2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

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Data deposits

Atomic coordinates and structure factors for the crystal structures have been deposited in the PDB under accession numbers 2XDV, 4DIQ, 4BU2, 4BXF, 4CCJ, 4CCK, 4CCL, 4CCM, 4CCN, 4CCO, 4LIU, 4LIT, 4LIV, 4CSW and 4CUG.


  1. 1.

    & Regulation of histone methylation by demethylimination and demethylation. Nature Rev. Mol. Cell Biol. 8, 307–318 (2007)

  2. 2.

    , & Mechanisms of human histone and nucleic acid demethylases. Curr. Opin. Chem. Biol. 16, 525–534 (2012)

  3. 3.

    & Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol. Cell 30, 393–402 (2008)

  4. 4.

    et al. Jmjd6 catalyses lysyl-hydroxylation of U2AF65, a protein associated with RNA splicing. Science 325, 90–93 (2009)

  5. 5.

    et al. The AlkB domain of mammalian ABH8 catalyzes hydroxylation of 5-methoxycarbonylmethyluridine at the wobble position of tRNA. Angew. Chem. Int. Edn Engl. 49, 8885–8888 (2010)

  6. 6.

    et al. Crystal structure of a novel JmjC-domain-containing protein, TYW5, involved in tRNA modification. Nucleic Acids Res. 39, 1576–1585 (2011)

  7. 7.

    et al. ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA. Nature Commun. 2, 172 (2011)

  8. 8.

    et al. Oxygenase-catalyzed ribosome hydroxylation occurs in prokaryotes and humans. Nature Chem. Biol. 8, 960–962 (2012)

  9. 9.

    et al. Optimal translational termination requires C4 lysyl hydroxylation of eRF1. Mol. Cell 53, 645–654 (2014)

  10. 10.

    et al. Structural and functional insights into Saccharomyces cerevisiae Tpa1, a putative prolylhydroxylase influencing translation termination and transcription. J. Biol. Chem. 285, 30767–30778 (2010)

  11. 11.

    et al. Hydroxylation of the eukaryotic ribosomal decoding center affects translational accuracy. Proc. Natl Acad. Sci. USA 111, 4019–4024 (2014)

  12. 12.

    et al. Dynamic regulatory network controlling TH17 cell differentiation. Nature 496, 461–468 (2013)

  13. 13.

    , & Structural and functional aspects of winged-helix domains at the core of transcription initiation complexes. Transcription 3, 2–7 (2012)

  14. 14.

    FeII/α-ketoglutarate-dependent hydroxylases and related enzymes. Crit. Rev. Biochem. Mol. Biol. 39, 21–68 (2004)

  15. 15.

    et al. Structural studies on 2-oxoglutarate oxygenases and related double-stranded β-helix fold proteins. J. Inorg. Biochem. 100, 644–669 (2006)

  16. 16.

    & Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey. Structure 8, 493–504 (2000)

  17. 17.

    et al. Structure of factor-inhibiting hypoxia-inducible factor (HIF) reveals mechanism of oxidative modification of HIF-1α. J. Biol. Chem. 278, 1802–1806 (2003)

  18. 18.

    , & Crystal structure and functional analysis of JMJD5 indicate an alternate specificity and function. Mol. Cell. Biol. 32, 4044–4052 (2012)

  19. 19.

    et al. Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA. Nature 452, 961–965 (2008)

  20. 20.

    et al. Linking of 2-oxoglutarate and substrate binding sites enables potent and highly selective inhibition of JmjC histone demethylases. Angew. Chem. Int. Edn Engl. 51, 1631–1634 (2012)

  21. 21.

    et al. Enzymatic and structural insights for substrate specificity of a family of jumonji histone lysine demethylases. Nature Struct. Mol. Biol. 17, 38–43 (2010)

  22. 22.

    et al. Crystal structures of histone demethylase JMJD2A reveal basis for substrate specificity. Nature 448, 87–91 (2007)

  23. 23.

    et al. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature 488, 404–408 (2012)

  24. 24.

    & Structural basis for histone H3 Lys 27 demethylation by UTX/KDM6A. Genes Dev. 25, 2266–2277 (2011)

  25. 25.

    , , , & Regulation of the osteoblast-specific transcription factor Osterix by NO66, a Jumonji family histone demethylase. EMBO J. 29, 68–79 (2010)

  26. 26.

    et al. Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains. FEBS J. 278, 1086–1097 (2011)

  27. 27.

    , , , & Direct spectroscopic detection of a C-H-cleaving high-spin Fe(IV) complex in a prolyl-4-hydroxylase. Proc. Natl Acad. Sci. USA 103, 14738–14743 (2006)

  28. 28.

    et al. Elucidation of the Fe(IV) = O intermediate in the catalytic cycle of the halogenase SyrB2. Nature 499, 320–323 (2013)

  29. 29.

    et al. Substrate selectivity analyses of factor inhibiting hypoxia-inducible factor. Angew. Chem. Int. Edn Engl. 52, 1700–1704 (2013)

  30. 30.

    , , & Origin and evolution of peptide-modifying dioxygenases and identification of the wybutosine hydroxylase/hydroperoxidase. Nucleic Acids Res. 38, 5261–5279 (2010)

  31. 31.

    , , , & Production of selenomethionine-labelled proteins using simplified culture conditions and generally applicable host/vector systems. Appl. Microbiol. Biotechnol. 56, 718–723 (2001)

  32. 32.

    et al. On the application of the minimal principle to solve unknown structures. Science 259, 1430–1433 (1993)

  33. 33.

    , , , & Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D 59, 2023–2030 (2003)

  34. 34.

    & Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996)

  35. 35.

    et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2012)

  36. 36.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  37. 37.

    Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479–485 (2010)

  38. 38.

    et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

  39. 39.

    The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006)

  40. 40.

    et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

  41. 41.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

  42. 42.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  43. 43.

    et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

  44. 44.

    Automated main-chain model building by template matching and iterative fragment extension. Acta Crystallogr. D 59, 38–44 (2003)

  45. 45.

    , & Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

  46. 46.

    et al. Selective small molecule probes for the hypoxia inducible factor (HIF) prolyl hydroxylases. ACS Chem. Biol. 8, 1488–1496 (2013)

  47. 47.

    TopDraw: a sketchpad for protein structure topology cartoons. Bioinformatics 19, 311–312 (2003)

  48. 48.

    et al. Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH). Proc. Natl Acad. Sci. USA 103, 14767–14772 (2006)

  49. 49.

    et al. Structural insights into histone demethylase NO66 in interaction with osteoblast specific transcription factor Osterix and gene repression. J. Biol. Chem. 288, 16430–16437 (2013)

  50. 50.

    et al. Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity. Biochem. J. 383, 429–437 (2004)

  51. 51.

    & Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010)

  52. 52.

    et al. How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic. EMBO J. 28, 1953–1964 (2009)

  53. 53.

    et al. The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA. Science 326, 688–694 (2009)

  54. 54.

    , & Structure of the DNA-binding domain of the response regulator PhoP from Mycobacterium tuberculosis. Biochemistry 46, 14751–14761 (2007)

  55. 55.

    The pleiotropic two-component regulatory system PhoP-PhoQ. J. Bacteriol. 183, 1835–1842 (2001)

  56. 56.

    , , , & Role of the jelly-roll fold in substrate binding by 2-oxoglutarate oxygenases. Curr. Opin. Struct. Biol. 22, 691–700 (2012)

  57. 57.

    , , , & Structural studies on human 2-oxoglutarate dependent oxygenases. Curr. Opin. Struct. Biol. 20, 659–672 (2010)

  58. 58.

    & phyloXML: XML for evolutionary biology and comparative genomics. BMC Bioinformatics 10, 356 (2009)

  59. 59.

    et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

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We thank the Biotechnology and Biological Sciences Research Council, the Wellcome Trust, European Research Council, Medical Research Council, Oxford NIHR Biomedical Research Unit, Cancer Research UK, Arthritis Research UK, Bayer Healthcare, the Rosetree Foundation and the Slovenian Academy of Sciences and Arts (R.S.) for funding. We thank the scientists of beamlines X10SA (Swiss Light Source) and I02, I03, I04, I04-1 (Diamond Light Source) for assistance. The Structural Genomics Consortium is a registered charity (number 1097737) funded by Abbvie, Boehringer Ingelheim, the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Eli Lilly, Genome Canada, GlaxoSmithKline, the Ontario Ministry of Economic Development and Innovation, Janssen, Novartis Research Foundation, Pfizer, Takeda and the Wellcome Trust.

Author information

Author notes

    • Rok Sekirnik
    •  & Nigel C. Brissett

    These authors contributed equally to this work.


  1. The Department of Chemistry and Oxford Centre for Integrative Systems Biology, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK

    • Rasheduzzaman Chowdhury
    • , Rok Sekirnik
    • , Chia-hua Ho
    • , Ian J. Clifton
    • , Wei Ge
    • , Nadia J. Kershaw
    • , Michael A. McDonough
    •  & Christopher J. Schofield
  2. Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, UK

    • Nigel C. Brissett
    •  & Aidan J. Doherty
  3. Structural Genomics Consortium, University of Oxford, Headington, Oxford OX3 7DQ, UK

    • Tobias Krojer
    • , Stanley S. Ng
    • , Joao R. C. Muniz
    • , Melanie Vollmar
    • , Claire Phillips
    • , Ewa S. Pilka
    • , Kathryn L. Kavanagh
    • , Frank von Delft
    •  & Udo Oppermann
  4. Synchrotron SOLEIL, Saint Aubin, 91192 Gif-sur-Yvette Cedex, France

    • Gavin C. Fox
  5. NIHR Oxford Biomedical Research Unit, Botnar Research Centre, Oxford OX3 7LD, UK

    • Udo Oppermann


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R.C., R.S., N.C.B., C.-h.H., W.G., N.J.K., C.P., S.S.N. and E.S.P. cloned the constructs and purified proteins; R.C. and R.S. performed assays; R.C., R.S., N.C.B., S.S.N., C.-h.H. and E.S.P. crystallized the protein–ligand/substrate complexes; R.C., N.C.B., T.K., S.S.N., I.J.C., G.C.F., K.L.K., F.v.D. and M.A.M. collected and processed X-ray data; R.C., N.C.B., T.K., J.R.C.M., M.V. and M.A.M. solved and refined complex structures; R.C., R.S., M.A.M. and C.J.S. analysed data; R.C., U.O., A.J.D. and C.J.S. designed the studies; R.C. and C.J.S. wrote the paper with the help of others. See Supplementary Table 5 for further details.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Rasheduzzaman Chowdhury or Christopher J. Schofield.

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