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Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB

Abstract

Nucleic acid damage by environmental and endogenous alkylation reagents creates lesions that are both mutagenic and cytotoxic, with the latter effect accounting for their widespread use in clinical cancer chemotherapy1,2. Escherichia coli AlkB3,4,5,6,7,8,9 and the homologous human proteins ABH2 and ABH3 (refs 5, 7) promiscuously repair DNA and RNA bases damaged by SN2 alkylation reagents, which attach hydrocarbons to endocyclic ring nitrogen atoms (N1 of adenine and guanine and N3 of thymine and cytosine)3,4,10,11,12,13,14,15. Although the role of AlkB in DNA repair has long been established based on phenotypic studies, its exact biochemical activity was only elucidated recently after sequence profile analysis revealed it to be a member of the Fe-oxoglutarate-dependent dioxygenase superfamily. These enzymes use an Fe(ii) cofactor and 2-oxoglutarate co-substrate to oxidize organic substrates. AlkB hydroxylates an alkylated nucleotide base to produce an unstable product that releases an aldehyde to regenerate the unmodified base. Here we have determined crystal structures of substrate and product complexes of E. coli AlkB at resolutions from 1.8 to 2.3 Å. Whereas the Fe-2-oxoglutarate dioxygenase core matches that in other superfamily members, a unique subdomain holds a methylated trinucleotide substrate into the active site through contacts to the polynucleotide backbone. Amide hydrogen exchange studies and crystallographic analyses suggest that this substrate-binding ‘lid’ is conformationally flexible, which may enable docking of diverse alkylated nucleotide substrates in optimal catalytic geometry. Different crystal structures show open and closed states of a tunnel putatively gating O2 diffusion into the active site. Exposing crystals of the anaerobic Michaelis complex to air yields slow but substantial oxidation of 2-oxoglutarate that is inefficiently coupled to nucleotide oxidation. These observations suggest that protein dynamics modulate redox chemistry and that a hypothesized migration of the reactive oxy-ferryl ligand on the catalytic Fe ion may be impeded when the protein is constrained in the crystal lattice.

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Figure 1: Crystal structure of the anaerobic Michaelis complex of E. coli AlkB-ΔN11 with Fe( ii ), 2OG and a methylated trinucleotide.
Figure 2: Nucleotide substrate recognition and active site cavities in the anaerobic Michaelis complex.
Figure 3: Stereo pairs showing active site stereochemistry in alternative ligand complexes of AlkB-ΔN11.

References

  1. 1

    Drablos, F. et al. Alkylation damage in DNA and RNA-repair mechanisms and medical significance. DNA Repair (Amst.) 3, 1389–1407 (2004)

    CAS  Article  Google Scholar 

  2. 2

    Sedgwick, B. Repairing DNA-methylation damage. Nature Rev. Mol. Cell Biol. 5, 148–157 (2004)

    CAS  Article  Google Scholar 

  3. 3

    Falnes, P. O., Johansen, R. F. & Seeberg, E. AlkB-mediated oxidative demethylation reverses DNA damage in Escherichia coli. Nature 419, 178–182 (2002)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Trewick, S. C., Henshaw, T. F., Hausinger, R. P., Lindahl, T. & Sedgwick, B. Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage. Nature 419, 174–178 (2002)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Aas, P. A. et al. Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature 421, 859–863 (2003)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Ougland, R. et al. AlkB restores the biological function of mRNA and tRNA inactivated by chemical methylation. Mol. Cell 16, 107–116 (2004)

    CAS  Article  Google Scholar 

  7. 7

    Falnes, P. O., Bjoras, M., Aas, P. A., Sundheim, O. & Seeberg, E. Substrate specificities of bacterial and human AlkB proteins. Nucleic Acids Res. 32, 3456–3461 (2004)

    CAS  Article  Google Scholar 

  8. 8

    Kataoka, H., Yamamoto, Y. & Sekiguchi, M. A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate. J. Bacteriol. 153, 1301–1307 (1983)

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Chen, B. J., Carroll, P. & Samson, L. The Escherichia coli AlkB protein protects human cells against alkylation-induced toxicity. J. Bacteriol. 176, 6255–6261 (1994)

    CAS  Article  Google Scholar 

  10. 10

    Koivisto, P., Robins, P., Lindahl, T. & Sedgwick, B. Demethylation of 3-methylthymine in DNA by bacterial and human DNA dioxygenases. J. Biol. Chem. 279, 40470–40474 (2004)

    CAS  Article  Google Scholar 

  11. 11

    Delaney, J. C. & Essigmann, J. M. Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli. Proc. Natl Acad. Sci. USA 101, 14051–14056 (2004)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Falnes, P. O. Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins. Nucleic Acids Res. 32, 6260–6267 (2004)

    CAS  Article  Google Scholar 

  13. 13

    Duncan, T. et al. Reversal of DNA alkylation damage by two human dioxygenases. Proc. Natl Acad. Sci. USA 99, 16660–16665 (2002)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Koivisto, P., Duncan, T., Lindahl, T. & Sedgwick, B. Minimal methylated substrate and extended substrate range of Escherichia coli AlkB protein, a 1-methyladenine-DNA dioxygenase. J. Biol. Chem. 278, 44348–44354 (2003)

    CAS  Article  Google Scholar 

  15. 15

    Delaney, J. C. et al. AlkB reverses etheno DNA lesions caused by lipid oxidation in vitro and in vivo. Nature Struct. Mol. Biol. 12, 855–860 (2005)

    CAS  Article  Google Scholar 

  16. 16

    Elkins, J. M. et al. X-ray crystal structure of Escherichia coli taurine/alpha-ketoglutarate dioxygenase complexed to ferrous iron and substrates. Biochemistry 41, 5185–5192 (2002)

    CAS  Article  Google Scholar 

  17. 17

    Muller, I. et al. Crystal structure of the alkylsulfatase AtsK: insights into the catalytic mechanism of the Fe(II) alpha-ketoglutarate-dependent dioxygenase superfamily. Biochemistry 43, 3075–3088 (2004)

    Article  Google Scholar 

  18. 18

    Zhang, Z. et al. Crystal structure of a clavaminate synthase-Fe(II)-2-oxoglutarate-substrate-NO complex: evidence for metal centered rearrangements. FEBS Lett. 517, 7–12 (2002)

    CAS  Article  Google Scholar 

  19. 19

    Murzin, A. G., Brenner, S. E., Hubbard, T. & Chothia, C. SCOP: a structural classification of proteins database for the investigation of sequences and structures. J. Mol. Biol. 247, 536–540 (1995)

    CAS  Google Scholar 

  20. 20

    Mishina, Y. & He, C. Probing the structure and function of the Escherichia coli DNA alkylation repair AlkB protein through chemical cross-linking. J. Am. Chem. Soc. 125, 8730–8731 (2003)

    CAS  Article  Google Scholar 

  21. 21

    Krebs, C. et al. Rapid freeze-quench 57Fe Mossbauer spectroscopy: monitoring changes of an iron-containing active site during a biochemical reaction. Inorg. Chem. 44, 742–757 (2005)

    CAS  Article  Google Scholar 

  22. 22

    Hegg, E. L. et al. Herbicide-degrading alpha-keto acid-dependent enzyme TfdA: metal coordination environment and mechanistic insights. Biochemistry 38, 16714–16726 (1999)

    CAS  Article  Google Scholar 

  23. 23

    Schofield, C. J. & Zhang, Z. Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes. Curr. Opin. Struct. Biol. 9, 722–731 (1999)

    CAS  Article  Google Scholar 

  24. 24

    Wilmouth, R. C. et al. Structure and mechanism of anthocyanidin synthase from Arabidopsis thaliana. Structure 10, 93–103 (2002)

    CAS  Article  Google Scholar 

  25. 25

    Rohde, J. U. et al. Crystallographic and spectroscopic characterization of a nonheme Fe(IV)-O complex. Science 299, 1037–1039 (2003)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Que, L. Jr. The oxo/peroxo debate: a nonheme iron perspective. J. Biol. Inorg. Chem. 9, 684–690 (2004)

    CAS  Article  Google Scholar 

  27. 27

    Price, J. C., Barr, E. W., Hoffart, L. M., Krebs, C. & Bollinger, J. M. Jr. Kinetic dissection of the catalytic mechanism of taurine:alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli. Biochemistry 44, 8138–8147 (2005)

    CAS  Article  Google Scholar 

  28. 28

    Valegard, K. et al. The structural basis of cephalosporin formation in a mononuclear ferrous enzyme. Nature Struct. Mol. Biol. 11, 95–101 (2004)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank R. Xiao, T. B. Acton and G. T. Montelione for provision of full-length AlkB protein, and J. C. Arbing, Y. Chen, M. Cheng, R. Farid, F. Forouhar, S. Handelman, A. Itkin, A. Kuzin, A. Petros, C. Prives, J. Schwanof, P. and W. Smith, D. Thompson, G. Verdon, W. Yong and A. Zupnick for advice and technical assistance. This work was supported by an NIH Protein Structure Initiative grant to the Northeast Structural Genomics Consortium and an Established Investigator Award from the American Heart Association to J.F.H.

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Correspondence to John F. Hunt.

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Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank under accession codes 2FD8, 2FDF, 2FDH, 2FDG, 2FDJ, 2FDI and 2FDK. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

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Supplementary Notes

This file contains the following sections: Supplementary Methods and Supplementary Discussion, Supplementary Tables 1–3 and Supplementary Figures 1–6. (PDF 2233 kb)

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Yu, B., Edstrom, W., Benach, J. et al. Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB. Nature 439, 879–884 (2006). https://doi.org/10.1038/nature04561

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