Tetrahydrofuran ring formation from dibenzylbutyrolactone lignans is a key step in the biosynthesis of aryltetralin lignans including deoxypodophyllotoxin and podophyllotoxin. Previously, Fe (II)- and 2-oxoglutarate-dependent dioxygenase (2-ODD) from Podophyllum hexandrum (Himalayan mayapple, Berberidaceae) was found to catalyze cyclization of a dibenzylbutyrolactone lignan, yatein, to give deoxypodophyllotoxin and designated as deoxypodophyllotoxin synthase (DPS). Recently, we reported that the biosynthesis of deoxypodophyllotoxin and podophyllotoxin evolved in a lineage-specific manner in phylogenetically unrelated plant species such as P. hexandrum and Anthriscus sylvestris (cow parsley, Apiaceae). Therefore, a comprehensive understanding of the characteristics of DPSs that catalyze the cyclization of yatein to deoxypodophyllotoxin in various plant species is important. However, for other plant species than P. hexandrum, the isolation of the DPS enzyme gene and the type of the enzyme, e.g., whether it is 2-ODD or another type of enzyme such as cytochrome P-450 has not been reported. Here, we report the identification and characterization of A. sylvestris DPS (AsDPS). Phylogenetic analysis showed that AsDPS belonged to the 2-ODD superfamily and shared moderate amino acid sequence identity (40.8%) with PhDPS. Recombinant protein assay indicated that AsDPS and PhDPS differ in terms of the selectivity of substrate enantiomers. Protein modeling using Alpha-Fold2 and site-directed mutagenesis indicated that the Tyr305 residue of AsDPS probably contributes to substrate recognition. This study advances our understanding of the podophyllotoxin biosynthetic pathway in A. sylvestris and provides new insight into 2-ODD involved in plant secondary (specialized) metabolism.

中文翻译：

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参与鬼臼毒素生物合成的脱氧鬼臼毒素合酶

由二苄基丁内酯木脂素形成四氢呋喃环是芳基四氢化萘木脂素（包括脱氧鬼臼毒素和鬼臼毒素）生物合成的关键步骤。此前，人们发现来自鬼臼（喜马拉雅Mayapple，小檗科）的Fe（II）-和2-酮戊二酸依赖性双加氧酶（2-ODD）催化二苄基丁内酯木脂素yatein的环化，产生脱氧鬼臼毒素，并命名为脱氧鬼臼毒素合酶（DPS） ）。最近，我们报道了脱氧鬼臼毒素和鬼臼毒素的生物合成在系统发育无关的植物物种中以谱系特异性方式进化，例如 P. hexandrum 和 Anthriscus sylvestris（牛欧芹，伞形科）。因此，全面了解不同植物物种中催化yatein环化为脱氧鬼臼毒素的DPS特性非常重要。然而，对于除六颗星以外的其他植物物种，DPS酶基因的分离和酶的类型，例如是否是2-ODD或另一种类型的酶如细胞色素P-450尚未见报道。在这里，我们报告了 A. sylvestris DPS (AsDPS) 的鉴定和表征。系统发育分析表明AsDPS属于2-ODD超家族，与PhDPS具有中等的氨基酸序列一致性（40.8%）。重组蛋白分析表明AsDPS 和PhDPS 在底物对映异构体的选择性方面存在差异。使用 Alpha-Fold2 和定点诱变进行的蛋白质建模表明 AsDPS 的 Tyr305 残基可能有助于底物识别。这项研究增进了我们对樟子松中鬼臼毒素生物合成途径的理解，并为参与植物次生（专门）代谢的 2-ODD 提供了新的见解。