Prenylation of cyclodipeptides contributes largely to the structure diversification and biological activity. The prenylated products can be further metabolized by modifications like hydroxylation with cytochrome P450 enzymes or nonheme Fe^II/2-oxoglutarate-dependent oxygenases. Herein, we cloned and overexpressed NFIA_045530 from Neosartorya fischeri, which shares high sequence similarity with the nonheme Fe^II/2-oxoglutarate-dependent oxygenase FtmOx1_Af from Aspergillus fumigatus on the amino acid level. FtmOx1_Af is a member of the biosynthetic enzymes for fumitremorgin-type mycotoxins and catalyzes the conversion of fumitremorgin B to verruculogen by insertion of an oxygen molecule into the two prenyl moieties. The recombinant protein EAW25734 encoded by NFIA_045530 was purified to apparent homogeneity and then was used for incubation with intermediates of the fumitremorgin biosynthetic pathway. LC-MS analysis revealed no consumption of fumitremorgin B but good conversion with its biosynthetic precursor tryprostatin B in the presence of Fe^II and 2-oxoglutarate. Structure elucidation confirmed 22-hydroxylisotryprostatin B and 14α, 22-dihydroxylisotryprostatin B as the major enzyme products. Further detailed biochemical characterization led to the identification of a novel enzyme, which catalyzes a double bond migration within the dimethylallyl moiety of tryprostatin B with concomitant hydroxylation. Incubation with ¹⁸O₂-enriched atmosphere confirmed O₂ as the major origin of the hydroxyl groups. Solvent exchange was also observed for that at C22. LC-MS analysis confirmed the presence of 22-hydroxylisotryprostatin B in a Neosartorya fischeri extract, highlighting the role of this enzyme in the metabolism of intermediates of the fumitremorgin/verruculogen pathway. A plausible reaction mechanism implementing a radical rearrangement prior to accepting a hydroxyl radical from Fe^III is discussed.

中文翻译：

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非血红素Fe ^II / 2-Oxoglutarate依赖加氧酶催化二甲基烯丙基部分伴随羟基化的双键迁移。

环二肽的烯丙基化主要有助于结构多样化和生物活性。可以通过修饰，例如用细胞色素P450酶或非血红素Fe ^II / 2-氧代戊二酸酯依赖性加氧酶进行羟基化，使异戊二烯基化产物进一步代谢。在这里，我们克隆和过度表达从费氏新孢子虫的NFIA_045530，其与来自烟曲霉的非血红素Fe ^II / 2-氧戊二酸依赖性加氧酶FtmOx1 _Af在氨基酸水平上具有高度的序列相似性。FtmOx1_房颤是Fumitremorgin型霉菌毒素生物合成酶的成员，并且通过将氧分子插入两个异戊二烯基部分来催化Fumitremorgin B转化为疣蛋白原。纯化由NFIA_045530编码的重组蛋白EAW25734，使其具有明显的同质性，然后用于与泛黄褐藻生物合成途径的中间体一起孵育。LC-MS分析表明，在存在Fe ^II的情况下，未消耗fumitremorgin B，但其生物合成前体Tryprostatin B具有良好的转化率和2-氧戊二酸。结构鉴定证实了22-羟基赖脯氨酸前列腺素B和14α，22-二羟基赖脯氨酸前列腺素B是主要的酶产物。进一步详细的生化表征导致鉴定了一种新型酶，该酶催化胰蛋白酶素B的二甲基烯丙基部分内的双键迁移并伴有羟基化作用。在富含¹⁸ O _2的气氛中温育证实了O ₂是羟基的主要来源。在C22处也观察到溶剂交换。LC-MS分析确认了费氏新肉球菌中存在22-羟基赖脯前列腺素B提取物，突显了这种酶在褐藻/ verruculogen途径的中间体代谢中的作用。讨论了在接受来自Fe ^III的羟基自由基之前实施自由基重排的可能的反应机理。