA isolated from methyl jasmonate-induced cell-suspension cultures of the California poppy Eschscholzia californica. Partial clones generated by RT-PCR with cytochrome P-450-specific primers were used as hybridization probes. RNA gel-blot hybridization indicated that the transcripts for CYP80B1 accumulate in response to the addition of methyl jasmonate to the cell culture medium. Both alleles were functionally expressed in Saccharomyces cerevisiae and in Spodoptera frugiperda Sf9 cells in the presence and absence of the E. californica cytochrome P-450 reductase. The enzyme was found to hydroxylate exclusively (S)-N-methylcoclaurine with a pH optimum of 7.5, temperature optimum of 35 degrees C and K(m) of 15 microns. In addition to the CYP80B1 alleles, another cytochrome P-450 with an inducible transcript (CYP82B1) was isolated and expressed in the same manner, but was not found to be involved in alkaloid biosynthesis in this plant.
源自四氢苄基异喹啉生物碱（S）-N-甲基荷包牡丹碱的生物碱代表了一系列生理活性分子，其结构广泛且多样。这些化合物包括二聚体双苄基异喹啉，如肌肉松弛剂（+）-筒箭毒碱，以及强效麻醉性阿片类药物吗啡、抗菌剂小檗碱和抗微生物的苯并[c]菲啶血根碱。（S）-N-甲基荷包牡丹碱的3'-羟基化是一个分支点，是中央生物碱中间体（S）-网状番荔枝碱生物合成的倒数第二步。本研究将该酶鉴定为一种细胞色素P-450依赖性单加氧酶，迄今为止，使用体外酶测定法一直未能鉴定出该酶。从加利福尼亚罂粟花菱叶罂粟经茉莉酸甲酯诱导的细胞悬浮培养物中分离的聚腺苷酸（poly(A)+）RNA制备的cDNA文库中，分离出编码这种新酶（S）-N-甲基荷包牡丹碱3'-羟化酶（CYP80B1）的两个等位基因。用细胞色素P-450特异性引物通过逆转录聚合酶链反应（RT-PCR）产生的部分克隆用作杂交探针。RNA凝胶印迹杂交表明，CYP80B1的转录本在向细胞培养基中添加茉莉酸甲酯后积累。在存在和不存在菱叶罂粟细胞色素P-450还原酶的情况下，两个等位基因均在酿酒酵母和草地贪夜蛾Sf9细胞中功能性表达。发现该酶仅将（S）-N-甲基荷包牡丹碱羟基化，最适pH为7.5，最适温度为35℃，米氏常数（K(m)）为15微摩尔。除了CYP80B1等位基因外，还以相同方式分离并表达了另一种具有可诱导转录本的细胞色素P-450（CYP82B1），但未发现其参与该植物的生物碱生物合成。
