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. The first step is the formation of a C-C bond between the histidine residue and the 3-amino-3carboxylpropyl group of S-adenosylmethionine (SAM), which is catalyzed by four enzymes Dph1-Dph4 in eukaryotic or only one enzyme Dph2 in archaea; the second step is the trimethylation of the amino group by Dph5; and the last step is an ATP depended amidation of the carboxyl group by an unknown enzyme. We have recently found that in an archaeal species 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 enzymes, biochemical evidence suggests that a 3-amino-3-carboxypropyl radical is generated in PhDph2. Further evidence shows that the 3-amino-3-carboxypropyl radical does not undergo hydrogen ion reaction, which was 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, the chemistry requires only one [4Fe-4S] cluster to be present in the PhDph2 dimer. The successful reconstitution of the first step of diphthamide biosynthesis provides the substrate for the second step. We then reconstituted the second step using P. horikoshii PhDph5 in vitro. The results demonstrate that PhDph5 is sufficient to catalyze the mono-, di-, and trimethylation of PhEF2. Interestingly, the trimethylated product from the PhDph5-catalyzed reaction can easily eliminate the trimethylamino group even in the very mild reaction conditions. This unexpected finding on the diphthamide biosynthesis pathway may suggest that the last amidation step occurs very quickly in cells to avoid the elimination reaction or the amidation step occurs before the trimethylation step.