Hu Zhimin, Ren Li, Bu Junling, Liu Xiuyu, Li Qishuang, Guo Wending, Ma Ying, Wang Jian, Chen Tong, Wang Ling, Jin Baolong, Tang Jinfu, Cui Guanghong, Guo Juan, Huang Luqi
State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.
School of Pharmaceutical Sciences, Henan University of Chinese Medicine, Zhengzhou, China.
Front Plant Sci. 2022 Jul 7;13:947674. doi: 10.3389/fpls.2022.947674. eCollection 2022.
is one of the most commonly used Chinese medicinal herbs. Tanshinones, the most abundant lipid-soluble bioactive constituents of , are a class of structural highly oxidized abietane-type diterpenoids with multiple pharmacological activities. Although several enzymes, including diterpene synthase, cytochrome P450, and Fe(II)/2-oxoglutarate-dependent dioxygenase (2OGD), have been functionally characterized in biosynthesis of abietane-type diterpenoids, the highly oxidized structure and complex secondary metabolic network of tanshinones imply that more oxidases should be characterized. Here, we identified a new 2OGD (Sm2OGD25) from . Molecular cloning and functional studies showed that Sm2OGD25 could catalyze the hydroxylation of sugiol at C-15 and C-16 positions to produce hypargenin B and crossogumerin C, respectively. The phylogenetic analysis of the DOXC family demonstrated that Sm2OGD25 belongs to the DOXC54 clade. Furthermore, structural modeling and site-directed mutagenesis characterization revealed the importance of the hydrogen-bonding residue Y339 and the hydrophobic residues (V122, F129, A144, A208, F303, and L344) in substrate binding and enzyme activity. This study will promote further studies on the catalytic characterization of plant 2OGDs and the secondary metabolic biosynthesis network of diterpenoids.
是最常用的中药材之一。丹参酮是其最丰富的脂溶性生物活性成分，是一类结构高度氧化的松香烷型二萜类化合物，具有多种药理活性。尽管包括二萜合酶、细胞色素P450和铁（II）/2-氧戊二酸依赖性双加氧酶（2OGD）在内的几种酶已在松香烷型二萜类化合物的生物合成中得到功能表征，但丹参酮的高度氧化结构和复杂的次生代谢网络表明，应该鉴定出更多的氧化酶。在这里，我们从 中鉴定出一种新的2OGD（Sm2OGD25）。分子克隆和功能研究表明，Sm2OGD25可以分别催化苏乔醇在C-15和C-16位的羟基化反应，生成海波根宁B和交沙古菌素C。DOXC家族的系统发育分析表明，Sm2OGD25属于DOXC54进化枝。此外，结构建模和定点诱变表征揭示了氢键残基Y339和疏水残基（V122、F129、A144、A208、F303和L344）在底物结合和酶活性中的重要性。本研究将促进对植物2OGD催化特性和二萜类次生代谢生物合成网络的进一步研究。
