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Diphthamide biosynthesis requires an organic radical generated by an iron–sulphur enzyme

A Corrigendum to this article was published on 26 May 2011


Archaeal and eukaryotic translation elongation factor 2 contain a unique post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide was proposed to involve three steps, with the first being the formation of a C–C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosyl-l-methionine (SAM). However, further details of the biosynthesis remain unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron–sulphur-cluster enzyme, Dph2. Dph2 is a homodimer and each of its monomers can bind a [4Fe–4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5′-deoxyadenosyl radical. Instead, it breaks the Cγ,Met–S bond of SAM and generates a 3-amino-3-carboxypropyl radical. Our results suggest that P. horikoshii Dph2 represents a previously unknown, SAM-dependent, [4Fe–4S]-containing enzyme that catalyses unprecedented chemistry.

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Figure 1: The structure of diphthamide and its proposed biosynthesis pathway.
Figure 2: Structure of the PhDPH2 homodimer.
Figure 3: In vitro reconstitution of PhDph2 activity.
Figure 4: Spectroscopic characterization of the [4Fe–4S] cluster in PhDph2.
Figure 5: Identification of SAM-derived small-molecule products in PhDph2-catalysed reactions.
Figure 6: The proposed reaction mechanism for PhDph2.

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Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the crystal structures reported here have been deposited with the Protein Data Bank under accession codes 3LZC for iron-free PhDph2 and 3LZD for reconstituted PhDph2.


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We thank L. Kinsland for assistance with manuscript preparation, the Dreyfus Foundation for a New Faculty Award to H.L., NIH/NIGMS R01GM088276 to H.L. and S.E.E., and NIH/NCRR P41-RR016292 for the ACERT Center Grant to J.F. This work is based upon research conducted at the Advanced Photon Source (APS), Argonne National Laboratory, on the Northeastern Collaborative Access Team beamlines, which are supported by award RR-15301 from the US National Center for Research Resources at the US National Institutes of Health. Use of the APS is supported by the US Department of Energy, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357.

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Authors and Affiliations



Y.Z. determined the crystal structure of iron-free PhDph2, X.Z. performed the biochemical studies and prepared protein samples for spectroscopic and structural studies, A.T.T. determined the crystal structure of anaerobically purified PhDph2, M.L. and C.K. performed the Mössbauer spectroscopy, B.D. and J.F. performed the EPR spectroscopy, R.M.K. prepared the initial PhDph2 crystals, E.W. prepared the PhEF2 mutant proteins, S.E.E. supervised the crystallographic studies, H.L. supervised the biochemical studies and H.L., S.E.E. and C.K. prepared the manuscript.

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Correspondence to Steven E. Ealick or Hening Lin.

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Zhang, Y., Zhu, X., Torelli, A. et al. Diphthamide biosynthesis requires an organic radical generated by an iron–sulphur enzyme. Nature 465, 891–896 (2010).

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