CoChat
Sign Up
Cited by 1Metalloenzymes and iron-sulfur proteinsEnzyme Structure and FunctionRNA and protein synthesis mechanisms

The DOI matches its Crossref record; 1 of the 1 references it cites were found in a registry or an index.

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines

Open access
Ütkür, Koray, Mayer, Klaus, Liu, Shihui, Brinkmann, Ulrich, Schaffrath, Raffael·2024·Biomolecules
DOI 10.3390/biom14040470PMC PMC11048331

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed DPH1 and DPH2 mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.3390/biom14040470

Cites

1 of 1 references verified0 citation errors · 0 not checkable

Statistics

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Flagged first, then by citations

Research works printed but not linked: 58

  1. Frey, 2008, The radical SAM superfamily, Crit. Rev. Biochem. Mol. Biol., 43, 63, 10.1080/10409230701829169 · DOI
  2. Hoffman, 2023, Mechanism of radical initiation in the radical SAM enzyme superfamily, Annu. Rev. Biochem., 92, 333, 10.1146/annurev-biochem-052621-090638 · DOI
  3. Yokoyama, 2018, C-C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products, Nat. Prod. Rep., 35, 660, 10.1039/C8NP00006A · DOI
  4. Nicolet, 2020, Structure–function relationships of radical SAM enzymes, Nat. Catal., 3, 337, 10.1038/s41929-020-0448-7 · DOI
  5. Broderick, 2014, Radical S-adenosylmethionine enzymes, Chem. Rev., 114, 4229, 10.1021/cr4004709 · DOI
  6. Haldar, S., Paul, S., Joshi, N., Dasgupta, A., and Chattopadhyay, K. (2012). The presence of the iron-sulfur motif is important for the conformational stability of the antiviral protein, Viperin. PLoS ONE, 7. · DOI
  7. Zhang, 2010, Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme, Nature, 465, 891, 10.1038/nature09138 · DOI
  8. Broderick, 2010, Biochemistry: A radically different enzyme, Nature, 465, 877, 10.1038/465877a · DOI
  9. Dong, 2019, The asymmetric function of Dph1-Dph2 heterodimer in diphthamide biosynthesis, J. Biol. Inorg. Chem., 24, 777, 10.1007/s00775-019-01702-0 · DOI
  10. Ütkür, K., Schmidt, S., Mayer, K., Klassen, R., Brinkmann, U., and Schaffrath, R. (2023). DPH1 Gene mutations identify a candidate SAM pocket in radical enzyme Dph1•Dph2 for diphthamide synthesis on EF2. Biomolecules, 13. · DOI
  11. Lin, 2011, S-Adenosylmethionine-dependent alkylation reactions: When are radical reactions used?, Bioorg. Chem., 39, 161, 10.1016/j.bioorg.2011.06.001 · DOI
  12. Zhu, 2011, Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] enzyme required for diphthamide biosynthesis, Mol. Biosyst., 7, 74, 10.1039/C0MB00076K · DOI
  13. Dong, 2018, Organometallic and radical intermediates reveal mechanism of diphthamide biosynthesis, Science, 359, 1247, 10.1126/science.aao6595 · DOI
  14. Su, 2013, The biosynthesis and biological function of diphthamide, Crit. Rev. Biochem. Mol. Biol., 48, 515, 10.3109/10409238.2013.831023 · DOI
  15. Liu, 2004, Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2, Mol. Cell. Biol., 24, 9487, 10.1128/MCB.24.21.9487-9497.2004 · DOI
  16. Miura, 2019, The hidden nature of protein translational control by diphthamide: The secrets under the leather, J. Biochem., 165, 1, 10.1093/jb/mvy071 · DOI
  17. Arend, 2023, Yeast gene KTI13 (alias DPH8) operates in the initiation step of diphthamide synthesis on elongation factor 2, Microb. Cell, 10, 195, 10.15698/mic2023.09.804 · DOI
  18. Urreizti, 2020, DPH1 syndrome: Two novel variants and structural and functional analyses of seven missense variants identified in syndromic patients, Eur. J. Hum. Genet., 28, 64, 10.1038/s41431-019-0374-9 · DOI
  19. Hawer, 2020, Diphthamide-deficiency syndrome: A novel human developmental disorder and ribosomopathy, Eur. J. Hum. Genet., 28, 1497, 10.1038/s41431-020-0668-y · DOI
  20. Shankar, 2022, A novel DPH5-related diphthamide-deficiency syndrome causing embryonic lethality or profound neurodevelopmental disorder, Genet. Med., 24, 1567, 10.1016/j.gim.2022.03.014 · DOI
  21. Mayer, 2023, DPH1 and DPH2 variants that confer susceptibility to diphthamide deficiency syndrome in human cells and yeast models, Dis. Models Mech., 16, dmmm050207, 10.1242/dmm.050207 · DOI
  22. Schaffrath, 2024, Diphthamide—A conserved modification of eEF2 with clinical relevance, Trends Mol. Med., 30, 164, 10.1016/j.molmed.2023.11.008 · DOI
  23. Merrill, 2006, The life and death of translation elongation factor 2, Biochem. Soc. Trans., 34, 1, 10.1042/BST0340001 · DOI
  24. Uthman, S., Bär, C., Scheidt, V., Liu, S., ten Have, S., Giorgini, F., Stark, M.J.R., and Schaffrath, R. (2013). The amidation step of diphthamide biosynthesis in yeast requires DPH6, a gene identified through mining the DPH1-DPH5 interaction network. PLoS Genet., 9. · DOI
  25. Toulmay, 2006, A Two-step method for the introduction of single or multiple defined point mutations into the genome of Saccharomyces cerevisiae, Yeast, 23, 825, 10.1002/yea.1397 · DOI
  26. Janke, 2004, A versatile toolbox for PCR-based tagging of yeast genes: New fluorescent proteins, more markers and promoter substitution cassettes, Yeast, 21, 947, 10.1002/yea.1142 · DOI
  27. Woods, 2002, Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method, Methods Enzymol., 350, 87, 10.1016/S0076-6879(02)50957-5 · DOI
  28. Sherman, 2002, Getting started with yeast, Methods Enzymol., 350, 3, 10.1016/S0076-6879(02)50954-X · DOI · Paper page
  29. Mirdita, 2022, ColabFold: Making protein folding accessible to all, Nat. Methods, 19, 679, 10.1038/s41592-022-01488-1 · DOI · Paper page
  30. Stahl, 2015, Loss of diphthamide pre-activates NF-ΚB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor, Proc. Natl. Acad. Sci. USA, 112, 10732, 10.1073/pnas.1512863112 · DOI
  31. Hawer, H., Ütkür, K., Arend, M., Mayer, K., Adrian, L., Brinkmann, U., and Schaffrath, R. (2018). Importance of diphthamide modified EF2 for translational accuracy and competitive cell growth in yeast. PLoS ONE, 13. · DOI
  32. Zhang, 2022, Translational fidelity and growth of Arabidopsis require stress-sensitive diphthamide biosynthesis, Nat. Comm., 13, 4009, 10.1038/s41467-022-31712-7 · DOI
  33. Zachariae, 1996, Identification of subunits of the anaphase-promoting complex of Saccharomyces cerevisiae, Science, 274, 201, 10.1126/science.274.5290.1201 · DOI
  34. Bradford, 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Chem., 72, 248
  35. Buchanan, 2016, Cycloheximide chase analysis of protein degradation in Saccharomyces cerevisiae, J. Vis. Exp., 110, 53957
  36. Mayer, K., Schröder, A., Schnitger, J., Stahl, S., and Brinkmann, U. (2017). Influence of DPH1 and DPH5 protein variants on the synthesis of diphthamide, the target of ADP-ribosylating toxins. Toxins, 9. · DOI
  37. Botet, 2008, A chemical genomic screen in Saccharomyces cerevisiae reveals a role for diphthamidation of translation elongation factor 2 in inhibition of protein synthesis by sordarin, Antimicrob. Agents Chemother., 52, 1623, 10.1128/AAC.01603-07 · DOI
  38. Zabel, 2008, A versatile partner of eukaryotic protein complexes that is involved in multiple biological processes: Kti11/Dph3, Mol. Microbiol., 69, 1221, 10.1111/j.1365-2958.2008.06350.x · DOI
  39. Shao, 2022, Sordarin—An anti-fungal antibiotic with a unique modus operandi, Br. J. Pharmacol., 179, 1125, 10.1111/bph.15724 · DOI
  40. Scheidt, 2013, Insights into diphthamide, key diphtheria toxin effector, Toxins, 5, 958, 10.3390/toxins5050958 · DOI
  41. Fenwick, 2019, The crystal structure of Dph2 in complex with elongation factor 2 reveals the structural basis for the first step of diphthamide biosynthesis, Biochemistry, 58, 4343, 10.1021/acs.biochem.9b00718 · DOI
  42. Prokhorova, 2014, Structural basis for the inhibition of the eukaryotic ribosome, Nature, 513, 517, 10.1038/nature13737 · DOI
  43. Susorov, 2018, Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome, J. Biol. Chem., 293, 5220, 10.1074/jbc.RA117.000761 · DOI
  44. Schimpf, 2022, Structure of the peripheral arm of a minimalistic respiratory complex I, Structure, 30, 80, 10.1016/j.str.2021.09.005 · DOI
  45. Duarte, 2002, Disruption of iron-sulphur cluster N2 from NADH: Ubiquinone oxidoreductase by site-directed mutagenesis, Biochem. J., 364, 833, 10.1042/bj20011750 · DOI
  46. Sun, 2005, Solution structure of Kti11p from Saccharomyces cerevisiae reveals a novel zinc-binding module, Biochemistry, 44, 8801, 10.1021/bi0504714 · DOI
  47. Proudfoot, 2008, Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain, J. Mol. Biol., 375, 301, 10.1016/j.jmb.2007.10.060 · DOI
  48. Dong, 2014, Dph3 is an electron donor for Dph1-Dph2 in the first step of eukaryotic diphthamide biosynthesis, J. Am. Chem. Soc., 136, 1754, 10.1021/ja4118957 · DOI
  49. Zhang, 2021, Dph3 enables aerobic diphthamide biosynthesis by donating one iron atom to transform a [3Fe-4S] to a [4Fe-4S] cluster in Dph1-Dph2, J. Am. Chem. Soc., 143, 9314, 10.1021/jacs.1c03956 · DOI
  50. Lin, 2016, Cbr1 is a Dph3 reductase required for the tRNA wobble uridine modification, Nat. Chem. Biol., 12, 995, 10.1038/nchembio.2190 · DOI
  51. Glatt, 2015, Structure of the Kti11/Kti13 heterodimer and its double role in modification of tRNA and eukaryotic elongation factor 2, Structure, 23, 149, 10.1016/j.str.2014.11.008 · DOI
  52. McEwen, 2015, Structure of the Elongator cofactor complex Kti11/Kti13 provides insight into the role of Kti13 in Elongator-dependent tRNA modification, FEBS J., 282, 819, 10.1111/febs.13199 · DOI
  53. Landgraf, 2016, Radical S-adenosylmethionine enzymes in human health and disease, Annu. Rev. Biochem., 85, 485, 10.1146/annurev-biochem-060713-035504 · DOI
  54. Pellegrino, 2018, Structural insights into the role of diphthamide on elongation factor 2 in mRNA reading-frame maintenance, J. Mol. Biol., 430, 2677, 10.1016/j.jmb.2018.06.006 · DOI
  55. Djumagulov, 2021, Accuracy mechanism of eukaryotic ribosome translocation, Nature, 600, 543, 10.1038/s41586-021-04131-9 · DOI
  56. Milicevic, 2024, mRNA reading frame maintenance during eukaryotic ribosome translocation, Nature, 625, 393, 10.1038/s41586-023-06780-4 · DOI
  57. Shin, 2023, eEF2 diphthamide modification restrains spurious frameshifting to maintain translational fidelity, Nucleic Acids Res., 51, 6899, 10.1093/nar/gkad461 · DOI
  58. Yao, Y., He, J., and Dong, M. (2024). Arsinothricin biosynthesis involving a non-canonical radical SAM enzyme for C-As bond formation. bioRxiv, bioRxiv: 2024.02.01.577332. · DOI

Metadata

Authors
Ütkür, K., Mayer, K., Liu, S., Brinkmann, U., & Schaffrath, R.
Affiliations
  • University of Kassel (DE)
  • Roche Pharma AG (Germany) (DE)
  • University of Pittsburgh (US)
Corresponding author
Schaffrath, Raffael
ORCID
Venue
Biomolecules
Publisher
MDPI AG
Published
2024-04-11
Volume
14
Issue
4
Pages
470
ISSN
2218-273X (ISSN-L)
DOI
10.3390/biom14040470
PMC
PMC11048331
OpenAlex
W4394764816
Cited by
1
References
1
Citations by year
2026: 1
FWCI
0.07
Type
journal article
Language
en
Open access
Licence
Locations
  • Biomolecules (publishedVersion, cc-by)
  • Preprints.org (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Zentraler Forschungsfonds (ZFF, Universität Kassel, Kassel, Germany) (2887, SCHA750/21-1)
  • Deutsche Forschungsgemeinschaft (DFG, Bonn, Germany), Priority Program 1927 Iron-Sulfur for Life (2887, SCHA750/21-1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Metalloenzymes and iron-sulfur proteins
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
Keywords
radical SAM enzymes, diphthamide biosynthesis, iron-sulfur clusters, protein stability, protein degradation, site-directed mutagenesis, Saccharomyces cerevisiae
MeSH terms
Cysteine, Histidine, Repressor Proteins, Saccharomyces cerevisiae, Carbon-Sulfur Lyases, Mutagenesis, Site-Directed, Amino Acid Motifs, Saccharomyces cerevisiae Proteins, Protein Multimerization

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review