Cysteine methylation controls radical generation in the Cfr radical AdoMet rRNA methyltransferase.
Cysteine methylation controls radical generation in the Cfr radical AdoMet rRNA methyltransferase.
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DOI：
10.1371/journal.pone.0067979
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Spencer J
中科院分区：
文献类型：
--
作者：
Challand MR;Salvadori E;Driesener RC;Kay CW;Roach PL;Spencer J
‘自由基S-腺苷-L-蛋氨酸(ADO-Met)’酶Cfr甲基化细菌核糖体肽基转移酶中心(P位)的23S rRNA的腺苷2503。这种修饰保护宿主细菌，特别是耐甲氧西林金黄色葡萄球菌(MRSA)免受多种抗生素的影响，包括为治疗这些细菌而开发的药物(例如利奈唑胺、瑞帕巴林)。CFR包含一个[4Fe-4S]团簇，在反应周期中将两个单独的ADOMet分子结合在一起。它们被顺序地用来首先甲基化半胱氨酸残基Cys338；然后产生氧化自由基中间体，该中间体促进甲基转移到腺苷2503的非活性C8(和/或C2)碳中心。CFR活性中心及其单一的[4Fe-4S]簇如何催化这两种不同的活性，每种活性都利用ADOMet作为底物，仍有待确定。在这里，我们使用吸光度和电子顺磁共振(EPR)光谱来研究ADOMet与野生型CFR的[4Fe-4S]簇和无法接受甲基的Cys338 Ala突变体的相互作用。尽管没有核糖核酸共底物，CFR仍能以高亲和力(∼10微米)结合ADOMet。在野生型CFR中，Cys338甲基化，ADOMet结合导致[4Fe-4S]簇的快速氧化和5‘-脱氧腺苷(DOA)的产生。相反，虽然Cys338 Ala CFR以相当的亲和力结合Adobe Met，但没有观察到[4Fe-4S]团簇的氧化。我们的结果表明，CFR Cys338上甲基的存在是该酶对ADOMet活性的关键决定因素，从而使单个活性位点能够支持两种不同的ADOMet切割模式。
The ‘radical S-adenosyl-L-methionine (AdoMet)’ enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), from numerous antibiotics, including agents (e.g. linezolid, retapamulin) that were developed to treat such organisms. Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of AdoMet during the reaction cycle. These are used sequentially to first methylate a cysteine residue, Cys338; and subsequently generate an oxidative radical intermediate that facilitates methyl transfer to the unreactive C8 (and/or C2) carbon centres of adenosine 2503. How the Cfr active site, with its single [4Fe-4S] cluster, catalyses these two distinct activities that each utilise AdoMet as a substrate remains to be established. Here, we use absorbance and electron paramagnetic resonance (EPR) spectroscopy to investigate the interactions of AdoMet with the [4Fe-4S] clusters of wild-type Cfr and a Cys338 Ala mutant, which is unable to accept a methyl group. Cfr binds AdoMet with high (∼ 10 µM) affinity notwithstanding the absence of the RNA cosubstrate. In wild-type Cfr, where Cys338 is methylated, AdoMet binding leads to rapid oxidation of the [4Fe-4S] cluster and production of 5'-deoxyadenosine (DOA). In contrast, while Cys338 Ala Cfr binds AdoMet with equivalent affinity, oxidation of the [4Fe-4S] cluster is not observed. Our results indicate that the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards AdoMet, thus enabling a single active site to support two distinct modes of AdoMet cleavage.
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影响因子：
2.9
作者：
Hinckley, GT;Frey, PA
通讯作者：
Frey, PA
自由基 SAM 酶进行甲基转移的结构基础。
DOI：
10.1126/science.1205358
发表时间：
2011-05-27
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Boal AK;Grove TL;McLaughlin MI;Yennawar NH;Booker SJ;Rosenzweig AC
通讯作者：
Rosenzweig AC
影响因子：
16.6
作者：
Driesener, Rebecca C.;Challand, Martin R.;Roach, Peter L.
通讯作者：
Roach, Peter L.
影响因子：
3.2
作者：
Dos Santos, Patricia C.;Johnson, Deborah C.;Dean, Dennis R.
通讯作者：
Dean, Dennis R.
影响因子：
2.9
作者：
Chandramouli, Kala;Unciuleac, Mihaela-Carmen;Johnson, Michael K.
通讯作者：
Johnson, Michael K.