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YBR246W is required for the third step of diphthamide biosynthesis.

ABSTRACT: 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.

SUBMITTER: Su X

PROVIDER: S-EPMC3264676 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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YBR246W is required for the third step of diphthamide biosynthesis.

Su Xiaoyang X Chen Wei W Lee Wankyu W Jiang Hong H Zhang Sheng S Lin Hening H

Journal of the American Chemical Society 20111221 2

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 demonstrat ...[more]

PMID: 22188241

Similar Datasets

Dph7 catalyzes a previously unknown demethylation step in diphthamide biosynthesis.

Project description: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.

| S-EPMC4015618 | biostudies-literature

DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

Project description:A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant. A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.

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Dph3 is an electron donor for Dph1-Dph2 in the first step of eukaryotic diphthamide biosynthesis.

Project description:Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. The biosynthesis of diphthamide was proposed to involve three steps. The first step is the transfer of the 3-amino-3-carboxypropyl group from S-adenosyl-l-methionine (SAM) to the histidine residue of EF2, forming a C-C bond. Previous genetic studies showed this step requires four proteins in eukaryotes, Dph1-Dph4. However, the exact molecular functions for the four proteins are unknown. Previous study showed that Pyrococcus horikoshii Dph2 (PhDph2), a novel iron-sulfur cluster-containing enzyme, forms a homodimer and is sufficient for the first step of diphthamide biosynthesis in vitro. Here we demonstrate by in vitro reconstitution that yeast Dph1 and Dph2 form a complex (Dph1-Dph2) that is equivalent to the homodimer of PhDph2 and is sufficient to catalyze the first step in vitro in the presence of dithionite as the reductant. We further demonstrate that yeast Dph3 (also known as KTI11), a CSL-type zinc finger protein, can bind iron and in the reduced state can serve as an electron donor to reduce the Fe-S cluster in Dph1-Dph2. Our study thus firmly establishes the functions for three of the proteins involved in eukaryotic diphthamide biosynthesis. For most radical SAM enzymes in bacteria, flavodoxins and flavodoxin reductases are believed to serve as electron donors for the Fe-S clusters. The finding that Dph3 is an electron donor for the Fe-S clusters in Dph1-Dph2 is thus interesting and opens up new avenues of research on electron transfer to Fe-S proteins in eukaryotic cells.

| S-EPMC3985478 | biostudies-literature

The biosynthesis and biological function of diphthamide.

Project description:Eukaryotic and archaeal elongation factor 2 contains a unique post-translationally modified histidine residue, named diphthamide. Genetic and biochemical studies have revealed that diphthamide biosynthesis involves a multi-step pathway that is evolutionally conserved among lower and higher eukaryotes. During certain bacterial infections, diphthamide is specifically recognized by bacterial toxins, including diphtheria toxin, Pseudomonas exotoxin A and cholix toxin. Although the pathological relevance is well studied, the physiological function of diphthamide is still poorly understood. Recently, many new interesting developments in understanding the biosynthesis have been reported. Here, we review the current understanding of the biosynthesis and biological function of diphthamide.

| S-EPMC4280834 | biostudies-literature

The amidation step of diphthamide biosynthesis in yeast requires DPH6, a gene identified through mining the DPH1-DPH5 interaction network.

Project description:Diphthamide is a highly modified histidine residue in eukaryal translation elongation factor 2 (eEF2) that is the target for irreversible ADP ribosylation by diphtheria toxin (DT). In Saccharomyces cerevisiae, the initial steps of diphthamide biosynthesis are well characterized and require the DPH1-DPH5 genes. However, the last pathway step-amidation of the intermediate diphthine to diphthamide-is ill-defined. Here we mine the genetic interaction landscapes of DPH1-DPH5 to identify a candidate gene for the elusive amidase (YLR143w/DPH6) and confirm involvement of a second gene (YBR246w/DPH7) in the amidation step. Like dph1-dph5, dph6 and dph7 mutants maintain eEF2 forms that evade inhibition by DT and sordarin, a diphthamide-dependent antifungal. Moreover, mass spectrometry shows that dph6 and dph7 mutants specifically accumulate diphthine-modified eEF2, demonstrating failure to complete the final amidation step. Consistent with an expected requirement for ATP in diphthine amidation, Dph6 contains an essential adenine nucleotide hydrolase domain and binds to eEF2. Dph6 is therefore a candidate for the elusive amidase, while Dph7 apparently couples diphthine synthase (Dph5) to diphthine amidation. The latter conclusion is based on our observation that dph7 mutants show drastically upregulated interaction between Dph5 and eEF2, indicating that their association is kept in check by Dph7. Physiologically, completion of diphthamide synthesis is required for optimal translational accuracy and cell growth, as indicated by shared traits among the dph mutants including increased ribosomal -1 frameshifting and altered responses to translation inhibitors. Through identification of Dph6 and Dph7 as components required for the amidation step of the diphthamide pathway, our work paves the way for a detailed mechanistic understanding of diphthamide formation.

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Noncanonical Radical SAM Enzyme Chemistry Learned from Diphthamide Biosynthesis.

Project description:Radical S-adenosylmethionine (SAM) enzymes are a superfamily of enzymes that use SAM and reduced [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical to catalyze numerous challenging reactions. We have reported a type of noncanonical radical SAM enzymes in the diphthamide biosynthesis pathway. These enzymes also use SAM and reduced [4Fe-4S] clusters, but generate a 3-amino-3-carboxypropyl (ACP) radical to modify the substrate protein, translation elongation factor 2. The regioselective cleavage of a different C-S bond of the sulfonium center of SAM in these enzymes comparing to canonical radical SAM enzymes is intriguing. Here, we highlight some recent findings in the mechanism of these types of enzymes, showing that the diphthamide biosynthetic radial SAM enzymes bound SAM with a distinct geometry. In this way, the unique iron of the [4Fe-4S] cluster in the enzyme can only attack the carbon on the ACP group to form an organometallic intermediate. The homolysis of the organometallic intermediate releases the ACP radical and generates the EF2 radial.

| S-EPMC6893904 | biostudies-literature

Organometallic and radical intermediates reveal mechanism of diphthamide biosynthesis.

Project description:Diphthamide biosynthesis involves a carbon-carbon bond-forming reaction catalyzed by a radical S-adenosylmethionine (SAM) enzyme that cleaves a carbon-sulfur (C-S) bond in SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. Using rapid freezing, we have captured an organometallic intermediate with an iron-carbon (Fe-C) bond between ACP and the enzyme's [4Fe-4S] cluster. In the presence of the substrate protein, elongation factor 2, this intermediate converts to an organic radical, formed by addition of the ACP radical to a histidine side chain. Crystal structures of archaeal diphthamide biosynthetic radical SAM enzymes reveal that the carbon of the SAM C-S bond being cleaved is positioned near the unique cluster Fe, able to react with the cluster. Our results explain how selective C-S bond cleavage is achieved in this radical SAM enzyme.

| S-EPMC6066404 | biostudies-literature

The Crystal Structure of Dph2 in Complex with Elongation Factor 2 Reveals the Structural Basis for the First Step of Diphthamide Biosynthesis.

Project description:Elongation factor 2 (EF-2), a five-domain, GTP-dependent ribosomal translocase of archaebacteria and eukaryotes, undergoes post-translational modification to form diphthamide on a specific histidine residue in domain IV prior to binding the ribosome. The first step of diphthamide biosynthesis in archaebacteria is catalyzed by Dph2, a homodimeric radical S-adenosylmethionine (SAM) enzyme having a noncanonical architecture. Here, we describe a 3.5 Å resolution crystal structure of the Methanobrevibacter smithii (Ms) Dph2 homodimer bound to two molecules of MsEF-2, one of which is ordered and the other largely disordered. MsEF-2 is bound to both protomers of MsDph2, with domain IV bound to the active site of one protomer and domain III bound to a surface ?-helix of an adjacent protomer. The histidine substrate of domain IV is inserted into the active site, which reveals for the first time the architecture of the Dph2 active site in complex with its target substrate. We also determined a high-resolution crystal structure of isolated MsDph2 bound to 5'-methylthioadenosine that shows a conserved arginine residue preoriented by conserved phenylalanine and aspartate residues for binding the carboxylate group of SAM. Mutagenesis experiments suggest that the arginine plays an important role in the first step of diphthamide biosynthesis.

| S-EPMC7857147 | biostudies-literature

Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2.

Project description:Diphthamide, a posttranslational modification of translation elongation factor 2 that is conserved in all eukaryotes and archaebacteria and is the target of diphtheria toxin, is formed in yeast by the actions of five proteins, Dph1 to -5, and a still unidentified amidating enzyme. Dph2 and Dph5 were previously identified. Here, we report the identification of the remaining three yeast proteins (Dph1, -3, and -4) and show that all five Dph proteins have either functional (Dph1, -2, -3, and -5) or sequence (Dph4) homologs in mammals. We propose a unified nomenclature for these proteins (e.g., HsDph1 to -5 for the human proteins) and their genes based on the yeast nomenclature. We show that Dph1 and Dph2 are homologous in sequence but functionally independent. The human tumor suppressor gene OVCA1, previously identified as homologous to yeast DPH2, is shown to actually be HsDPH1. We show that HsDPH3 is the previously described human diphtheria toxin and Pseudomonas exotoxin A sensitivity required gene 1 and that DPH4 encodes a CSL zinc finger-containing DnaJ-like protein. Other features of these genes are also discussed. The physiological function of diphthamide and the basis of its ubiquity remain a mystery, but evidence is presented that Dph1 to -3 function in vivo as a protein complex in multiple cellular processes.

| S-EPMC522255 | biostudies-literature

Translational fidelity and growth of Arabidopsis require stress-sensitive diphthamide biosynthesis.

Project description:Diphthamide, a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR2 (eEF2), is the human host cell-sensitizing target of diphtheria toxin. Diphthamide biosynthesis depends on the 4Fe-4S-cluster protein Dph1 catalyzing the first committed step, as well as Dph2 to Dph7, in yeast and mammals. Here we show that diphthamide modification of eEF2 is conserved in Arabidopsis thaliana and requires AtDPH1. Ribosomal -1 frameshifting-error rates are increased in Arabidopsis dph1 mutants, similar to yeast and mice. Compared to the wild type, shorter roots and smaller rosettes of dph1 mutants result from fewer formed cells. TARGET OF RAPAMYCIN (TOR) kinase activity is attenuated, and autophagy is activated, in dph1 mutants. Under abiotic stress diphthamide-unmodified eEF2 accumulates in wild-type seedlings, most strongly upon heavy metal excess, which is conserved in human cells. In summary, our results suggest that diphthamide contributes to the functionality of the translational machinery monitored by plants to regulate growth.

| S-EPMC9273596 | biostudies-literature

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