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The crystal structure of class II ribonucleotide reductase reveals how an allosterically regulated monomer mimics a dimer

Nature Structural Biology volume 9, pages 293300 (2002) | Download Citation

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Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. Here we present the crystal structure of class II (coenzyme B12-dependent) ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii in the apo enzyme form and in complex with the B12 analog adeninylpentylcobalamin at 1.75 and 2.0 Å resolution, respectively. This monomeric, allosterically regulated class II RNR retains all the key structural features associated with the catalytic and regulatory machinery of oligomeric RNRs. Surprisingly, the dimer interface responsible for effector binding in class I RNR is preserved through a single 130-residue insertion in the class II structure. Thus, L. leichmannii RNR is a paradigm for the simplest structural entity capable of ribonucleotide reduction, a reaction linking the RNA and DNA worlds.

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References

  1. 1.

    & The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439 (2000).

  2. 2.

    & Ribonucleotide reductases. Annu. Rev. Biochem. 67, 71–98 (1998).

  3. 3.

    et al. International Commission for Protection Against Environmental Mutagens and Carcinogens. Deoxyribonucleoside triphosphate levels: a critical factor in the maintenance of genetic stability. Mutat. Res. 318, 1–64 (1994).

  4. 4.

    , , & Gemcitabine: preclinical pharmacology and mechanisms of action. Semin. Oncol. 23, 3–15 (1996).

  5. 5.

    et al. Antiviral activity of a selective ribonucleotide reductase inhibitor against acyclovir-resistant herpes simplex virus type 1 in vivo. Antimicrob. Agents Chemother. 42, 1629–1635 (1998).

  6. 6.

    Ribonucleotide reductases: the link between an RNA and a DNA world? Curr. Opin. Struct. Biol. 10, 731–736 (2000).

  7. 7.

    From RNA to DNA, why so many ribonucleotide reductases? Science 260, 1773–1777 (1993).

  8. 8.

    et al. Ribonucleotide reduction in Pseudomonas species: simultaneous presence of active enzymes from different classes. J. Bacteriol. 181, 3974–3980 (1999).

  9. 9.

    & Protein radicals in enzyme catalysis. Chem. Rev. 98, 705–762 (1998).

  10. 10.

    & Direct spectrophotometric observation of an intermediate formed from deoxyadenosylcobalamin in ribonucleotide reduction. Biochemistry 12, 24–34 (1973).

  11. 11.

    , & Three-dimensional structure of the free radical protein of ribonucleotide reductase. Nature 345, 593–598 (1990).

  12. 12.

    & Structure of ribonucleotide reductase protein R1. Nature 370, 533–539 (1994).

  13. 13.

    , , & A glycyl radical site in the crystal structure of a class III ribonucleotide reductase. Science 283, 1499–1504 (1999).

  14. 14.

    et al. Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding. Structure 5, 1077–1092 (1997).

  15. 15.

    et al. Binding of Cob(II)alamin to the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii. Identification of dimethylbenzimidazole as the axial ligand. J. Biol. Chem. 274, 7039–7042. (1999).

  16. 16.

    et al. A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase. Structure Fold. Des. 7, 997–1008 (1999).

  17. 17.

    , , , & How a protein binds B12: a 3.0 Å X-ray structure of B12-binding domains of methionine synthase. Science 266, 1669–1674 (1994).

  18. 18.

    et al. How coenzyme B12 radicals are generated: the crystal structure of methylmalonyl-coenzyme A mutase at 2 Å resolution. Structure 4, 339–350 (1996).

  19. 19.

    et al. Glutamate mutase from Clostridium cochlearium: the structure of a coenzyme B12-dependent enzyme provides new mechanistic insights. Structure Fold. Des. 7, 891–902 (1999).

  20. 20.

    & Ribonucleotide reductases: metal and free radical interplay. Coordin. Chem. Rev. 186, 775–793 (1999).

  21. 21.

    , , , & Electron paramagnetic resonance investigations of a kinetically competent intermediate formed in ribonucleotide reduction: evidence for a thiyl radical-Cob(II)alamin interaction. J. Am. Chem. Soc. 118, 8192–8197 (1996).

  22. 22.

    & Ion-dependent activation and inhibition of ribonucleotide reductase from Lactobacillus leichmannii. Biochem. Biophys. Res. Commun. 37, 793–800 (1969).

  23. 23.

    , , , & Ribonucleoside diphosphate reductase. Purification of the two subunits, proteins B1 and B2. Eur. J. Biochem. 9, 561–573 (1969).

  24. 24.

    , , & Allosteric control of three B12-dependent (class II) ribonucleotide reductases. Implications for the evolution of ribonucleotide reduction. J. Biol. Chem. 274, 7182–7189. (1999).

  25. 25.

    et al. Preserved catalytic activity in an engineered ribonucleotide reductase R2 protein with a nonphysiological radical transfer pathway. The importance of hydrogen bond connections between the participating residues. J. Biol. Chem. 273, 21003–21008 (1998).

  26. 26.

    , , & Two conserved tyrosine residues in protein R1 participate in an intermolecular electron transfer in ribonucleotide reductase. J. Biol. Chem. 271, 20655–20659 (1996).

  27. 27.

    , , , & Structural basis for allosteric substrate specificity regulation in anaerobic ribonucleotide reductases. Structure 9, 739–750 (2001).

  28. 28.

    & Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii. Proc. Natl. Acad. Sci. USA 90, 8352–8356 (1993).

  29. 29.

    , & The interaction of adeninylalkylcobalamins with ribonucleotide reductase. Biochim. Biophys. Acta 428, 228–232 (1976).

  30. 30.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  31. 31.

    Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997).

  32. 32.

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

  33. 33.

    The CCP4 Suite — programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  34. 34.

    & Density modification for macromolecular phase improvement. Prog. Biophys. Mol. Biol. 72, 245–270 (1999).

  35. 35.

    , , & Coenzyme B12-dependent ribonucleotide reductase: evidence for the participation of five cysteine residues in ribonucleotide reduction. Biochemistry 33, 12676–12685 (1994).

  36. 36.

    , , , & Formate is the hydrogen donor for the anaerobic ribonucleotide reductase from Escherichia coli. Proc. Natl. Acad. Sci. USA 92, 8759–8762 (1995).

  37. 37.

    Ribbons. Methods Enzymol. 277, 493–505 (1997).

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Acknowledgements

We thank T. Earnest (ALS), G. McDermott (ALS), R. Sweet (NSLS) and M. Becker (NSLS) for help with data collection, and C.C. Lawrence for help with protein purification. Support has been provided by the Surdna and Searle foundations (C.L.D.) and an NIH Grant (J.S.). The data collection facilities at ALS and NSLS are funded by the U.S. Department of Energy, Office of Basic Energy Sciences.

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  1. From the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

    • Michael D. Sintchak
    • , Gitrada Arjara
    • , Brenda A. Kellogg
    • , JoAnne Stubbe
    •  & Catherine L. Drennan

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The authors declare no competing financial interests.

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Correspondence to Catherine L. Drennan.

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https://doi.org/10.1038/nsb774

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