The Multifunctional Enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) Converts Cysteine-Indole-3-Acetonitrile to Camalexin in the Indole-3-Acetonitrile Metabolic Network of Arabidopsis thaliana
The Multifunctional Enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) Converts Cysteine-Indole-3-Acetonitrile to Camalexin in the Indole-3-Acetonitrile Metabolic Network of Arabidopsis thaliana
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DOI：
10.1105/tpc.109.066670
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发表时间：
2009-06-01
期刊：
影响因子：
11.6
通讯作者：
Glawischnig, Erich
中科院分区：
文献类型：
--
作者：
Boettcher, Christoph;Westphal, Lore;Glawischnig, Erich
山茶毒素是拟南芥特有的植物抗毒素，其积累受多种植物病原菌的诱导。其来源于Trp，Trp在细胞色素P450酶CYP 79 B2/B3和CYP 71 A13的连续作用下转化为吲哚-3-乙腈（IAN）。采用超高效液相色谱-电喷雾四极杆飞行时间质谱法对经银处理或接种致病疫霉的野生型植物和camalexin生物合成突变体的提取物进行了综合分析。该代谢组学方法与前体饲喂实验相结合，以表征IAN代谢网络，并鉴定Camalexin的新型生物合成中间体和代谢产物。吲哚-3-甲醛和吲哚-3-羧酸衍生物被证明是来自IAN。IAN与谷胱甘肽、γ-谷氨酰半胱氨酸和半胱氨酸[Cys（IAN）]的结合物在受挑战的植物抗毒素缺陷3（pad 3）突变体中积累。Cys（IAN）拯救了cyp 79 b2 cyp 79 b3的camalexin缺陷表型，并且自身通过从银处理的拟南芥叶中分离的微粒体转化为二氢camalexic酸（DHCA），DHCA是CYP 71 B15（PAD 3）的已知底物。令人惊讶的是，酵母表达的CYP 71 B15也催化噻唑啉环闭合、DHCA形成和以Cys（IAN）为底物的氰化物释放。总之，在Camalexin生物合成途径中，IAN衍生为中间体Cys（IAN），其作为多功能细胞色素P450酶CYP 71 B15的底物。
Accumulation of camalexin, the characteristic phytoalexin of Arabidopsis thaliana, is induced by a great variety of plant pathogens. It is derived from Trp, which is converted to indole-3-acetonitrile (IAN) by successive action of the cytochrome P450 enzymes CYP79B2/B3 and CYP71A13. Extracts from wild-type plants and camalexin biosynthetic mutants, treated with silver nitrate or inoculated with Phytophthora infestans, were comprehensively analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. This metabolomics approach was combined with precursor feeding experiments to characterize the IAN metabolic network and to identify novel biosynthetic intermediates and metabolites of camalexin. Indole-3-carbaldehyde and indole-3-carboxylic acid derivatives were shown to originate from IAN. IAN conjugates with glutathione, gamma-glutamylcysteine, and cysteine [Cys(IAN)] accumulated in challenged phytoalexin deficient3 (pad3) mutants. Cys(IAN) rescued the camalexin-deficient phenotype of cyp79b2 cyp79b3 and was itself converted to dihydrocamalexic acid (DHCA), the known substrate of CYP71B15 (PAD3), by microsomes isolated from silver nitrate-treated Arabidopsis leaves. Surprisingly, yeast-expressed CYP71B15 also catalyzed thiazoline ring closure, DHCA formation, and cyanide release with Cys(IAN) as substrate. In conclusion, in the camalexin biosynthetic pathway, IAN is derivatized to the intermediate Cys(IAN), which serves as substrate of the multifunctional cytochrome P450 enzyme CYP71B15.