Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853, USA.
Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
J Biol Inorg Chem. 2019 Sep;24(6):777-782. doi: 10.1007/s00775-019-01702-0. Epub 2019 Aug 28.
Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue found in archaeal and eukaryotic translation elongation factor 2 (EF2). In the first step of diphthamide biosynthesis, a [4Fe-4S] cluster-containing radical SAM enzyme, Dph1-Dph2 heterodimer in eukaryotes or Dph2 homodimer in archaea, cleaves S-adenosylmethionine and transfers the 3-amino-3-carboxypropyl group to EF2. It was demonstrated previously that for the archaeal Dph2 homodimer, only one [4Fe-4S] cluster is necessary for the in vitro activity. Here, we demonstrate that for the eukaryotic Dph1-Dph2 heterodimer, the [4Fe-4S] cluster-binding cysteine residues in each subunit are required for diphthamide biosynthesis to occur in vivo. Furthermore, our in vitro reconstitution experiments with Dph1-Dph2 mutants suggested that the Dph1 cluster serves a catalytic role, while the Dph2 cluster facilitates the reduction of the Dph1 cluster by the physiological reducing system Dph3/Cbr1/NADH. Our results reveal the asymmetric functional roles of the Dph1-Dph2 heterodimer and may help to understand how the Fe-S clusters in radical SAM enzymes are reduced in biology.
二氢假噻吩，白喉毒素的靶标，是一种在古菌和真核翻译延伸因子 2（EF2）中发现的翻译后修饰组氨酸残基。在二氢假噻吩生物合成的第一步中，一种含有[4Fe-4S]簇的自由基 SAM 酶，即真核生物中的 Dph1-Dph2 异二聚体或古菌中的 Dph2 同二聚体，切割 S-腺苷甲硫氨酸并将 3-氨基-3-羧丙基转移到 EF2 上。先前已经证明，对于古菌的 Dph2 同二聚体，体外活性只需要一个[4Fe-4S]簇。在这里，我们证明对于真核生物的 Dph1-Dph2 异二聚体，每个亚基中的[4Fe-4S]簇结合半胱氨酸残基对于体内二氢假噻吩生物合成的发生是必需的。此外，我们用 Dph1-Dph2 突变体进行的体外重组实验表明，Dph1 簇起催化作用，而 Dph2 簇促进生理还原系统 Dph3/Cbr1/NADH 还原 Dph1 簇。我们的结果揭示了 Dph1-Dph2 异二聚体的不对称功能作用，并可能有助于理解生物体内自由基 SAM 酶中的 Fe-S 簇如何被还原。
