Key Laboratory of National Forestry and Grassland Administration/Beijing for Bamboo & Rattan Science and Technology, Beijing 100102, China.
Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China.
J Agric Food Chem. 2022 May 11;70(18):5728-5737. doi: 10.1021/acs.jafc.2c01281. Epub 2022 Apr 27.
All -methylated derivatives of emodin, including physcion, questin, and 1--methylemodin, show potential antifungal activities. Notably, emodin and questin are two pivotal intermediates of geodin biosynthesis in . Although most of the geodin biosynthetic steps have been investigated, the key -methyltransferase (OMT) responsible for the -methylation of emodin to generate questin has remained unidentified. Herein, through phylogenetic tree analysis and biochemical assays, the long-sought class II emodin--methyltransferase GedA has been functionally characterized. Additionally, the catalytic mechanism and key residues at the catalytic site of GedA were elucidated by enzyme-substrate-methyl donor analogue ternary complex crystal structure determination and site-directed mutagenesis. As we demonstrate, GedA adopts a typical general acid/base (E446/H373)-mediated transmethylation mechanism. In particular, residue D374 is also crucial for efficient catalysis through blocking the formation of intramolecular hydrogen bonds in emodin. This study will facilitate future engineering of GedA for the production of physcion or other site-specific -methylated anthraquinone derivatives with potential applications as biopesticides.
大黄素的所有甲基化衍生物，包括大黄酸、大黄素甲醚和 1--甲基大黄素，都显示出潜在的抗真菌活性。值得注意的是，大黄素和大黄素甲醚是大黄素在 生物合成中的两个关键中间体。尽管已经研究了大部分大黄素生物合成步骤，但负责将大黄素甲基化为大黄素甲醚的关键 -甲基转移酶（OMT）仍然未被鉴定。在此，通过系统发育树分析和生化测定，功能表征了长期以来被追寻的 II 类大黄素 -甲基转移酶 GedA。此外，通过酶-底物-甲基供体类似物三元复合物晶体结构测定和定点突变，阐明了 GedA 的催化机制和催化位点的关键残基。正如我们所证明的，GedA 采用了典型的酸碱（E446/H373）介导的转甲基化机制。特别是，残基 D374 也通过阻止大黄素中分子内氢键的形成对高效催化至关重要。本研究将有助于未来对 GedA 的工程改造，以生产大黄酸或其他具有生物农药应用潜力的特定位置 -甲基化蒽醌衍生物。
