Law Brian J C, Struck Anna-Winona, Bennett Matthew R, Wilkinson Barrie, Micklefield Jason
School of Chemistry and Manchester Institute of Biotechnology , The University of Manchester , 131 Princess Street , Manchester , M1 7DN , UK . Email:
Department of Molecular Microbiology , John Innes Centre , Norwich , NR4 7UH , UK.
Chem Sci. 2015 May 1;6(5):2885-2892. doi: 10.1039/c5sc00164a. Epub 2015 Mar 2.
The methylation of natural products by -adenosyl methionine (AdoMet, also known as SAM)-dependent methyltransferase enzymes is a common tailoring step in many biosynthetic pathways. The introduction of methyl substituents can affect the biological and physicochemical properties of the secondary metabolites produced. Recently it has become apparent that some AdoMet-dependent methyltransferases exhibit promiscuity and will accept AdoMet analogues enabling the transfer of alternative alkyl groups. In this study we have characterised a methyltransferase, RapM, which is involved in the biosynthesis of the potent immunosuppressive agent rapamycin. We have shown that recombinant RapM regioselectively methylates the C16 hydroxyl group of desmethyl rapamycin precursors and is promiscuous in accepting alternative co-factors in addition to AdoMet. A coupled enzyme system was developed, including a mutant human enzyme methionine adenosyl transferase (MAT), along with RapM, which was used to prepare alkylated rapamycin derivatives (rapalogs) with alternative ethyl and allyl ether groups, derived from simple -ethyl or -allyl methionine analogues. There are two other methyltransferases RapI and RapQ which provide methyl substituents of rapamycin. Consequently, using the enzymatic approach described here, it should be possible to generate a diverse array of alkylated rapalogs, with altered properties, that would be difficult to obtain by traditional synthetic approaches.
由依赖S-腺苷甲硫氨酸（AdoMet，也称为SAM）的甲基转移酶对天然产物进行甲基化是许多生物合成途径中常见的修饰步骤。甲基取代基的引入会影响所产生的次生代谢产物的生物学和物理化学性质。最近已变得明显的是，一些依赖AdoMet的甲基转移酶表现出选择性，并且会接受AdoMet类似物，从而实现替代烷基的转移。在本研究中，我们对一种参与强效免疫抑制剂雷帕霉素生物合成的甲基转移酶RapM进行了表征。我们已经表明，重组RapM区域选择性地将去甲基雷帕霉素前体的C16羟基甲基化，并且除了AdoMet之外还能选择性地接受替代辅因子。我们开发了一种偶联酶系统，包括一种突变型人酶甲硫氨酸腺苷转移酶（MAT）以及RapM，该系统用于制备具有替代乙基和烯丙基醚基团的烷基化雷帕霉素衍生物（rapalogs），这些基团衍生自简单的S-乙基或S-烯丙基甲硫氨酸类似物。还有另外两种甲基转移酶RapI和RapQ，它们为雷帕霉素提供甲基取代基。因此，使用本文所述的酶促方法，应该有可能生成一系列具有不同性质的烷基化rapalogs，而这些通过传统合成方法很难获得。
