Stereospecific Radical-Mediated B12-Dependent Methyl Transfer by the Fosfomycin Biosynthesis Enzyme Fom3
Stereospecific Radical-Mediated B12-Dependent Methyl Transfer by the Fosfomycin Biosynthesis Enzyme Fom3
复制标题
DOI：
10.1021/acs.biochem.8b00616
复制
发表时间：
2018-08-21
期刊：
影响因子：
2.9
通讯作者：
van der Donk, Wilfred A.
中科院分区：
文献类型：
--
作者：
McLaughlin, Martin, I;van der Donk, Wilfred A.
form3是链霉菌（Streptomyces spp.）中磷霉素生物合成途径的前位酶，是一种B类钴胺依赖的自由基SAM甲基转移酶，可催化(5'- cytidyyl)-2-羟乙基膦酸盐（2-HEP-CMP）甲基化形成（5'- cytidyyl）2-羟丙基膦酸盐（2-HPP-CMP）。在此之前，Fom3与2-HEP-CMP反应产生了2-HPPCMP在C2处具有混合立体化学。由于在大肠杆菌中不溶性表达和钴胺素（B-12）结合不良，机制表征一直具有挑战性。最近，通过过表达BtuCEDFB钴胺摄取系统，在大肠杆菌中实现了钴胺素在Fom3中的可溶性表达和整合。本文采用这种新方法从wedmorensis链霉菌中获得了form3。我们证明了引发剂5'-脱氧腺苷基自由基立体特异性地从2-HEP-CMP的C2位置提取了前r氢原子，并使用下游酶FomD和Fom4证明了我们制备的Fom3只产生(2S)-2-HPP-CMP。此外，我们发现在多次周转条件下，添加的甲基来自SAM，但第一次周转使用已经存在于酶上的甲基供体；此外，从form3反应混合物中分离的钴胺素含有源自SAM的甲基。这些结果与一个模型一致，在这个模型中，Fom3催化甲基从SAM转移到钴胺素，产生的甲基钴胺素（MeCbl）是该反应的最终甲基来源。
Fom3, the antepenultimate enzyme in the fosfomycin biosynthetic pathway in Streptomyces spp., is a class B cobalamin-dependent radical SAM methyltransferase that catalyzes methylation of (5'-cytidylyl)-2-hydroxyethylphosphonate (2-HEP-CMP) to form (5'-cytidylyl)2-hydroxypropylphosphonate (2-HPP-CMP). Previously, the reaction of Fom3 with 2-HEP-CMP produced 2-HPPCMP with mixed stereochemistry at C2. Mechanistic characterization has been challenging because of insoluble expression and poor cobalamin (B-12) incorporation in Escherichia coli. Recently, soluble E. coli expression and incorporation of cobalamin into Fom3 were achieved by overexpression of the BtuCEDFB cobalamin uptake system. Herein, we use this new method to obtain Fom3 from Streptomyces wedmorensis. We show that the initiator 5'-deoxyadenosyl radical stereospecifically abstracts the pro-R hydrogen atom from the C2 position of 2-HEP-CMP and use the downstream enzymes FomD and Fom4 to demonstrate that our preparation of Fom3 produces only (2S)-2-HPP-CMP. Additionally, we show that the added methyl group originates from SAM under multiple-turnover conditions, but the first turnover uses a methyl donor already present on the enzyme; furthermore, cobalamin isolated from Fom3 reaction mixtures contains methyl groups derived from SAM. These results are consistent with a model in which Fom3 catalyzes methyl transfer from SAM to cobalamin and the resulting methylcobalamin (MeCbl) is the ultimate methyl source for the reaction.