Heterodimeric Non-heme Iron Enzymes in Fungal Meroterpenoid Biosynthesis
Heterodimeric Non-heme Iron Enzymes in Fungal Meroterpenoid Biosynthesis
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DOI：
10.1021/jacs.1c11548
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发表时间：
2021-12-09
影响因子：
15
通讯作者：
Abe, Ikuro
中科院分区：
文献类型：
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作者：
Li, Xinyang;Awakawa, Takayoshi;Abe, Ikuro
Talaromyoliides (1-6) 是一组不寻常的 6/6/6/6/6/6 六环类萜类化合物，具有 (3R)-6-羟基梅素和 4,5-仲二丙烷亚结构，从海洋真菌 Talaromyces purpureogenus 中分离出来。我们通过曲霉中的异源表达、体外酶测定和基于 CRISPR-Cas9 的基因失活鉴定了生物合成基因簇 tlxA-J。值得注意的是，非血红素铁 (NHI) 酶的异二聚体 TlxJ-TlxI 催化三个氧化步骤，包括关键反应，即 12 的 C-5 和 C-9 处的羟基化（具有 3-酮羟基二甲基苯胺支架的中间体），以促进逆羟醛反应，从而构建 4,5-司科二甲基苯胺骨架和 1-6 的特征缩酮骨架。 TlxJ-TlxI 的产物 1 和 4 在 C-4'β 处被另一种 NHI 异二聚体 TlxA-TlxC 进一步羟基化，并被 TlxB 乙酰化，得到最终产物 3 和 6。X 射线结构分析与定点诱变相结合，为异二聚体 TlxJ-TlxI 的形成及其催化提供了深入的了解。这是第一份报告表明两种 NHI 蛋白形成异二聚体进行催化，并利用一种新的方法在次级代谢物生物合成中创建功能性加氧酶结构。
Talaromyolides (1-6) are a group of unusual 6/6/6/6/6/6 hexacyclic meroterpenoids with (3R)-6-hydroxymellein and 4,5-seco-drimane substructures, isolated from the marine fungus Talaromyces purpureogenus. We have identified the biosynthetic gene cluster tlxA-J by heterologous expression in Aspergillus, in vitro enzyme assays, and CRISPR-Cas9-based gene inactivation. Remarkably, the heterodimer of non-heme iron (NHI) enzymes, TlxJ-TlxI, catalyzes three steps of oxidation including a key reaction, hydroxylation at C-5 and C-9 of 12, the intermediate with 3-ketohydroxydrimane scaffold, to facilitate a retro-aldol reaction, leading to the construction of the 4,5-secodrimane skeleton and characteristic ketal scaffold of 1-6. The products of TlxJ-TlxI, 1 and 4, were further hydroxylated at C-4'beta by another NHI heterodimer, TlxA-TlxC, and acetylated by TlxB to yield the final products, 3 and 6. The X-ray structural analysis coupled with site-directed mutagenesis provided insights into the heterodimer TlxJ-TlxI formation and its catalysis. This is the first report to show that two NHI proteins form a heterodimer for catalysis and utilizes a novel methodology to create functional oxygenase structures in secondary metabolite biosynthesis.