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Mechanistic understanding of Pyrococcus horikoshiiDph2, a [4Fe–4S] enzyme required for diphthamidebiosynthesis

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Abstract

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). The proposed biosynthesis of diphthamide involves three steps and we have recently found that in Pyrococcus horikoshii (P. horikoshii), the first step uses an S-adenosyl-L-methionine (SAM)-dependent [4Fe–4S] enzyme, PhDph2, to catalyze the formation of a C–C bond. Crystal structure shows that PhDph2 is a homodimer and each monomer contains three conserved cysteine residues that can bind a [4Fe–4S] cluster. In the reduced state, the [4Fe–4S] cluster can provide one electron to reductively cleave the bound SAM molecule. However, different from classical radical SAM family of enzymes, biochemical evidence suggest that a 3-amino-3-carboxypropyl radical is generated in PhDph2. Here we present evidence supporting that the 3-amino-3-carboxypropyl radical does not undergo hydrogen abstraction reaction, which is observed for the deoxyadenosyl radical in classical radical SAM enzymes. Instead, the 3-amino-3-carboxypropyl radical is added to the imidazole ring in the pathway towards the formation of the product. Furthermore, our data suggest that the chemistry requires only one [4Fe–4S] cluster to be present in the PhDph2 dimer.

Graphical abstract: Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe–4S] enzyme required for diphthamide biosynthesis

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Article information


Submitted
29 Jun 2010
Accepted
16 Sep 2010
First published
08 Oct 2010

Mol. BioSyst., 2011,7, 74-81
Article type
Paper

Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe–4S] enzyme required for diphthamide biosynthesis

X. Zhu, B. Dzikovski, X. Su, A. T. Torelli, Y. Zhang, S. E. Ealick, J. H. Freed and H. Lin, Mol. BioSyst., 2011, 7, 74 DOI: 10.1039/C0MB00076K

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