The Formation of a Camalexin Biosynthetic Metabolon
The Formation of a Camalexin Biosynthetic Metabolon
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骆驼蓬苷生物合成代谢物的形成
DOI：
10.1105/tpc.19.00403
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发表时间：
2019-11-01
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：
Mucha, Stefanie;Heinzlmeir, Stephanie;Glawischnig, Erich
拟南芥（Arabidopsis thaliana）有效地合成抗真菌植物防御素camalexin，而没有明显释放生物活性中间体，如吲哚-3-乙醛肟，这表明该化合物的生物合成途径通过形成酶复合物来引导。为了鉴定此类蛋白质相互作用，我们使用了两种独立的非靶向免疫共沉淀（co-IP）方法，以生物合成酶CYP 71 B15和CYP 71 A13作为诱饵，并确定山茶素生物合成P450酶与这些酶共纯化。这些相互作用得到了证实，有针对性的co-IP和福斯特共振能量转移测量荧光寿命显微镜（FRET FLIM）的基础上。此外，还观察到CYP 71 A13与拟南芥P450还原酶1的相互作用。我们检测到CYP 71 A13存在时CYP 79 B2的底物亲和力增加，表明存在变构相互作用。Camalexin生物合成涉及中间体吲哚-3-氰醇的谷胱甘肽化，吲哚-3-氰醇由CYP 71 A12特别是CYP 71 A13合成。FRET-FLIM和co-IP表明，谷胱甘肽转移酶GSTU 4，这是与色氨酸和骆驼蛋白特异性酶共表达，是物理招募到复杂的。令人惊讶的是，Camalexin浓度在敲除中升高，而在GSTU 4过表达植物中降低。这表明GSTU 4不直接参与Camalexin生物合成，而是在竞争机制中发挥作用。
Arabidopsis (Arabidopsis thaliana) efficiently synthesizes the antifungal phytoalexin camalexin without the apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting that the biosynthetic pathway of this compound is channeled by the formation of an enzyme complex. To identify such protein interactions, we used two independent untargeted coimmunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B15 and CYP71A13 as baits and determined that the camalexin biosynthetic P450 enzymes copurified with these enzymes. These interactions were confirmed by targeted co-IP and Forster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, the interaction of CYP71A13 and Arabidopsis P450 Reductase1 was observed. We detected increased substrate affinity of CYP79B2 in the presence of CYP71A13, indicating an allosteric interaction. Camalexin biosynthesis involves glutathionylation of the intermediary indole-3-cyanohydrin, which is synthesized by CYP71A12 and especially CYP71A13. FRET-FLIM and co-IP demonstrated that the glutathione transferase GSTU4, which is coexpressed with Trp- and camalexin-specific enzymes, is physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knockout and reduced in GSTU4-overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather plays a role in a competing mechanism.