Luo Ling, Li Dan, Long Bi, Huang Xiaoling, Wan Ying, Long Hongping, Wang Wenxuan, Li Jing, Xu Kangping, Tan Guishan, Yu Xia
Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, People's Republic of China; Hunan Key Laboratory of Diagnostic and Therapeutic Drug Research for Chronic Diseases, Central South University, Changsha, People's Republic of China.
Pharmacy Department, Jiujiang City Key Laboratory of Cell Therapy, JiuJiang NO.1 People's Hospital, Jiujiang, Jingxi, People's Republic of China.
J Biol Chem. 2025 Jul;301(7):110319. doi: 10.1016/j.jbc.2025.110319. Epub 2025 May 31.
Isoindolinone scaffold is widely presented in bioactive natural products and serves as a fundamental structure in pharmaceutical compounds. However, the key step of the isoindolinone core formation is not well-defined. In this study, we elucidated the complete biosynthetic pathway of the phytotoxic isoindolinone alkaloid zinnimidine, which was isolated from the cultures of various phytopathogenic fungi of the genus Alternaria. This was achieved through a systematic approach that integrated heterologous expression, feeding experiments, and enzymatic characterizations. Enzymes ZinADEF were identified as key catalysts involved in the biosynthesis of isoindolinone scaffold, directing the transformation from the 2-methyl benzoic acid-like tetraketide precursor to form the isoindolinone scaffold, while ZinB and ZinE catalyzes methylation and prenylation modification, respectively. Detailed enzymatic studies of the flavin-dependent oxidoreductase ZinD uncovered the key catalytic processes underlying the conversion from 1,2-benzenediol to the isoindolinone core, accompanied by the generation of hydrogen peroxide. Through enzymatic reactions of ZinD with various amino-containing compounds, porritoxin and several novel isoindolinones were synthesized. The analysis utilizing cblaster highlighted the conservation of ZinADEF genes across different fungi, indicating the existence of common biosynthetic pathways and mechanisms among these fungal species. Additionally, our study explained the late-stage biosynthetic branch details of isoindolinone and isobenzofuranone natural products. Our research unveils the biosynthetic mechanism of fungal isoindolinones, providing a theoretical foundation for the biocontrol of phytotoxins such as zinnimidine, and establishes a foundation for the biosynthesis of isoindolinone alkaloid derivatives.
异吲哚啉酮骨架广泛存在于生物活性天然产物中，是药物化合物的基本结构。然而，异吲哚啉酮核心形成的关键步骤尚不清楚。在本研究中，我们阐明了植物毒性异吲哚啉酮生物碱津尼米定的完整生物合成途径，该生物碱是从链格孢属各种植物致病真菌的培养物中分离得到的。这是通过整合异源表达、饲喂实验和酶学表征的系统方法实现的。酶ZinADEF被鉴定为参与异吲哚啉酮骨架生物合成的关键催化剂，指导从2-甲基苯甲酸样四酮前体转化形成异吲哚啉酮骨架，而ZinB和ZinE分别催化甲基化和异戊烯基化修饰。对黄素依赖性氧化还原酶ZinD的详细酶学研究揭示了从1,2-苯二醇转化为异吲哚啉酮核心的关键催化过程，同时伴随着过氧化氢的产生。通过ZinD与各种含氨基化合物的酶促反应，合成了波里毒素和几种新型异吲哚啉酮。利用cblaster进行的分析突出了ZinADEF基因在不同真菌中的保守性，表明这些真菌物种中存在共同的生物合成途径和机制。此外，我们的研究解释了异吲哚啉酮和异苯并呋喃酮天然产物后期生物合成分支的细节。我们的研究揭示了真菌异吲哚啉酮的生物合成机制，为津尼米定等植物毒素的生物防治提供了理论基础，并为异吲哚啉酮生物碱衍生物的生物合成奠定了基础。
