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Cofactor mobility determines reaction outcome in the IMPDH and GMPR (β-α)8 barrel enzymes

Nature Chemical Biology volume 7, pages 950–958 (2011)Cite this article

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Abstract

Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 Å from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.

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Figure 1: The reactions catalyzed by IMPDH and GMPR.
Figure 2: Formation of E-XMP* during the GMPR reaction.
Figure 3: Structure of the IMP or GMP binding site in IMPDH and GMPR.
Figure 4: NADPH has two conformations in the E–IMP complex of hGMPR2.
Figure 5: Reactions of EcGMPR.
Figure 6

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  1. Glasner, M.E., Gerlt, J.A. & Babbitt, P.C. Evolution of enzyme superfamilies. Curr. Opin. Chem. Biol. 10, 492–497 (2006).

    Article  CAS  Google Scholar 

  2. Soskine, M. & Tawfik, D.S. Mutational effects and the evolution of new protein functions. Nat. Rev. Genet. 11, 572–582 (2010).

    Article  CAS  Google Scholar 

  3. Zalatan, J.G. & Herschlag, D. The far reaches of enzymology. Nat. Chem. Biol. 5, 516–520 (2009).

    Article  CAS  Google Scholar 

  4. Nagano, N., Orengo, C.A. & Thornton, J.M. One fold with many functions: the evolutionary relationships between TIM barrel families based on their sequences, structures and functions. J. Mol. Biol. 321, 741–765 (2002).

    Article  CAS  Google Scholar 

  5. Gerlt, J.A. & Raushel, F.M. Evolution of function in (β/α)8-barrel enzymes. Curr. Opin. Chem. Biol. 7, 252–264 (2003).

    Article  CAS  Google Scholar 

  6. Wise, E.L. & Rayment, I. Understanding the importance of protein structure to nature's routes for divergent evolution in TIM barrel enzymes. Acc. Chem. Res. 37, 149–158 (2004).

    Article  CAS  Google Scholar 

  7. Lo Conte, L., Brenner, S.E., Hubbard, T.J., Chothia, C. & Murzin, A.G. SCOP database in 2002: refinements accommodate structural genomics. Nucleic Acids Res. 30, 264–267 (2002).

    Article  CAS  Google Scholar 

  8. Orengo, C.A. et al. CATH—a hierarchic classification of protein domain structures. Structure 5, 1093–1108 (1997).

    Article  CAS  Google Scholar 

  9. Anantharaman, V., Aravind, L. & Koonin, E.V. Emergence of diverse biochemical activities in evolutionarily conserved structural scaffolds of proteins. Curr. Opin. Chem. Biol. 7, 12–20 (2003).

    Article  CAS  Google Scholar 

  10. Taylor, J.S. & Raes, J. Duplication and divergence: the evolution of new genes and old ideas. Annu. Rev. Genet. 38, 615–643 (2004).

    Article  CAS  Google Scholar 

  11. Bergthorsson, U., Andersson, D.I. & Roth, J.R. Ohno's dilemma: evolution of new genes under continuous selection. Proc. Natl. Acad. Sci. USA 104, 17004–17009 (2007).

    Article  CAS  Google Scholar 

  12. Conant, G.C. & Wolfe, K.H. Turning a hobby into a job: how duplicated genes find new functions. Nat. Rev. Genet. 9, 938–950 (2008).

    Article  CAS  Google Scholar 

  13. Innan, H. & Kondrashov, F. The evolution of gene duplications: classifying and distinguishing between models. Nat. Rev. Genet. 11, 97–108 (2010).

    Article  CAS  Google Scholar 

  14. Khersonsky, O. & Tawfik, D.S. Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu. Rev. Biochem. 79, 471–505 (2010).

    Article  CAS  Google Scholar 

  15. Hedstrom, L. IMP dehydrogenase: structure, mechanism and inhibition. Chem. Rev. 109, 2903–2928 (2009).

    Article  CAS  Google Scholar 

  16. Weber, G., Nakamura, H., Natsumeda, Y., Szekeres, T. & Nagai, M. Regulation of GTP biosynthesis. Adv. Enzyme Regul. 32, 57–69 (1992).

    Article  CAS  Google Scholar 

  17. Gan, L., Petsko, G.A. & Hedstrom, L. Crystal structure of a ternary complex of Tritrichomonas foetus inosine 5′-monophosphate dehydrogenase: NAD+ orients the active site loop for catalysis. Biochemistry 41, 13309–13317 (2002).

    Article  CAS  Google Scholar 

  18. Prosise, G.L. & Luecke, H. Crystal structures of Tritrichomonas foetus inosine monophosphate dehydrogenase in complex with substrate, cofactor and analogs: a structural basis for the random-in ordered-out kinetic mechanism. J. Mol. Biol. 326, 517–527 (2003).

    Article  CAS  Google Scholar 

  19. Colby, T.D., Vanderveen, K., Strickler, M.D., Markham, G.D. & Goldstein, B.M. Crystal structure of human type II inosine monophosphate dehydrogenase: implications for ligand binding and drug design. Proc. Natl. Acad. Sci. USA 96, 3531–3536 (1999).

    Article  CAS  Google Scholar 

  20. Riera, T.V., Wang, W., Josephine, H.R. & Hedstrom, L. A kinetic alignment of orthologous inosine-5′-monophosphate dehydrogenases. Biochemistry 47, 8689–8696 (2008).

    Article  CAS  Google Scholar 

  21. Gan, L. et al. The immunosuppressive agent mizoribine monophosphate forms a transition state analog complex with IMP dehydrogenase. Biochemistry 42, 857–863 (2003).

    Article  CAS  Google Scholar 

  22. Guillén Schlippe, Y.V., Riera, T.V., Seyedsayamdost, M.R. & Hedstrom, L. Substitution of the conserved Arg-Tyr dyad selectively disrupts the hydrolysis phase of the IMP dehydrogenase reaction. Biochemistry 43, 4511–4521 (2004).

    Article  Google Scholar 

  23. Guillén Schlippe, Y.V. & Hedstrom, L. Is Arg418 the catalytic base required for the hydrolysis step of the IMP dehydrogenase reaction? Biochemistry 44, 11700–11707 (2005).

    Article  Google Scholar 

  24. Min, D. et al. An enzymatic atavist revealed in dual pathways for water activation. PLoS Biol. 6, e206 (2008).

    Article  Google Scholar 

  25. Li, J. et al. Crystal structure of human guanosine monophosphate reductase 2 (GMPR2) in complex with GMP. J. Mol. Biol. 355, 980–988 (2006).

    Article  CAS  Google Scholar 

  26. Spector, T., Jones, T.E. & Miller, R.L. Reaction mechanism and specificity of human GMP reductase. Substrates, inhibitors, activators, and inactivators. J. Biol. Chem. 254, 2308–2315 (1979).

    CAS  PubMed  Google Scholar 

  27. Deng, Y. et al. NADPH-dependent GMP reductase isoenzyme of human (GMPR2). Expression, purification, and kinetic properties. Int. J. Biochem. Cell Biol. 34, 1035–1050 (2002).

    Article  CAS  Google Scholar 

  28. Martinelli, L.K. et al. Recombinant Escherichia coli GMP reductase: kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme-ligand binary complex formation. Mol. Biosyst. 7, 1289–1305 (2011).

    Article  CAS  Google Scholar 

  29. Bellamacina, C.R. The nicotinamide dinucleotide binding motif: a comparison of nucleotide binding proteins. FASEB J. 10, 1257–1269 (1996).

    Article  CAS  Google Scholar 

  30. Hughes, R., Magee, E.A. & Bingham, S. Protein degradation in the large intestine: relevance to colorectal cancer. Curr. Issues Intest. Microbiol. 1, 51–58 (2000).

    CAS  PubMed  Google Scholar 

  31. Roberts, R.E., Lienhard, C.I., Gaines, C.G., Smith, J.M. & Guest, J.R. Genetic and molecular characterization of the guaC-nadC-aroP region of Escherichia coli K-12. J. Bacteriol. 170, 463–467 (1988).

    Article  CAS  Google Scholar 

  32. Macpherson, I.S. et al. The structural basis of Cryptosporidium-specific IMP dehydrogenase inhibitor selectivity. J. Am. Chem. Soc. 132, 1230–1231 (2010).

    Article  CAS  Google Scholar 

  33. Talfournier, F., Pailot, A., Stines-Chaumeil, C. & Branlant, G. Stabilization and conformational isomerization of the cofactor during the catalysis in hydrolytic ALDHs. Chem. Biol. Interact. 178, 79–83 (2009).

    Article  CAS  Google Scholar 

  34. Minegishi, S. & Mayr, H. How constant are Ritchie′s “constant selectivity relationships”? A general reactivity scale for n-, pi-, and sigma-nucleophiles. J. Am. Chem. Soc. 125, 286–295 (2003).

    Article  CAS  Google Scholar 

  35. Musa-Aziz, R., Chen, L.M., Pelletier, M.F. & Boron, W.F. Relative CO2/NH3 selectivities of AQP1, AQP4, AQP5, AmtB, and RhAG. Proc. Natl. Acad. Sci. USA 106, 5406–5411 (2009).

    Article  CAS  Google Scholar 

  36. Ji, R. & Brune, A. Nitrogen mineralization, ammonia accumulation, and emission of gaseous NH3 by soil-feeding termites. Biogeochemistry 78, 267–283 (2006).

    Article  Google Scholar 

  37. van Ham, R.C. et al. Reductive genome evolution in Buchnera aphidicola. Proc. Natl. Acad. Sci. USA 100, 581–586 (2003).

    Article  CAS  Google Scholar 

  38. Hansen, A.K. & Moran, N.A. Aphid genome expression reveals host-symbiont cooperation in the production of amino acids. Proc. Natl. Acad. Sci. USA 108, 2849–2854 (2011).

    Article  CAS  Google Scholar 

  39. Zahnle, K., Schaefer, L. & Fegley, B. Earth's earliest atmospheres. Cold Spring Harb. Perspect. Biol. 2, a004895 (2010).

    Article  CAS  Google Scholar 

  40. Kim, H.S., Mittenthal, J.E. & Caetano-Anolles, G. MANET: tracing evolution of protein architecture in metabolic networks. BMC Bioinformatics 7, 351 (2006).

    Article  Google Scholar 

  41. Zhang, R. et al. Differential signatures of bacterial and mammalian IMP dehydrogenase enzymes. Curr. Med. Chem. 6, 537–543 (1999).

    CAS  PubMed  Google Scholar 

  42. Leslie, A.G.W. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 and ESF-EAMCB Newsl. Protein Crystallogr. 26, (1992).

  43. Kabsch, W. Evaluation of single-crystal X-ray diffraction data from a position-sensitive detector. J. Appl. Crystallogr. 21, 916–924 (1988).

    Article  CAS  Google Scholar 

  44. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  45. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  47. Frisch, M.J. et al. Gaussian 03, Revision C.02. (ed. Gaussian, I.) (2004).

  48. Brady, K. & Abeles, R.H. Inhibition of chymotrypsin by peptidyl trifluoromethyl ketones: determinants of slow-binding kinetics. Biochemistry 29, 7608–7617 (1990).

    Article  CAS  Google Scholar 

  49. Zhang, R. et al. Characteristics and crystal structure of bacterial inosine-5′-monophosphate dehydrogenase. Biochemistry 38, 4691–4700 (1999).

    Article  CAS  Google Scholar 

  50. Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Andrews at the University of Sheffield for supplying the pUC plasmid carrying the E. coli K12 GMPR gene. We also are grateful for construction of pET28a GMPR by I.S. MacPherson. We also thank the staff at BESSY (Berlin, Germany) for support at beamline BL14.1. This work was supported by the US National Institutes of Health (NIH) GM54403 (L.H.) and by grants from the Swedish Research Council (P.N. and P.S.), the Swedish Cancer Society (P.N.), the Wenner-Gren Foundations (P.S.) and the Swedish Foundation for Strategic Research (P.S.). The Q-Tof Ultima mass spectrometer at the University of Illinois was purchased in part with a grant from the National Science Foundation, Division of Biological Infrastructure (DBI-0100085). Molecular graphics images were produced using the University of California, San Fransisco (UCSF) Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the UCSF (supported by NIH P41 RR001081). The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust.

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  1. Petri Kursula

    Present address: Current address: Department of Biochemistry, University of Oulu, Oulu, Finland.,

Authors and Affiliations

  1. Department of Biology, Brandeis University, Waltham, Massachusetts, USA

    Gregory C Patton, Deviprasad R Gollapalli & Lizbeth Hedstrom

  2. Department of Medical Biochemistry and Biophysics, Structural Genomics Consortium, The Karolinska Institute, Stockholm, Sweden

    Pål Stenmark, Petri Kursula, Susanne Flodin, Herwig Schuler & Pär Nordlund

  3. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

    Pål Stenmark

  4. Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, Stockholm, Sweden

    Robin Sevastik & Fahmi Himo

  5. Department of Chemistry, Brandeis University, Waltham, Massachusetts, USA

    Colin T Swales & Lizbeth Hedstrom

  6. Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Uppsala, Sweden

    Hans Eklund

  7. School of Biological Sciences, Nanyang Technological University, Singapore

    Pär Nordlund

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Contributions

G.C.P., D.R.G., C.T.S. and L.H. conceived and designed the mechanism experiments. G.C.P., D.R.G. and C.T.S. performed and analyzed these experiments. P.S., H.S. and P.N. conceived and designed the structural experiments. P.S., H.S., S.F. and P.K. performed these experiments, and P.S., H.S., P.K., P.N. and H.E. analyzed the data. R.S. and F.H. conceived, performed and analyzed DFT computations. L.H., G.C.P., H.E., P.S. and P.N. wrote the paper.

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Correspondence to Pär Nordlund or Lizbeth Hedstrom.

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Patton, G., Stenmark, P., Gollapalli, D. et al. Cofactor mobility determines reaction outcome in the IMPDH and GMPR (β-α)8 barrel enzymes. Nat Chem Biol 7, 950–958 (2011). https://doi.org/10.1038/nchembio.693

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