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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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Both the Alpha_ANH_like_IV and YjgF-YER057c-UK114 domains in Dph6 are essential for its functionality.(A) Diagram showing the DPH6 wild-type and mutant constructs tested in (B), indicating the Alpha_ANH_like_IV (ANH) and YjgF-YER057c-UK114 (UK114) domains and the position of point mutations, an in-frame deletion (- - - - -) and triple myc epitope tag (myc3) as appropriate. (B) Ten-fold serial cell dilutions of a dph6 deletion strain carrying the constructs shown in (A) or the corresponding empty vector (top panel, pSU6 [wt DPH6]; lower panel, pSU7 [wt DPH6]: Table S3) were spotted onto SCD-Leu plates with or without 10 µg/ml sordarin and grown at 30°C for 3 days.
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pgen-1003334-g007: Both the Alpha_ANH_like_IV and YjgF-YER057c-UK114 domains in Dph6 are essential for its functionality.(A) Diagram showing the DPH6 wild-type and mutant constructs tested in (B), indicating the Alpha_ANH_like_IV (ANH) and YjgF-YER057c-UK114 (UK114) domains and the position of point mutations, an in-frame deletion (- - - - -) and triple myc epitope tag (myc3) as appropriate. (B) Ten-fold serial cell dilutions of a dph6 deletion strain carrying the constructs shown in (A) or the corresponding empty vector (top panel, pSU6 [wt DPH6]; lower panel, pSU7 [wt DPH6]: Table S3) were spotted onto SCD-Leu plates with or without 10 µg/ml sordarin and grown at 30°C for 3 days.

Mentions: Dph6 contains three conserved domains consistent with it functioning as an enzyme (Figure S8). The amino-terminal 225 residues constitute an Alpha_ANH_like_IV domain (cd1994 in the NCBI Conserved Domain Database [62], also known as DUF71), a member of the adenine nucleotide alpha hydrolase superfamily that is predicted to bind ATP. Many DUF71 proteins from archaea to mammals contain the highly conserved motif –E215GG(D/E)XE220– (Dph6 numbering), which has been proposed to be involved in substrate binding and catalysis and which is replaced by –ENGE(F/Y)H– in a group of related DUF71 proteins implicated in biotin synthesis [60]. Based on this we generated a dph6 allele encoding two substitutions in this region (G216N, E220A) and tested its functionality by monitoring complementation of sordarin resistance in a yeast dph6 knockout strain. Figure 7 clearly shows that this small change completely inactivates the function of Dph6, demonstrating that the Alpha_ANH_like_IV domain is critical for the conversion of diphthine to diphthamide. The C-terminal portion of Dph6 contains two domains related to the YjgF-YER057c-UK114 protein family (eu_AANH_C1: cd06155 and eu_AANH_C2: cd06166) that may promote homotrimerisation and formation of an inter-subunit cleft that has been proposed to bind small molecule ligands [63]–[65]. Several key residues in human UK114 required for homotrimerisation and ligand binding [66] are present in Dph6 (Figure S8) including arg-107, which in E. coli TdcF forms a bidentate salt bridge with the carboxylic acid group of bound ligands [63]. Deletion of residues 335–415 encompassing much of the YjgF-YER057c-UK114 region abolished the function of Dph6 as monitored by sordarin resistance (Figure 7), while truncation of Dph6 at the first of the two conserved domains by insertion of a myc tag also eliminated Dph6 function (Figure 7) despite detectable expression of the truncated polypeptide (data not shown), indicating that the YjgF-YER057c-UK114 domains are also important for Dph6 function and that the Alpha_ANH_like_IV domain is nonfunctional on its own. Since Salmonella enterica YjgF has an enamine/imine deaminase activity that is conserved in human UK114 [67] it is possible that the YjgF-YER057c-UK114 domains in Dph6 are used to generate ammonia for diphthamide formation.


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)

Both the Alpha_ANH_like_IV and YjgF-YER057c-UK114 domains in Dph6 are essential for its functionality.(A) Diagram showing the DPH6 wild-type and mutant constructs tested in (B), indicating the Alpha_ANH_like_IV (ANH) and YjgF-YER057c-UK114 (UK114) domains and the position of point mutations, an in-frame deletion (- - - - -) and triple myc epitope tag (myc3) as appropriate. (B) Ten-fold serial cell dilutions of a dph6 deletion strain carrying the constructs shown in (A) or the corresponding empty vector (top panel, pSU6 [wt DPH6]; lower panel, pSU7 [wt DPH6]: Table S3) were spotted onto SCD-Leu plates with or without 10 µg/ml sordarin and grown at 30°C for 3 days.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003334-g007: Both the Alpha_ANH_like_IV and YjgF-YER057c-UK114 domains in Dph6 are essential for its functionality.(A) Diagram showing the DPH6 wild-type and mutant constructs tested in (B), indicating the Alpha_ANH_like_IV (ANH) and YjgF-YER057c-UK114 (UK114) domains and the position of point mutations, an in-frame deletion (- - - - -) and triple myc epitope tag (myc3) as appropriate. (B) Ten-fold serial cell dilutions of a dph6 deletion strain carrying the constructs shown in (A) or the corresponding empty vector (top panel, pSU6 [wt DPH6]; lower panel, pSU7 [wt DPH6]: Table S3) were spotted onto SCD-Leu plates with or without 10 µg/ml sordarin and grown at 30°C for 3 days.
Mentions: Dph6 contains three conserved domains consistent with it functioning as an enzyme (Figure S8). The amino-terminal 225 residues constitute an Alpha_ANH_like_IV domain (cd1994 in the NCBI Conserved Domain Database [62], also known as DUF71), a member of the adenine nucleotide alpha hydrolase superfamily that is predicted to bind ATP. Many DUF71 proteins from archaea to mammals contain the highly conserved motif –E215GG(D/E)XE220– (Dph6 numbering), which has been proposed to be involved in substrate binding and catalysis and which is replaced by –ENGE(F/Y)H– in a group of related DUF71 proteins implicated in biotin synthesis [60]. Based on this we generated a dph6 allele encoding two substitutions in this region (G216N, E220A) and tested its functionality by monitoring complementation of sordarin resistance in a yeast dph6 knockout strain. Figure 7 clearly shows that this small change completely inactivates the function of Dph6, demonstrating that the Alpha_ANH_like_IV domain is critical for the conversion of diphthine to diphthamide. The C-terminal portion of Dph6 contains two domains related to the YjgF-YER057c-UK114 protein family (eu_AANH_C1: cd06155 and eu_AANH_C2: cd06166) that may promote homotrimerisation and formation of an inter-subunit cleft that has been proposed to bind small molecule ligands [63]–[65]. Several key residues in human UK114 required for homotrimerisation and ligand binding [66] are present in Dph6 (Figure S8) including arg-107, which in E. coli TdcF forms a bidentate salt bridge with the carboxylic acid group of bound ligands [63]. Deletion of residues 335–415 encompassing much of the YjgF-YER057c-UK114 region abolished the function of Dph6 as monitored by sordarin resistance (Figure 7), while truncation of Dph6 at the first of the two conserved domains by insertion of a myc tag also eliminated Dph6 function (Figure 7) despite detectable expression of the truncated polypeptide (data not shown), indicating that the YjgF-YER057c-UK114 domains are also important for Dph6 function and that the Alpha_ANH_like_IV domain is nonfunctional on its own. Since Salmonella enterica YjgF has an enamine/imine deaminase activity that is conserved in human UK114 [67] it is possible that the YjgF-YER057c-UK114 domains in Dph6 are used to generate ammonia for diphthamide formation.

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