Abstract

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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Accessions

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.

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Acknowledgements

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.

Affiliations

  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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Contributions

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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https://doi.org/10.1038/nature13263

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