Institute for Plant Biology, TU Braunschweig, Mendelssohnstr. 4, D-38106 Braunschweig, Germany.
Yasouj University, Yasouj, Kohgiluyeh Va Boyer Ahmad, Iran.
Plant Cell Physiol. 2017 Aug 1;58(8):1421-1430. doi: 10.1093/pcp/pcx077.
The most prominent alkaloid of Chelidonium majus is dihydrocoptisine, revealing the characteristic benzophenanthridine skeleton. To date, any informationon on the enzymes responsible for its biosynthesis and the related genes in C. majus is lacking. Based on sequence similarities to the corresponding methylenedioxy bridge-forming Cyt P450 enzymes involved in isoquinoline alkaloid biosynthesis in Eschscholzia californica, genes for a cheilanthifoline synthase and a stylopine synthase from C. majus were isolated, sequenced and heterologously expressed in yeast. The activity of the heterologously expressed Cyt P450 enzymes was determined in situ as well as on the basis of microsomal fractions. It was shown that cheilanthifoline synthase (c8931) converts scoulerine into cheilanthifoline, the latter subsequently being converted to stylopine by the action of a stylopine synthase (c1128). Based on the well-known instability of stylopine, it can be assumed that in vivo-under the acidic conditions in the vacuole-this alkaloid is converted to dihydrocoptisine, which accumulates in C. majus leaves. Both methylenedioxy bridge-forming Cyt P450 enzymes from C. majus are characterized by their high substrate specificity. Apart from their genuine substrates, i.e. scoulerine and cheilanthifoline, cheilanthifoline synthase and stylopine synthase do not accept other substrates tested; the only alternative substrate identified was scoulerine, which is converted by stylopine synthase to yield minor amounts of nandinine. Quantitative real-time PCR revealed that the expression of cheilanthifoline synthase and stylopine synthase genes is very similar in both roots and leaves from C. majus, although the alkaloid accumulation patterns in these organs are quite different.
白屈菜的主要生物碱是二氢血根碱，具有特征性的苯并菲啶骨架。迄今为止，关于其生物合成的酶以及白屈菜中相关基因的任何信息都缺乏。基于与参与加利福尼亚罂粟异喹啉生物碱生物合成的相应亚甲二氧基桥形成 Cyt P450 酶的序列相似性，从白屈菜中分离、测序并异源表达了一个白屈菜菲林合成酶和一个血根碱合成酶的基因。在酵母中，通过原位和基于微粒体部分的方法测定了异源表达 Cyt P450 酶的活性。结果表明，白屈菜菲林合成酶（c8931）将胡椒乙碱转化为白屈菜菲林，后者随后被血根碱合成酶（c1128）转化为血根碱。基于血根碱众所周知的不稳定性，可以假设在体内-在液泡中的酸性条件下-这种生物碱转化为二氢血根碱，后者在白屈菜中积累。白屈菜中的这两种亚甲二氧基桥形成 Cyt P450 酶的特点是其高底物特异性。除了它们真正的底物，即胡椒乙碱和白屈菜菲林外，白屈菜菲林合成酶和血根碱合成酶都不接受其他测试的底物；唯一鉴定出的替代底物是胡椒乙碱，它被血根碱合成酶转化为少量的那丁宁。定量实时 PCR 显示，白屈菜菲林合成酶和血根碱合成酶基因在白屈菜的根和叶中的表达非常相似，尽管这些器官中的生物碱积累模式有很大差异。
