Diphthamide is a unique post-translational modification on eukaryotic and archaeal elongation factor 2 (eEF2). Like diphthamide, hypusine is a unique post-translational modification on eukaryotic initiation factor 5a (eIF5A). These modifications were identified decades ago. Deletion of these modifications in animal model cause severe developmental defects. However, the biosynthesis and biological function of the modifications are not fully understood.The biosynthesis of diphthamide involves 4 steps and at least 7 proteins. The first step biosynthesis of diphthamide in Saccharomyces cerevisiae requires a unique radical SAM complex, Dph1-Dph2. The function of Dph1-Dph2 requires an oxygen-sensitive [4Fe-4S] cluster. However, the cluster is easily degraded into [3Fe-4S] in cytosol. We found that the small iron containing protein Dph3 can provide additional Fe during the reaction. The Dph1-Dph2-Dph3 is the first radical SAM enzyme system to maintain activity in the presence of oxygen. Dph3-like protein is likely to present to help other radical SAM enzymes to keep activity in the cytosol. The biological function of diphthamide was not understood. Diphthamide was proposed to suppress -1 frameshift in translation. Through genome wide profiling for potential -1 frameshift site, we identified that TORC1/mTORC1 signaling pathway is affected by the deletion of diphthamide. Diphthamide deletion suppresses translation of TORC1 activating proteins. Our results suggest diphthamide is essential for translating complex proteome and provide an explanation why diphthamide is evolutionary conserved and why diphthamide deletion can cause sever developmental defects. Deoxyhypusine hydroxylation is the second step biosynthesis of hypusine. The biological function of this step is not known. The enzymatic reaction for deoxyhypusine hydroxylation requires oxygen. We found that deletion of deoxyhypusine hydroxylase compromised yeast respiration through translation downregulation of protein involved in respiration. The translation suppression is through the N-terminal coding sequence. Deletion of the modification affects a set of N-terminal sequence translation and affects mitochondrial proteins, proteins essential in oxidative stress response and heat shock response. The discovery of the function of post-translation modification through scrutinizing substrate in biochemical reactions can be generalized. During the investigation of the function of diphthamide, we discovered that treatment of rapamycin can cause different regulations for isozymes that perform the same enzymatic function. Through enzymological and biochemical studies, we demonstrate that a rapamycin-upregulated enolase isozyme (Eno1) favors gluconeogenesis, and a rapamycin-upregulated alcohol dehydrogenase isozyme (Ald4) promotes the reduction of NAD+ to NADH. Gene deletion study in yeast showed that the Eno1 and Ald4 are important for yeast survival in less favorable growth conditions. Our study thus highlights the different metabolic needs of cells under different conditions and how nature chooses different isozymes to fit the metabolic needs. Furthermore, we discovered that the active site cysteine of Ald4 can form disulfide bond with the adjacent cysteine under oxidative stress. This response promotes yeast survival under oxidative stress.