Dph3 is an electron donor for Dph1-Dph2 in the first step of eukaryotic diphthamide biosynthesis.
Dph3 is an electron donor for Dph1-Dph2 in the first step of eukaryotic diphthamide biosynthesis.
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DOI：
10.1021/ja4118957
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发表时间：
2014-02-05
影响因子：
15
通讯作者：
Lin H
中科院分区：
文献类型：
--
作者：
Dong M;Su X;Dzikovski B;Dando EE;Zhu X;Du J;Freed JH;Lin H
白喉毒素的作用靶点是白喉酰胺，它是古细菌和真核生物中翻译延伸因子2（EF 2）的一种独特的翻译后修饰。提出了双苯二甲酰胺的生物合成包括三个步骤。第一步是将3-氨基-3-羧丙基从S-腺苷-L-甲硫氨酸（SAM）转移到EF 2的组氨酸残基上，形成C-C键。以前的遗传研究表明，这一步骤需要真核生物中的四种蛋白质Dph 1-Dph 4。然而，这四种蛋白质的确切分子功能尚不清楚。以往的研究表明，Pyrococcus horikoshii Dph 2（PhDph 2），一个新的铁硫簇含酶，形成一个同源二聚体，是足够的第一步，在体外合成二苯二甲酰胺。在这里，我们证明了在体外重建，酵母Dph 1和Dph 2形成一个复合物（Dph 1-Dph 2），相当于PhDph 2的同型二聚体，并足以在体外催化的第一步中存在的连二亚硫酸盐作为还原剂。我们进一步证明酵母Dph 3（也称为KTI 11）是一种CSL型锌指蛋白，可以结合铁，并且在还原状态下可以作为电子供体来还原Dph 1-Dph 2中的Fe-S簇。因此，我们的研究坚定地建立了三个蛋白质参与真核生物合成的功能。对于细菌中的大多数自由基SAM酶，认为黄素氧还蛋白和黄素氧还蛋白还原酶充当Fe-S簇的电子供体。因此，Dph 3是Dph 1-Dph 2中Fe-S簇的电子供体的发现很有趣，并为真核细胞中Fe-S蛋白的电子转移研究开辟了新的途径。
Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. The biosynthesis of diphthamide was proposed to involve three steps. The first step is the transfer of the 3-amino-3-carboxypropyl group from S-adenosyl-l-methionine (SAM) to the histidine residue of EF2, forming a C–C bond. Previous genetic studies showed this step requires four proteins in eukaryotes, Dph1–Dph4. However, the exact molecular functions for the four proteins are unknown. Previous study showed that Pyrococcus horikoshii Dph2 (PhDph2), a novel iron-sulfur cluster-containing enzyme, forms a homodimer and is sufficient for the first step of diphthamide biosynthesis in vitro. Here we demonstrate by in vitro reconstitution that yeast Dph1 and Dph2 form a complex (Dph1-Dph2) that is equivalent to the homodimer of PhDph2 and is sufficient to catalyze the first step in vitro in the presence of dithionite as the reductant. We further demonstrate that yeast Dph3 (also known as KTI11), a CSL-type zinc finger protein, can bind iron and in the reduced state can serve as an electron donor to reduce the Fe-S cluster in Dph1-Dph2. Our study thus firmly establishes the functions for three of the proteins involved in eukaryotic diphthamide biosynthesis. For most radical SAM enzymes in bacteria, flavodoxins and flavodoxin reductases are believed to serve as electron donors for the Fe-S clusters. The finding that Dph3 is an electron donor for the Fe-S clusters in Dph1-Dph2 is thus interesting and opens up new avenues of research on electron transfer to Fe-S proteins in eukaryotic cells.
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影响因子：
5.3
作者：
MOEHRING, TJ;DANLEY, DE;MOEHRING, JM
通讯作者：
MOEHRING, JM
影响因子：
3.2
作者：
PAPPENHEIMER, AM;DUNLOP, PC;BODLEY, JW
通讯作者：
BODLEY, JW
DOI：
10.1073/pnas.1214346109
发表时间：
2012-12-04
影响因子：
11.1
作者：
Su, Xiaoyang;Lin, Zhewang;Lin, Hening
通讯作者：
Lin, Hening
影响因子：
5.6
作者：
Proudfoot, Michael;Sanders, Stephen A.;Yakunin, Alexander F.
通讯作者：
Yakunin, Alexander F.
影响因子：
3.6
作者：
Fichtner, L;Schaffrath, R
通讯作者：
Schaffrath, R