Substrate-Dependent Cleavage Site Selection by Unconventional Radical S-Adenosylmethionine Enzymes in Diphthamide Biosynthesis.
Substrate-Dependent Cleavage Site Selection by Unconventional Radical S-Adenosylmethionine Enzymes in Diphthamide Biosynthesis.
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DOI：
10.1021/jacs.7b01712
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发表时间：
2017-04-26
影响因子：
15
通讯作者：
Lin H
中科院分区：
文献类型：
--
作者：
Dong M;Horitani M;Dzikovski B;Freed JH;Ealick SE;Hoffman BM;Lin H
S-腺苷蛋氨酸含有一个带有三个不同C-S键的硫离子。传统的自由基SAM酶使用[4Fe-4S]簇来均裂SAM的C5‘，腺苷-S键，生成5’-脱氧腺苷自由基，从而催化各种下游化学反应。参与联苯二甲胺生物合成的自由基链霉菌Dph2(PhDph2)和酵母菌Dph1-Dph2代替裂解蛋氨酸的C-γ，Met-S键生成3-氨基-3-羧丙基。我们发现，通过改变SAM的结构，可以调节自由基SAM酶来裂解硫磺上的第三个C-S键。PhDph2与脱羧基SAM类似物(DC-SAM)一起裂解C-甲基-S键，形成5‘-脱氧-5’-(3-氨基丙硫基)腺苷(dAPTA，1)。与其他两个C-S键的裂解一样，甲基裂解活性依赖于[4Fe-4S]+簇的存在。电子-核双共振(ENDOR)和质谱学数据表明，从机理上讲，[4Fe-4S]团簇中的一个S原子从DC-SAM中捕获了甲基，形成了一个独特的EPR活性中间体，该中间体可以将甲基转移到二硫苏糖醇等亲核试剂上。这揭示了自由基SAM酶中的[4Fe-4S]簇可以被调节来切割SAM或类似物的硫磺上的三个键中的任何一个，并首次证明了自由基SAM酶可以底物依赖的方式从基于Fe的单电子转移反应切换到基于S的双电子转移反应。因此，这项研究为Fe-S团簇的多种反应性提供了一个显着的例证。
S -adenosylmethionine (SAM) has a sulfonium ion with three distinct C-S bonds. Conventional radical SAM enzymes use a [4Fe–4S] cluster to homolytically cleave the C5′,adenosine-S bond of SAM to generate a 5′-deoxyadenosyl radical, which catalyzes various downstream chemical reactions. Radical SAM enzymes involved in diphthamide biosynthesis, such as Pyrococcus horikoshii Dph2 (PhDph2) and yeast Dph1-Dph2 instead cleave the Cγ,Met–S bond of methionine to generate the 3-amino-3-carboxylpropyl radical. We here show that radical SAM enzymes can be tuned to cleave the third C-S bond to the sulfonium sulfur by changing the structure of SAM. With a decarboxyl SAM analogue (dc-SAM), PhDph2 cleaves the Cmethyl–S bond, forming 5′-deoxy-5′-(3-aminopropylthio) adenosine (dAPTA, 1). The methyl cleavage activity, like the cleavage of the other two C-S bonds, is dependent on the presence of a [4Fe-4S]+ cluster. Electron-nuclear double resonance (ENDOR) and mass spectroscopy data suggests that mechanistically, one of the S atoms in the [4Fe-4S] cluster captures the methyl group from dc-SAM, forming a distinct EPR-active intermediate, which can transfer the methyl group to nucleophiles such as dithiothreitol. This reveals that the [4Fe-4S] cluster in a radical SAM enzyme can be tuned to cleave any one of the three bonds to the sulfonium sulfur of SAM or analogues, and is the first demonstration that a radical SAM enzyme could switch from an Fe-based one electron transfer reaction to a S-based two electron transfer reaction in a substrate-dependent manner. This study thus provides a remarkable illustration of the versatile reactivity of Fe-S clusters.
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影响因子：
15
作者：
Dong M;Su X;Dzikovski B;Dando EE;Zhu X;Du J;Freed JH;Lin H
通讯作者：
Lin H
影响因子：
15
作者：
Bhandari, Dhananjay M.;Fedoseyenko, Dmytro;Begley, Tadhg P.
通讯作者：
Begley, Tadhg P.
影响因子：
15
作者：
Walsby, CJ;Ortillo, D;Hoffman, BM
通讯作者：
Hoffman, BM
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
15
作者：
Grove, Tyler L.;Radle, Matthew I.;Krebs, Carsten;Booker, Squire J.
通讯作者：
Booker, Squire J.