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

Abstract

Summary Archaeal and eukaryotic translation elongation factor 2 contain a unique posttranslationally modified histidine residue called “diphthamide”, the target of diphtheria toxin. The biosynthesis of diphthamide were proposed to involve three steps, with the first step being the formation of a C-C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosylmethionine (SAM). However, details of the biosynthesis have remained 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-sulfur cluster enzyme, Dph2. Dph2 is a homodimer and each monomer contains 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-carboxylpropyl radical. This work suggests that Pyrococcus horikoshii Dph2 represents a novel SAM-dependent [4Fe-4S]-containing enzyme that catalyzes unprecedented chemistry.

A Modified Form of Diphthamide Causes Immunotoxin Resistance in a Lymphoma Cell Line with a Deletion of the WDR85 Gene

“…Resistance-There are six proteins required for diphthamide biosynthesis: DPH1-5 and WDR85 (10,11,20). Using RT-PCR, we measured the level of the mRNA for each of these proteins and found that the level of WDR85 mRNA was extremely low in CA46-R cells (Fig.…”

Diphtheria Disease and Genes Involved in Formation of Diphthamide, Key Effector of the Diphtheria Toxin

“…Diphthamide modification on eukaryotic translation elongation factor 2 (EF2) in budding yeast operates through a multi-step pathway. The diphthamide pathway, modified after Zhang et al (2010), involves known and elusive (?) steps with the intermediates 2-(3-carboxyl-3-aminopropyl)-histidine and diphthine being generated.…”

Chemogenomic approach identified yeast YLR143W as diphthamide synthetase

“…The second step is a trimethylation reaction to form diphthine, which is catalyzed by a single methyltransferase DPH5. Both the first and second steps have been reconstituted in vitro using purified proteins from a thermophilic archaea, Pyrococcus horikoshii (10)(11)(12). The third step is the amidation of diphthine to form diphthamide.…”