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

Uthman S, Bär C, Scheidt V, Liu S, ten Have S, Giorgini F, Stark MJ, Schaffrath R - PLoS Genet. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, United Kingdom.

ABSTRACT
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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The biosynthetic pathway for modification of eEF2 by diphthamide.For roles played by the bona fide diphthamide genes DPH1–DPH5 in steps 1 and 2 of the pathway, see main text. The ill-defined step 3, conversion of diphthine to diphthamide by amidation, is highlighted (red label). It likely involves ATP and ammonium cofactors in a reaction catalyzed by unidentified DPH gene product(s). Step 4 indicates diphthamide can be hijacked for eEF2 inactivation and cell death induction by antifungals, i.e. sordarin and bacterial ADP ribosylase toxins (ADPRtox); alternatively, it has been reported to undergo cell growth related physiological ADP ribosylation (ADPRphys?) by elusive cellular modifier(s). ACP, 2-[3-amino-carboxyl-propyl]-histidine; SAM: S-adenosylmethionine.
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pgen-1003334-g001: The biosynthetic pathway for modification of eEF2 by diphthamide.For roles played by the bona fide diphthamide genes DPH1–DPH5 in steps 1 and 2 of the pathway, see main text. The ill-defined step 3, conversion of diphthine to diphthamide by amidation, is highlighted (red label). It likely involves ATP and ammonium cofactors in a reaction catalyzed by unidentified DPH gene product(s). Step 4 indicates diphthamide can be hijacked for eEF2 inactivation and cell death induction by antifungals, i.e. sordarin and bacterial ADP ribosylase toxins (ADPRtox); alternatively, it has been reported to undergo cell growth related physiological ADP ribosylation (ADPRphys?) by elusive cellular modifier(s). ACP, 2-[3-amino-carboxyl-propyl]-histidine; SAM: S-adenosylmethionine.

Mentions: Regulation of biological processes by posttranslational modification can involve the function, distribution and interaction capabilities of the modified protein [1]–[3]. Though most modification pathways such as phosphorylation and ubiquitin conjugation target many different proteins, some exceptional ones uniquely target just a single polypeptide [4]. One prominent example is diphthamide formation on eukaryal translation elongation factor 2 (eEF2) [5]. Strikingly, this modification is pathobiologically important because it is hijacked for eEF2 inhibition by sordarin fungicides and by diphtheria toxin (DT) produced by pathovarieties of Corynebacterium diphtheriae that cause the severe human disease syndrome diphtheria [6]–[8]. Both agents efficiently block protein synthesis by inactivating the essential function of the modified translation factor, via stalling the diphthamide modified form of eEF2 on ribosomes and irreversible ADP ribosylation of eEF2, respectively [9]–[12]. Diphthamide itself is a highly modified histidine residue on eEF2 – 2-[3-carboxyamido-3-(trimethylamino)-propyl]-histidine – which is conserved from yeast (H699) to man (H715) (Figure 1) [5], [8], [13]. Intriguingly, it is absent from the bacterial eEF2 analog, EF-G, thus conferring immunity on the DT producer.


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

Uthman S, Bär C, Scheidt V, Liu S, ten Have S, Giorgini F, Stark MJ, Schaffrath R - PLoS Genet. (2013)

The biosynthetic pathway for modification of eEF2 by diphthamide.For roles played by the bona fide diphthamide genes DPH1–DPH5 in steps 1 and 2 of the pathway, see main text. The ill-defined step 3, conversion of diphthine to diphthamide by amidation, is highlighted (red label). It likely involves ATP and ammonium cofactors in a reaction catalyzed by unidentified DPH gene product(s). Step 4 indicates diphthamide can be hijacked for eEF2 inactivation and cell death induction by antifungals, i.e. sordarin and bacterial ADP ribosylase toxins (ADPRtox); alternatively, it has been reported to undergo cell growth related physiological ADP ribosylation (ADPRphys?) by elusive cellular modifier(s). ACP, 2-[3-amino-carboxyl-propyl]-histidine; SAM: S-adenosylmethionine.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585130&req=5

pgen-1003334-g001: The biosynthetic pathway for modification of eEF2 by diphthamide.For roles played by the bona fide diphthamide genes DPH1–DPH5 in steps 1 and 2 of the pathway, see main text. The ill-defined step 3, conversion of diphthine to diphthamide by amidation, is highlighted (red label). It likely involves ATP and ammonium cofactors in a reaction catalyzed by unidentified DPH gene product(s). Step 4 indicates diphthamide can be hijacked for eEF2 inactivation and cell death induction by antifungals, i.e. sordarin and bacterial ADP ribosylase toxins (ADPRtox); alternatively, it has been reported to undergo cell growth related physiological ADP ribosylation (ADPRphys?) by elusive cellular modifier(s). ACP, 2-[3-amino-carboxyl-propyl]-histidine; SAM: S-adenosylmethionine.
Mentions: Regulation of biological processes by posttranslational modification can involve the function, distribution and interaction capabilities of the modified protein [1]–[3]. Though most modification pathways such as phosphorylation and ubiquitin conjugation target many different proteins, some exceptional ones uniquely target just a single polypeptide [4]. One prominent example is diphthamide formation on eukaryal translation elongation factor 2 (eEF2) [5]. Strikingly, this modification is pathobiologically important because it is hijacked for eEF2 inhibition by sordarin fungicides and by diphtheria toxin (DT) produced by pathovarieties of Corynebacterium diphtheriae that cause the severe human disease syndrome diphtheria [6]–[8]. Both agents efficiently block protein synthesis by inactivating the essential function of the modified translation factor, via stalling the diphthamide modified form of eEF2 on ribosomes and irreversible ADP ribosylation of eEF2, respectively [9]–[12]. Diphthamide itself is a highly modified histidine residue on eEF2 – 2-[3-carboxyamido-3-(trimethylamino)-propyl]-histidine – which is conserved from yeast (H699) to man (H715) (Figure 1) [5], [8], [13]. Intriguingly, it is absent from the bacterial eEF2 analog, EF-G, thus conferring immunity on the DT producer.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, United Kingdom.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus