Institute of Biomolecule Reconstruction (iBR), Department of Life Science and Biochemical Engineering, Sun Moon Universitygrid.412859.3, Asan-si, Chungnam, Republic of Korea.
Department of Biotechnology and Pharmaceutical Engineering, Sun Moon Universitygrid.412859.3, Asan-si, Chungnam, Republic of Korea.
Appl Environ Microbiol. 2022 Jul 12;88(13):e0075422. doi: 10.1128/aem.00754-22. Epub 2022 Jun 15.
Methyltransferases transfer a methyl group to a diverse group of natural products, thus providing structural diversity, stability, and altered pharmacological properties to the molecules. A limited number of regiospecific sugar--methyltransferases are functionally characterized. Thus, discovery of such an enzyme could solve the difficulties of biological production of methoxy derivatives of glycosylated molecules. In the current study, a regiospecific sugar--methyltransferase, ThnM1, belonging to the biosynthetic gene cluster (BGC) of 1-(α-L-(2--methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene produced by sp. strain CS682, was analyzed and functionally characterized. ThnM1 demonstrated promiscuity to diverse chemical structures such as rhamnose-containing anthraquinones and flavonoids with regiospecific methylation at the 2'-hydroxyl group of the sugar moiety. Compared with other compounds, anthraquinone rhamnosides were found to be the preferred substrates for methylation. Thus, the enzyme was further employed for whole-cell biotransformation using engineered Escherichia coli to produce a methoxy-rhamnosyl derivative of quinizarin, an anthraquinone derivative. The structure of the newly generated derivative from Escherichia coli fermentation was elucidated by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopic analyses and identified as quinizarin-4--α-l-2--methylrhamnoside (QRM). Further, the biological impact of methylation was studied by comparing the cytotoxicity of QRM with that of quinizarin against the U87MG, SNU-1, and A375SM cancer cell lines. ThnM1 is a putative sugar--methyltransferase produced by the sp. strain CS682 and is encoded by a gene belonging to the biosynthetic gene cluster (BGC) of 1-(α-l-(2--methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene. We demonstrated that ThnM1 is a promiscuous enzyme with regiospecific activity at the 2'-OH of rhamnose. As regiospecific methylation of sugars by chemical synthesis is a challenging step, ThnM1 may fill the gap in the potential diversification of natural products by methylating the rhamnose moiety attached to them.
甲基转移酶将甲基基团转移到各种天然产物上，从而为分子提供结构多样性、稳定性和改变的药理学性质。有少数几种区域特异性糖-甲基转移酶具有功能特征。因此，发现这样的酶可以解决生物生产糖基化分子的甲氧基衍生物的困难。在本研究中，分析并功能表征了属于 sp. CS682 产生的 1-(α-L-(2--甲基)-6-去氧甘露吡喃糖氧基)-3,6,8-三甲氧基萘的生物合成基因簇（BGC）的一种区域特异性糖-甲基转移酶 ThnM1。ThnM1 对各种化学结构表现出混杂性，如含有鼠李糖的蒽醌和黄酮类化合物，糖部分的 2'-羟基具有区域特异性甲基化。与其他化合物相比，蒽醌鼠李糖苷被发现是甲基化的首选底物。因此，该酶进一步用于使用工程大肠杆菌进行全细胞生物转化，以生产蒽醌衍生物喹啉嗪的甲氧基-鼠李糖苷衍生物。通过液质联用和核磁共振波谱分析阐明了大肠杆菌发酵产生的新衍生物的结构，并鉴定为喹啉嗪-4--α-l-2--甲基鼠李糖苷（QRM）。此外，通过比较 QRM 与喹啉嗪对 U87MG、SNU-1 和 A375SM 癌细胞系的细胞毒性，研究了甲基化的生物学影响。ThnM1 是由 sp. CS682 产生的一种假定的糖-甲基转移酶，由编码 1-(α-L-(2--甲基)-6-去氧甘露吡喃糖氧基)-3,6,8-三甲氧基萘的生物合成基因簇（BGC）中的基因编码。我们证明 ThnM1 是一种具有混杂性的酶，在鼠李糖的 2'-OH 上具有区域特异性活性。由于糖的区域特异性甲基化是化学合成的一个具有挑战性的步骤，因此 ThnM1 可以通过对它们附着的鼠李糖部分进行甲基化来填补潜在天然产物多样化的空白。
