| Popis: |
Diphthamide, the target of Diphtheria toxin, is a unique post-translational modification on His₆₉₉ (S. cerevisiae) of translation elongation factor 2 (eEF2) found in eukarya and archaea. It serves as the unique target for bacterial ADP-ribosylating toxins such as Diphtheria Toxin, Exotoxin A and Cholix toxin. So far six genes have been known to be involved in the complex three-step biosynthesis pathway: bona fide diphthamide genes DPH1-DPH5 and the recently identified YBR246w. While the latter was shown to be involved in the final step of the pathway, its exact role remains unclear. Dph1-Dph4 facilitate the initial step of the pathway and the methytransferase, Dph5, the second step. Surprisingly, after almost four decades of intensive research the enzyme catalyzing the final step, the conversion of the intermediate diphthine into the final product diphthamide, has remained elusive. We sought to exploit yeast genetic interaction and chemical genomic databases in order to identify novel diphthamide biosynthesis genes. A novel candidate gene YLR143w was identified and we here present genetic, phenotypic and biochemical analyses that clearly identify YLR143w as a novel diphthamide biosynthesis gene. Our observations implicate that YLR143w is the main catalytic enzyme necessary for the third step of the pathway, while YBR246w has a regulatory role involving Dph5-EF2 interaction. Furthermore, we demonstrate that Dph1 is likely the primary catalytic enzyme which generates the initial modification on the His[subscript 699] residue. In addition to the implications in bacterial pathogenesis, diphthamide and the biosynthesis genes DPH1, DPH3 and DPH4 are associated with cancer formation as well as defects in embryonic development and cell proliferation control. We here demonstrate that diphthamide deficient yeast cells display a significant increase in -1 frameshifting during translation and propose that this is the underlying cause of the phenotypes seen in mammalian organisms. |
