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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C--O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera.
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Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C--O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera.

机译：编码berbamunine合酶的cDNA的分子克隆和异源表达，其是来自高等植物小ber的C-O酚偶联细胞色素P450。

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摘要

A cDNA encoding a cytochrome P450-dependent oxidase, berbamunine synthase (EC 1.1.3.34; CYP80), from cell suspension cultures of the higher plant Berberis stolonifera Koehne and Wolf (barberry) has been isolated and heterologously expressed in functional form in insect cell culture using a baculovirus-based expression system. This cytochrome P450-dependent enzyme is unusual in that it catalyzes the regio- and stereoselective formation of a C--O phenol couple in bisbenzylisoquinoline alkaloid biosynthesis without concomitant incorporation of activated oxygen into the product. Consistent with the function of an oxidase rather than a monooxygenase, an essential glycine residue in the distal helix, which forms the oxygen-binding pocket in the well-studied bacterial enzyme P-450cam, is replaced by proline at the equivalent position in berbamunine synthase. This oxidase was accumulated in an active form in insect cell microsomes and accepted electrons from the endogenous NADPH-cytochrome P450 reductase. The heterologously expressed enzyme oxidatively couples either two molecules of (R)-N-methylcoclaurine to form the (R,R) dimer guattegaumerine or one molecule each of (R)- and (S)-N-methylcoclaurine to form the (R,S) dimer berbamunine. The ratio of the two bisbenzylisoquinolines formed could be altered by reductase source or by varying the enantiomer composition of the substrates.
机译：已从高等植物小ber菜Koehne和Wolf（伏牛花）的细胞悬浮培养物中分离了编码细胞色素P450依赖的氧化酶，小ba碱合酶（EC 1.1.3.34; CYP80）的cDNA，并在昆虫细胞培养中以功能形式异源表达使用基于杆状病毒的表达系统。这种依赖于细胞色素P450的酶是不寻常的，因为它催化双苄基异喹啉生物碱生物合成中C-O酚对的区域选择性和立体选择性形成，而不会伴随活性氧的结合。与氧化酶而不是单加氧酶的功能一致，远端螺旋结构中的必需甘氨酸残基在经过精心研究的细菌酶P-450cam中形成氧结合口袋，在berbamunine合酶中的等效位置被脯氨酸取代。该氧化酶以活性形式积累在昆虫细胞微粒体中，并接受来自内源性NADPH-细胞色素P450还原酶的电子。异源表达的酶氧化偶联两个分子（R）-N-甲基coclaurine形成（R，R）二聚体guattegaumerine或一个分子各自（R）-和（S）-N-甲基coclaurine形成（R， S）二聚体巴巴胺。可以通过还原酶源或通过改变底物的对映异构体组成来改变形成的两个双苄基异喹啉的比例。

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