Molecular basis for the P450-catalyzed C-N bond formation in indolactam biosynthesis
Molecular basis for the P450-catalyzed C-N bond formation in indolactam biosynthesis
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DOI：
10.1038/s41589-019-0380-9
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发表时间：
2019-12-01
影响因子：
14.8
通讯作者：
Abe, Ikuro
中科院分区：
文献类型：
--
作者：
He, Fei;Mori, Takahiro;Abe, Ikuro
细胞色素P450单加氧酶的催化多功能性是显著的。在这里，我们介绍了来自母芽链霉菌的TleB和来自印度斯坦链霉菌的同源物HinD的机制和结构特征，它们催化不同寻常的分子内C-N键形成，从二肽n -甲基戊酰色氨酸中生成吲哚内酰胺V。体外分析表明，这两种p450都表现出混杂底物特异性，n13 -甲基的修饰导致形成吲哚融合的6/5/6三环产物。此外，x射线晶体结构与底物和基于结构的诱变揭示了酶反应的内部结构细节。我们认为双自由基的生成对吲哚内酰胺的形成至关重要，并且分子内的C(sp(2))-H胺化是由N1吲哚氢的抽象引起的。吲哚自由基重新定位并去除N13氢后，正确折叠的双自由基的偶联导致吲哚内酰胺的C4-N13键的形成。
The catalytic versatility of cytochrome P450 monooxygenases is remarkable. Here, we present mechanistic and structural characterizations of TleB from Streptomyces blastmyceticus and its homolog HinD from Streptoalloteichus hindustanus, which catalyze unusual intramolecular C-N bond formation to generate indolactam V from the dipeptide N-methylvalyl-tryptophanol. In vitro analyses demonstrated that both P450s exhibit promiscuous substrate specificity, and modification of the N13-methyl group resulted in the formation of indole-fused 6/5/6 tricyclic products. Furthermore, X-ray crystal structures in complex with substrates and structure-based mutagenesis revealed the intimate structural details of the enzyme reactions. We propose that the generation of a diradical species is critical for the indolactam formation, and that the intramolecular C(sp(2))-H amination is initiated by the abstraction of the N1 indole hydrogen. After indole radical repositioning and subsequent removal of the N13 hydrogen, the coupling of the properly-folded diradical leads to the formation of the C4-N13 bond of indolactam.