Cytochrome P450 Mediated Cyclization in Eunicellane Derived Diterpenoid Biosynthesis.
Cytochrome P450 Mediated Cyclization in Eunicellane Derived Diterpenoid Biosynthesis.
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DOI：
10.1002/anie.202312490
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发表时间：
2023-09
影响因子：
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通讯作者：
Zengyuan Wang;Qian Yang;Jingyi He;Haixin Li;Xingming Pan;Zining Li;Hui-Min Xu;Jeffrey D Rudolf;D. Tantillo;Liao-Bin Dong
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作者：
Zengyuan Wang;Qian Yang;Jingyi He;Haixin Li;Xingming Pan;Zining Li;Hui-Min Xu;Jeffrey D Rudolf;D. Tantillo;Liao-Bin Dong
萜烯环化反应是自然界中最复杂的化学反应之一，通常由两类萜烯环化酶（TC）催化。细胞色素P450作为意想不到的TC样酶是已知的，但非常罕见。在这里，我们从嗜热放线菌菌株Arida无枝酸菌中挖掘了一个神秘的细菌萜类化合物基因簇，命名为ari。通过采用异源生产系统，我们分离并表征了三种高度氧化的尤尼花烷衍生的二萜类化合物，aridacins A-C（1 - 3），其具有6/7/5-稠合的三环骨架。体内和体外实验系统地建立了二萜骨架形成的非经典两步生物合成途径。首先，I类TC（AriE）将香叶基香叶基二磷酸（GGPP）环化成6/10-稠合双环顺式-尤尼凯烷骨架。接下来，细胞色素P450（AriF）通过C2-C6键的形成催化尤卡坦骨架环化为6/7/5-稠合的三环骨架。量子化学计算的结果的基础上，氢提取，然后通过电子转移耦合到无势垒的碳正离子环闭合被证明是一个可行的机制AriF介导的环化。aridacins中骨架构建的生物合成逻辑是前所未有的，扩大了P450的催化能力和多样性，并为研究P450在萜类化合物生物合成中产生碳阳离子的内在原理奠定了基础。
Terpene cyclization, one of the most complex chemical reactions in nature, is generally catalyzed by two classes of terpene cyclases (TCs). Cytochrome P450s that act as unexpected TC-like enzymes are known but are very rare. Here, we genome-mined a cryptic bacterial terpenoid gene cluster, named ari, from the thermophilic actinomycete strain Amycolatopsis arida. By employing a heterologous production system, we isolated and characterized three highly oxidized eunicellane derived diterpenoids, aridacins A-C (1-3), that possess a 6/7/5-fused tricyclic scaffold. In vivo and in vitro experiments systematically established a non-canonical two-step biosynthetic pathway for diterpene skeleton formation. First, a class I TC (AriE) cyclizes geranylgeranyl diphosphate (GGPP) into a 6/10-fused bicyclic cis-eunicellane skeleton. Next, a cytochrome P450 (AriF) catalyzes cyclization of the eunicellane skeleton into the 6/7/5-fused tricyclic scaffold via C2-C6 bond formation. Based on the results of quantum chemical computations, hydrogen abstraction followed by electron transfer coupled to barrierless carbocation ring-closure is shown to be a viable mechanism for AriF-mediated cyclization. The biosynthetic logic of skeleton construction in the aridacins is unprecedented, expanding the catalytic capacity and diversity of P450s and setting the stage to investigate the inherent principles of carbocation generation by P450s in the biosynthesis of terpenoids.