Stark Lab Paper: The Amidation Step of Diphthamide Biosynthesis in Yeast Requires DPH6, a Gene Identified through Mining the DPH1-DPH5 Interaction Network

Diphthamide is an unusual modified amino acid found uniquely in a single protein, eEF2, which is required for cells to synthesize new proteins. The name refers to its target function for eEF2 inactivation by diphtheria toxin, the disease-inducing agent produced by the pathogen Corynebacterium diphtheriae. Why cells require eEF2 to contain diphthamide is unclear, although mice unable to make it fail to complete embryogenesis. Cells generate diphthamide by modifying a specific histidine residue in eEF2 using a three-step biosynthetic pathway, the first two steps of which are well defined. However, the enzyme(s) involved in the final amidation step are unknown. Here we integrate genomic and molecular approaches to identify a candidate for the elusive amidase (Dph6) and confirm involvement of a second protein (Dph7) in the amidation step, showing that failure to synthesize diphthamide affects the accuracy of protein synthesis. In contrast to Dph6, however, Dph7 may be regulatory. Our data strongly suggest that it promotes dissociation of eEF2 from diphthine synthase (Dph5), which carries out the second step of diphthamide synthesis, and that Dph5 has a novel role as an eEF2 inhibitor when diphthamide synthesis is incomplete.

Professor Stark also made point mutations in DPH6 predicted to prevent its catalytic activity and showed that they inactivated the protein.