Hapalindole生物碱生物合成双酶系统的结构与催化机理研究

Hapalindole type alkaloids (HAPs) are a group of natural products which exhibit fascinating polycyclic structures with multiple chiral centers. These compounds show diverse important biological activities and thus are of great potentials in further development of novel pharmaceuticals. The biosynthesis of HAPs is mediated by two consecutive enzyme-mediated catalytic processes. First step is catalyzed by a novel prenyltransferase FamD2, which transfers a GPP onto an indole nitrile to produce a common precursor of all HAPs: compound 1. Stig cyclases catalyze then cyclize compound 1 to produce various HAPs. FamD2 can produce two distinct products but only compound 1 can be further reacted. Stig cyclases are a class of novel enzyme whose function and structure are previously unexplored. The underlying mechanism of stig cyclases is of great interests because their abilities of catalyzing cope rearrangement under physiological condition and regulating chiral center formation. The proposal aims to solve crystal structures and complex structures of FamD2 and stig cyclases to demonstrate protein folding, substrate-binding pattern, and catalytic mechanism of these novel enzymes. Furthermore, structure-based enzyme engineering will be performed to optimize HAPs biosynthetic process. These results shall provide fundamental information in further studies of both enzymes and HAPs biosynthesis.

Hapalindole型生物碱是一群具有多个手性中心的多环系统天然化合物，由于具有多种重要生物活性，其生物合成过程相关研究受到广泛重视。Hapalindole型生物碱核心结构由两个主要酶-介导催化过程合成，首先FamD2将一个分子的GPP转移到吲哚异腈上形成化合物1，化合物1接着再由不同stig环化酶环化成各种hapalindole型生物碱。FamD2为新型ABBA异戊烯基转移酶，其催化产物有两种，只有化合物1可继续反应成hapalindole型生物碱。Stig环化酶为结构完全未知的全新家族，为酶催化科普重排及调控手性中心形成之重要研究目标。本项目拟解析FamD2以及stig环化酶家族成员的X光复合体结构，阐明这两种新型酶种的底物结合模式及其催化机制，并基于结构对催化系统进行改造，以提高hapalindole型生物碱双酶系统的生产效率，为hapalindole型生物碱生物合成奠定重要基础。

Hapalindole 型生物碱包括包括 hapalindole、ambiguine、fischerindole 以及 welwitindolinone，是一群真枝藻目蓝绿藻 (Stigonematalean) 所产生的次 级代谢物，化学结构特征为吲哚及由单萜环化组成的多环系统。这些化合物具有多种重要生物活性，包括抗细菌活性，抗结核杆菌活性，抗霉菌活性，杀虫剂活性，以及抗肿瘤活性，其中数种化合物活性可达纳摩尔等级，甚至对抗药株细菌以及抗药性肿瘤细胞有显着的抑制效果。由于 hapalindole 型生物碱特殊复杂的分子结构，及成为新型药物的巨大潜力，其生物合成过程相关研究受到广泛重视。本项目解析了Hapalindole型生物碱合成关键酶FamD1和多种Stig环化酶的晶体结构及其与底物/类似物的复合体结构，通过结构分析和突变体验证，确定了几种位于活性口袋的芳香族氨基酸为催化关键氨基酸。除了通用的底物结合模式和催化机理外，本研究也发现不同亚种酶的变异也可能导致产物多样性的形成。这些结果将进一步扩大我们对酶学认识和研究范围，特别是对生物合成Cope重排的进一步研究。