diphthine and diphthamide

Present on archaeal and eukaryotic translation elongation factor 2, diphthamide represents one of the most intriguing post-translational modifications on proteins. The biosynthesis of diphthamide was proposed to occur in three steps requiring seven proteins, Dph1-7, in eukaryotes. The functional assignments of Dph1-5 in the first and second step have been well established. Recent studies suggest that Dph6 (yeast YLR143W or human ATPBD4) and Dph7 (yeast YBR246W or human WDR85) are involved in the last amidation step, with Dph6 being the actual diphthamide synthetase catalyzing the ATP-dependent amidation reaction. However, the exact molecular role of Dph7 is unclear. Here we demonstrate that Dph7 is an enzyme catalyzing a previously unknown step in the diphthamide biosynthesis pathway. This step is between the Dph5- and Dph6-catalyzed reactions. We demonstrate that the Dph5-catalyzed reaction generates methylated diphthine, a previously overlooked intermediate, and Dph7 is a methylesterase that hydrolyzes methylated diphthine to produce diphthine and allows the Dph6-catalyzed amidation reaction to occur. Thus, our study characterizes the molecular function of Dph7 for the first time and provides a revised diphthamide biosynthesis pathway.

Topics: Biosynthetic Pathways; Gene Deletion; Histidine; Humans; Methylation; Protein Processing, Post-Translational; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins

Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue that is found in archaeal and eukaryotic translation elongation factor 2. The biosynthesis and function of this modification has attracted the interest of many biochemists for decades. The biosynthesis has been known to proceed in three steps. Proteins required for the first and second steps have been identified, but the protein(s) required for the last step have remained elusive. Here we demonstrate that the YBR246W gene in yeast is required for the last step of diphthamide biosynthesis, as the deletion of YBR246W leads to the accumulation of diphthine, which is the enzymatic product of the second step of the biosynthesis. This discovery will provide important information leading to the complete elucidation of the full biosynthesis pathway of diphthamide.

Topics: Gene Deletion; Gene Expression Regulation, Fungal; Histidine; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). Although diphthamide modification was discovered three decades ago, in vitro reconstitution of diphthamide biosynthesis using purified proteins has not been reported. The proposed biosynthesis pathway of diphthamide involves three steps. Our laboratory has recently showed that in Pyrococcus horikoshii (P. horikoshii), the first step uses a [4Fe-4S] enzyme PhDph2 to generate a 3-amino-3-carboxypropyl radical from S-adenosyl-L-methionine (SAM) to form a C−C bond. The second step is the trimethylation of an amino group to form the diphthine intermediate. This step is catalyzed by a methyltransferase called diphthine synthase or Dph5. Here we report the in vitro reconstitution of the second step using P. horikoshii Dph5 (PhDph5). Our results demonstrate that PhDph5 is sufficient to catalyze the mono-, di-, and trimethylation of P. horikoshii EF2 (PhEF2). Interestingly, the trimethylated product from the PhDph5-catalyzed reaction can easily eliminate the trimethylamino group. The potential implication of this unexpected finding on the diphthamide biosynthesis pathway is discussed.

Topics: Archaeal Proteins; Biosynthetic Pathways; Histidine; Methyltransferases; Peptide Elongation Factor 2; Protein Processing, Post-Translational; Pyrococcus horikoshii; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Diphthamide, a unique amino acid, is a post-translational derivative of histidine that exists in protein synthesis elongation factor 2 at the site of diphtheria toxin-catalyzed ADP-ribosylation of elongation factor 2. We investigated steps in the biosynthesis of diphthamide with mutants of Chinese hamster ovary cells that were altered in different steps of this complex post-translational modification. Biochemical evidence indicates that this modification requires a minimum of three steps, two of which we accomplished in vitro. We identified a methyltransferase activity that transfers methyl groups from S-adenosyl methionine to an unmethylated form of diphthine (the deamidated form of diphthamide), and we tentatively identified an ATP-dependent synthetase activity involved in the biosynthesis of diphthamide from diphthine. Our results are in accord with the proposed structure of diphthamide (B. G. VanNess, et al., J. Biol. Chem. 255:10710-10716, 1980).

Topics: Animals; Cell Line; Cricetinae; Diphtheria Toxin; Drug Resistance; Female; Histidine; Methylation; Mutation; Peptide Elongation Factor 2; Peptide Elongation Factors; Peptide Fragments; Protein Processing, Post-Translational; S-Adenosylmethionine