S166: The unusual enzyme chemistry in diphthamide biosynthesis

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 C_(gamma,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.