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Ground-state destabilization by electrostatic repulsion is not a driving force in orotidine-5′-monophosphate decarboxylase catalysis

Nature Catalysis volume 5, pages 332–341 (2022)Cite this article

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Abstract

The origins of enzyme catalysis have been attributed to both transition-state stabilization as well as ground-state destabilization of the substrate. For the latter paradigm, the enzyme orotidine-5′-monophosphate decarboxylase (OMPDC) serves as a reference system as it contains a negatively charged residue at the active site that is thought to facilitate catalysis by exerting an electrostatic stress on the substrate carboxylate leaving group. Snapshots of how the substrate binds to the active site and interacts with the negative charge have remained elusive. Here we present crystallographic snapshots of human OMPDC in complex with the substrate, substrate analogues, transition-state analogues and product that defy the proposed ground-state destabilization by revealing that the substrate carboxylate is protonated and forms a favourable low-barrier hydrogen bond with a negatively charged residue. The catalytic prowess of OMPDC almost entirely results from the transition-state stabilization by electrostatic interactions of the enzyme with charges spread over the substrate. Our findings bear relevance for the design of (de)carboxylase catalysts.

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Fig. 1: Proposed mechanism and catalytic strategies of OMPDC.
Fig. 2: Structural snapshots of substrate binding in OMPDC.
Fig. 3: Structure of the transition-state analogue BMP bound to human OMPDC.
Fig. 4: Structure of the product UMP bound to human OMPDC and implications for the mechanism.

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Data availability

The refined structural protein models and corresponding structure-factor amplitudes are deposited under PDB accession codes 7OUZ (wild-type, BMP, crystal 1), 7OTU (wild-type, BMP, crystal 2), 6ZWY (wild-type, UMP, crystal 1), 7ASQ (wild-type, UMP, crystal 2), 6ZX1 (wild-type, aza-UMP), 7OV0 (wild-type, resting state), 6ZX2 (carboxamido-UMP), 6ZX3 (thiocarboxamido-UMP), 6ZWZ (variant K314AcK, resting state), 6YWU (variant K314AcK, UMP), 6YVK (variant K314AcK, 2 min soaking with OMP, 0.71 MGy dose), 6YVL (variant K314AcK, 2 min soaking with OMP, 1.42 MGy dose), 6YVM (variant K314AcK, 2 min soaking with OMP, 2.13 MGy dose), 6YVN (variant K314AcK, 2 min soaking with OMP, 2.84 MGy dose), 6YVO (variant K314AcK, 2 min soaking with OMP, 3.55 MGy dose), 6YWT (variant K314AcK, BMP), 7OQF (variant K314AcK, 5 min soaking with OMP), 7OQI (variant K314AcK, 10 min soaking with OMP), 7OQK (variant K314AcK, 15 min soaking with OMP), 7OQM (variant K314AcK, 20 min soaking with OMP), 7OQN (variant K314AcK, 30 min soaking with OMP), 7AM9 (variant K314AcK, 2 min soaking with OMP, merged dataset), 6ZX0 (variant K314AcK, OMP), 7Q1H (variant D312N, 2 min soaking with OMP) (Supplementary Table 1). The results of the quantum chemical calculations have been deposited in the GRO.data repository at https://doi.org/10.25625/6OOHE5. All other data are available on request.

References

  1. Radzicka, A. & Wolfenden, R. A proficient enzyme. Science 267, 90–93 (1995).

    Article  CAS  PubMed  Google Scholar 

  2. Lee, J. K. & Houk, K. N. A proficient enzyme revisited: the predicted mechanism for orotidine monophosphate decarboxylase. Science 276, 942–945 (1997).

    Article  CAS  PubMed  Google Scholar 

  3. Miller, B. G. & Wolfenden, R. Catalytic proficiency: the unusual case of OMP decarboxylase. Annu. Rev. Biochem. 71, 847–885 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Richard, J. P., Amyes, T. L. & Reyes, A. C. Orotidine 5′-monophosphate decarboxylase: probing the limits of the possible for enzyme catalysis. Acc. Chem. Res. 51, 960–969 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wu, N., Mo, Y., Gao, J. & Pai, E. F. Electrostatic stress in catalysis: structure and mechanism of the enzyme orotidine monophosphate decarboxylase. Proc. Natl Acad. Sci. USA 97, 2017–2022 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Appleby, T. C., Kinsland, C., Begley, T. P. & Ealick, S. E. The crystal structure and mechanism of orotidine 5′-monophosphate decarboxylase. Proc. Natl Acad. Sci. USA 97, 2005–2010 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Miller, B. G., Hassell, A. M., Wolfenden, R., Milburn, M. V. & Short, S. A. Anatomy of a proficient enzyme: the structure of orotidine 5′-monophosphate decarboxylase in the presence and absence of a potential transition state analog. Proc. Natl Acad. Sci. USA 97, 2011–2016 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Harris, P., Navarro Poulsen, J. C., Jensen, K. F. & Larsen, S. Structural basis for the catalytic mechanism of a proficient enzyme: orotidine 5′-monophosphate decarboxylase. Biochemistry 39, 4217–4224 (2000).

    Article  CAS  PubMed  Google Scholar 

  9. Amyes, T. L., Wood, B. M., Chan, K., Gerlt, J. A. & Richard, J. P. Formation and stability of a vinyl carbanion at the active site of orotidine 5′-monophosphate decarboxylase: pKa of the C-6 proton of enzyme-bound UMP. J. Am. Chem. Soc. 130, 1574–1575 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chan, K. K. et al. Mechanism of the orotidine 5′-monophosphate decarboxylase-catalyzed reaction: evidence for substrate destabilization. Biochemistry 48, 5518–5531 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Jencks, W. P. Binding energy, specificity, and enzymic catalysis: the Circe effect. Adv. Enzymol. Relat. Areas Mol. Biol. 43, 219–410 (1975).

    CAS  PubMed  Google Scholar 

  12. Warshel, A. & Florián, J. Computer simulations of enzyme catalysis: finding out what has been optimized by evolution. Proc. Natl.# Acad. Sci. USA 95, 5950–5955 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Warshel, A., Štrajbl, M., Villa, J. & Florián, J. Remarkable rate enhancement of orotidine 5′-monophosphate decarboxylase is due to transition-state stabilization rather than to ground-state destabilization. Biochemistry 39, 14728–14738 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Warshel, A., Florián, J., Štrajbl, M. & Villà, J. Circe effect versus enzyme preorganization: what can be learned from the structure of the most proficient enzyme? ChemBioChem 2, 109–111 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Goryanova, B., Amyes, T. L. & Richard, J. P. Role of the carboxylate in enzyme-catalyzed decarboxylation of orotidine 5′-monophosphate: transition state stabilization dominates over ground state destabilization. J. Am. Chem. Soc. 141, 13468–13478 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wu, N., Gillon, W. & Pai, E. F. Mapping the active site−ligand interactions of orotidine 5′-monophosphate decarboxylase by crystallography. Biochemistry 41, 4002–4011 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Wittmann, J. G. et al. Structures of the human orotidine-5′-monophosphate decarboxylase support a covalent mechanism and provide a framework for drug design. Structure 16, 82–92 (2008).

    Article  CAS  PubMed  Google Scholar 

  18. Neumann, P. & Tittmann, K. Marvels of enzyme catalysis at true atomic resolution: distortions, bond elongations, hidden flips, protonation states and atom identities. Curr. Opin. Struct. Biol. 29, 122–133 (2014).

    Article  CAS  PubMed  Google Scholar 

  19. Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C. & Schultz, P. G. A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244, 182–188 (1989).

    Article  CAS  PubMed  Google Scholar 

  20. Takusagawa, F. & Shimada, A. The crystal structure of orotic acid monohydrate (vitamin B13). Bull. Chem. Soc. Jpn 46, 2011–2019 (1973).

    Article  CAS  Google Scholar 

  21. Lüdtke, S. et al. Sub-ångström-resolution crystallography reveals physical distortions that enhance reactivity of a covalent enzymatic intermediate. Nat. Chem. 5, 762–767 (2013).

    Article  PubMed  CAS  Google Scholar 

  22. Asztalos, P. et al. Strain and near attack conformers in enzymic thiamin catalysis: X-ray crystallographic snapshots of bacterial transketolase in covalent complex with donor ketoses xylulose 5-phosphate and fructose 6-phosphate, and in noncovalent complex with acceptor aldose ribose 5-phosphate. Biochemistry 46, 12037–12052 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Gurusaran, M., Shankar, M., Nagarajan, R., Helliwell, J. R. & Sekar, K. Do we see what we should see? Describing non-covalent interactions in protein structures including precision. IUCrJ 1, 74–81 (2014).

    Article  CAS  PubMed  Google Scholar 

  24. Owen, R. L., Rudiño-Piñera, E. & Garman, E. F. Experimental determination of the radiation dose limit for cryocooled protein crystals. Proc. Natl Acad. Sci. USA 103, 4912–4917 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Frank, R. A., Titman, C. M., Pratap, J. V., Luisi, B. F. & Perham, R. N. A molecular switch and proton wire synchronize the active sites in thiamine enzymes. Science 306, 872–876 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Dai, S. et al. Low-barrier hydrogen bonds in enzyme cooperativity. Nature 573, 609–613 (2019).

    Article  CAS  PubMed  Google Scholar 

  27. Rabe von Pappenheim, F. et al. Structural basis for antibiotic action of the B1 antivitamin 2′-methoxy-thiamine. Nat. Chem. Biol. 16, 1237–1245 (2020).

    Article  CAS  PubMed  Google Scholar 

  28. Kluger, R. Decarboxylation, CO2 and the reversion problem. Acc. Chem. Res. 48, 2843–2849 (2015).

    Article  CAS  PubMed  Google Scholar 

  29. Kong, D., Moon, P. J., Lui, E. K., Bsharat, O. & Lundgren, R. J. Direct reversible decarboxylation from stable organic acids in dimethylformamide solution. Science 369, 557–561 (2020).

    Article  CAS  PubMed  Google Scholar 

  30. Desai, B. J. et al. Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution. Proc. Natl Acad. Sci. USA 111, 15066–15071 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Jencks, W. P. When is an intermediate not an intermediate? Enforced mechanisms of general acid–base, catalyzed, carbocation, carbanion, and ligand exchange reaction. Acc. Chem. Res. 13, 161–169 (1980).

    Article  CAS  Google Scholar 

  32. Schwander, T., von Borzyskowski, L. S., Burgener, S., Cortina, N. S. & Erb, T. J. A synthetic pathway for the fixation of carbon dioxide in vitro. Science 354, 900–904 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Stoffel, G. M. et al. Four amino acids define the CO2 binding pocket of enoyl-CoA carboxylases/reductases. Proc. Natl Acad. Sci. USA 116, 13964–13969 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Whitney, S. M., Houtz, R. L. & Alonso, H. Advancing our understanding and capacity to engineer nature’s CO2-sequestering enzyme, Rubisco. Plant Phys. 155, 27–35 (2011).

    Article  CAS  Google Scholar 

  35. Zhou, S., Nguyen, B. T., Richard, J. P., Kluger, R. & Gao, J. Origin of free energy barriers of decarboxylation and the reverse process of CO2 capture in dimethylformamide and in water. J. Am. Chem. Soc. 143, 137–141 (2021).

    Article  CAS  PubMed  Google Scholar 

  36. Ho, M. C., Ménétret, J. F., Tsuruta, H. & Allen, K. N. The origin of the electrostatic perturbation in acetoacetate decarboxylase. Nature 459, 393–397 (2009).

    Article  CAS  PubMed  Google Scholar 

  37. Fujihashi, M. et al. Substrate distortion contributes to the catalysis of orotidine 5′-monophosphate decarboxylase. J. Am. Chem. Soc. 135, 17432–17443 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Warshel, A. et al. Electrostatic basis for enzyme catalysis. Chem. Rev. 106, 3210–3235 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Warshel, A. Electrostatic origin of the catalytic power of enzymes and the role of preorganized active sites. J. Biol. Chem. 273, 27035–27038 (1998).

    Article  CAS  PubMed  Google Scholar 

  40. Prah, A., Frančišković, E., Mavri, J. & Stare, J. Electrostatics as the driving force behind the catalytic function of the monoamine oxidase A enzyme confirmed by quantum computations. ACS Catal. 9, 1231–1240 (2019).

    Article  CAS  Google Scholar 

  41. Pauling, L. Nature of forces between large molecules of biological interest. Nature 161, 707–709 (1948).

    Article  CAS  PubMed  Google Scholar 

  42. Wille, G. et al. The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography. Nat. Chem. Biol. 2, 324–328 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Meyer, D. et al. Double duty for a conserved glutamate in pyruvate decarboxylase: evidence of the participation in stereoelectronically controlled decarboxylation and in protonation of the nascent carbanion/enamine intermediate. Biochemistry 49, 8197–8212 (2010).

    Article  CAS  PubMed  Google Scholar 

  44. Kluger, R. & Tittmann, K. Thiamin diphosphate catalysis: enzymic and nonenzymic covalent intermediates. Chem. Rev. 108, 1797–1833 (2008).

    Article  CAS  PubMed  Google Scholar 

  45. Wittmann, J. G. & Rudolph, M. G. Pseudo-merohedral twinning in monoclinic crystals of human orotidine-5′-monophosphate decarboxylase. Acta Crystallogr. D 63, 744–749 (2007).

    Article  CAS  PubMed  Google Scholar 

  46. Studier, F. W. Protein production by auto-induction in high-density shaking cultures. Prot. Expres Purif. 41, 207–234 (2005).

    Article  CAS  Google Scholar 

  47. Gasteiger, E. et al. in The Proteomics Protocols Handbook (ed. Walker, J. M.) 571–607 (Springer, 2005).

  48. Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Genetically encoding Nε-acetyllysine in recombinant proteins. Nat. Chem. Biol. 4, 232–234 (2008).

    Article  CAS  PubMed  Google Scholar 

  49. Poduch, E. et al. Design of inhibitors of orotidine monophosphate decarboxylase using bioisosteric replacement and determination of inhibition kinetics. J. Med. Chem. 49, 4937–4945 (2006).

    Article  CAS  PubMed  Google Scholar 

  50. Keller, S. et al. High-precision isothermal titration calorimetry with automated peak-shape analysis. Anal. Chem. 84, 5066–5073 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Houtman, J. C. et al. Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: application to adaptor protein complexes in cell signaling. Protein Sci. 16, 30–42 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Zhao, H., Piszczek, G. & Schuck, P. SEDPHAT—a platform for global ITC analysis and global multi-method analysis of molecular interactions. Methods 76, 137–148 (2015).

    Article  CAS  PubMed  Google Scholar 

  53. Pemberton, T. A. et al. Proline: mother nature’s cryoprotectant applied to protein crystallography. Acta Crystallogr. D 68, 1010–1018 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Vonrhein, C. et al. Advances in automated data analysis and processing within autoPROC, combined with improved characterisation, mitigation and visualisation of the anisotropy of diffraction limits using STARANISO. Acta Crystallogr. A 74, a360 (2018).

    Article  Google Scholar 

  56. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).

    Article  CAS  PubMed  Google Scholar 

  59. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  61. The PyMOL Molecular Graphics System v.2.0 (Schrödinger, LLC).

  62. Vosko, S. H., Wilk, L. & Nusair, M. Accurate spin-dependent electron liquid correlation energies for local spin density calculations: a critical analysis. Can. J. Phys. 58, 1200–1211 (1980).

    Article  CAS  Google Scholar 

  63. Becke, A. D. Density‐functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652 (1993).

    Article  CAS  Google Scholar 

  64. Lee, C., Yang, W. & Parr, R. G. Development of the Colle–Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B 37, 785–789 (1988).

    Article  CAS  Google Scholar 

  65. Grimme, S., Antony, J., Ehrlich, S. & Krieg, H. A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H–Pu. J. Chem. Phys. 132, 154104 (2010).

    Article  PubMed  CAS  Google Scholar 

  66. Grimme, S., Ehrlich, S. & Goerigk, L. Effect of the damping function in dispersion corrected density functional theory. J. Comp. Chem. 32, 1456–1465 (2011).

    Article  CAS  Google Scholar 

  67. Weigend, F. & Ahlrichs, R. Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: design and assessment of accuracy. Phys. Chem. Chem. Phys. 7, 3297–3305 (2005).

    Article  CAS  PubMed  Google Scholar 

  68. Tomasi, J., Mennucci, B. & Cammi, R. Quantum mechanical continuum solvation models. Chem. Rev. 105, 2999–3094 (2005).

    Article  CAS  PubMed  Google Scholar 

  69. Frisch, M. J. et al. Gaussian 16 Revision A.03 (Gaussian, Inc., 2016).

  70. Neese, F. Software update: the ORCA program system, version 4.0. WIREs Comp. Mol. Sci. 8, e1327 (2018).

    Article  Google Scholar 

  71. Weigend, F. Accurate Coulomb-fitting basis sets for H to Rn. Phys. Chem. Chem. Phys. 8, 1057–1065 (2006).

    Article  CAS  PubMed  Google Scholar 

  72. Case, D. et al. AMBER 18 (Univ. California, 2018).

  73. Metz, S., Kästner, J., Sokol, A. A., Keal, T. W. & Sherwood, P. CHEMSHELL—a modular software package for QM/MM simulations. WIREs Comp. Mol. Sci. 4, 101–110 (2014).

    Article  CAS  Google Scholar 

  74. Maier, J. A. Improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Wang, J., Wang, W., Kollman, P. A. & Case, D. A. Automatic atom type and bond type perception in molecular mechanical calculations. J. Mol. Graph. Model. 25, 247–260 (2006).

    Article  PubMed  CAS  Google Scholar 

  76. Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general AMBER force field. J. Comput. Chem. 25, 1157–1174 (2004).

    Article  CAS  PubMed  Google Scholar 

  77. Bayly, C. I., Cieplak, P., Cornell, W. & Kollman, P. A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem. 97, 10269–10280 (1993).

    Article  CAS  Google Scholar 

  78. Gaus, M., Goez, A. & Elstner, M. Parametrization and benchmark of DFTB3 for organic molecules. J. Chem. Theory Comput. 9, 338–354 (2013).

    Article  CAS  PubMed  Google Scholar 

  79. Grossfield, A. WHAM: the weighted histogram analysis method v.2.0.9. (University of Rochester Medical Center, accessed 15 November 2013); http://membrane.urmc.rochester.edu/?page_id=126

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Acknowledgements

This study was supported by the Max-Planck Society and the DFG-funded Göttingen Graduate Center for Neurosciences, Biophysics, and Molecular Biosciences GGNB. We acknowledge access to beamline P14 at DESY/EMBL. We thank M. Rudolph for providing a plasmid for recombinant expression of human OMPDC and H. Neumann for providing the AMBER suppression system for the expression of acetyllysine residues. We thank J. Chin, G. Howe and A. Pearson for discussion. We thank G. Bricogne and his team from Global Phasing Limited for discussion regarding anisotropic data processing.

Author information

Author notes

  1. These authors contributed equally: Sören Rindfleisch, Matthias Krull, Jon Uranga.

  2. This publication is dedicated to our collaborator, colleague and friend Ulf Diederichsen (1963–2021).

Authors and Affiliations

  1. Department of Molecular Enzymology, Göttingen Center of Molecular Biosciences, Georg-August University Göttingen, Göttingen, Germany

    Sören Rindfleisch, Fabian Rabe von Pappenheim, Laura Liliana Kirck & Kai Tittmann

  2. Department of Structural Dynamics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany

    Sören Rindfleisch, Fabian Rabe von Pappenheim, Laura Liliana Kirck, Ashwin Chari & Kai Tittmann

  3. Institute for Organic and Biomolecular Chemistry, Georg-August University Göttingen, Göttingen, Germany

    Matthias Krull, Tobias Schmidt & Ulf Diederichsen

  4. Institute of Physical Chemistry, Georg-August University Göttingen, Göttingen, Germany

    Jon Uranga, Angeliki Balouri & Ricardo A. Mata

  5. European Molecular Biology Laboratory (EMBL), Hamburg Outstation c/o Deutsches Elektronen Synchrotron (DESY), Hamburg, Germany

    Thomas Schneider & Gleb Bourenkov

  6. Department of Chemistry, University of Toronto, Ontario, Canada

    Ronald Kluger

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Contributions

K.T., R.A.M. and U.D. designed and coordinated the project. S.R. expressed, purified, crystallized and enzymatically characterized proteins under the supervision of K.T. S.R. collected crystallographic datasets with support from A.C., G.B. and T.S. S.R. and F.R.v.P. refined the structures with support from A.C., G.B., T.S. and K.T. L.L.K. expressed, purified and crystallized variant Asp312Asn. S.R., F.R.v.P. and K.T. interpreted the crystallographic data. J.U. carried out the electronic structure calculations and molecular dynamics calculations. A.B. carried out molecular dynamics calculations under the supervision of J.U. J.U. and R.A.M. interpreted the calculations. M.K. and T.S. chemically synthesized the substrate and transition-state analogues under the supervision of U.D. S.R., J.U., R.K., U.D, R.A.M. and K.T. discussed the enzymatic reaction mechanism. S.R., J.U., T.S., R.A.M. and K.T. wrote the paper with input from all the other authors.

Corresponding authors

Correspondence to Ricardo A. Mata or Kai Tittmann.

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Extended data

Extended Data Fig. 1 Suggested mechanisms of OMPDC-catalysed decarboxylation of OMP.

Suggested mechanisms of OMPDC-catalysed decarboxylation of OMP. (a) protonation at 2-oxo, (b) attack of a nucleophile at C5, (c) protonation at 4-oxo, (d) electrophilic displacement, (e) protonation at C5. Scheme adapted from3. The currently accepted mechanism invoking a combination of transition-state stabilisation and ground-state destabilisation is shown in Fig. 1a of the main manuscript. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate.

Extended Data Fig. 2 Structure of human OMPDC variant Lys314Ac-Lys in complex with product UMP at a resolution of 1.10 Å.

Structure of human OMPDC variant Lys314Ac-Lys in complex with product UMP at a resolution of 1.10 Å. (a) Structure of the active site showing the bound product and interacting protein groups. UMP and mutated residue AcLys314 are highlighted in yellow color. The structural model is superposed with the corresponding 2mF-DFc electron density map at a contour level of 4σ (UMP: blue, protein residues: grey). H-bonding interactions are indicated. (b) Superposition of the active sites of human OMPDC wild-type in complex with UMP (green, this study) with variant Lys314AcLys (yellow). Shown are the bound product UMP and interacting protein groups. Note the conserved binding mode of the product and interactions with protein groups except for a flip of the Cδ-Cε bond of the Lys side-chain in the variant. (c) Superposition of the backbone of human OMPDC wild-type in complex with UMP (green, this study) with variant Lys314AcLys (yellow). The RMSD of the Cα carbons amounts to 0.15 Å. Crystallographic statistics are provided in Supplementary Table 1. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate; UMP, uridine-5’-monophosphate; RMSD, root mean square deviation.

Extended Data Fig. 3 Structures of human OMPDC wild-type in complex with substrate analogs.

Structures of human OMPDC wild-type in complex with substrate analogs. (a) Structure of human OMPDC in complex with substrate analog 6-thio-carboxyamido-UMP showing the active site with the bound analog and interacting protein groups. The structural model of the analog is super-posed with the corresponding 2mFo-DFc electron density map at a contour level of 3.3σ. Hydrogen-bonding interactions are indicated. Note the H-bond interaction between the N7 atom of the analog with Asp312 similar to the genuine substrate (see Fig. 2a of the main manuscript). (b) Physical distortion of the C6-C7 bond of the analog relative to the base ring plane shown in grey and tilt of the thio-carboxamido plane relative to the base ring plane shown in grey. (c) Structure of human OMPDC in complex with substrate analog 6-carboxyamido-UMP showing the active site with the bound analog and interacting protein groups. The structural model of the analog is superposed with the corresponding 2mFo-DFc electron density map at a contour level of 3σ. Hydrogen-bonding interactions are indicated. Note the H-bond interaction between the N7 atom of the analog with Asp312 similar to the genuine substrate (see Fig. 2a of the main manuscript). (d) Physical distortion of the C6-C7 bond of the analog relative to the base ring plane shown in grey and tilt of the carboxamido plane relative to the base ring plane shown in grey. Crystallographic statistics are provided in Supplementary Table 1. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase.

Extended Data Fig. 4 Dose-dependent structure analysis of human OMPDC in complex with substrate OMP.

Dose-dependent structure analysis of human OMPDC in complex with substrate OMP. Datasets of crystals of OMPDC variant Lys31AcLys soaked with OMP for 2 min were collected at beamline P14 at DESY/EMBL Hamburg depositing calibrated doses as indicated. The refined structural models of bound substrate and interacting residue Asp312 are shown for both active sites of the homodimer. The structural models are superposed with the corresponding 2mFo-DFc electron density maps at a contour level of 1σ. No evidence for a radiation-induced decarboxylation of either the substrate or side chain of Asp312 was obtained. Refinements were carried out in space group P21 (containing a functional homodimer in the asymmetric unit) for all datasets. Crystallographic statistics are provided in Supplementary Table 1. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate.

Extended Data Fig. 5 Time-resolved structural snapshots of OMPDC-catalysed conversion of substrate OMP into product UMP.

Time-resolved structural snapshots of OMPDC-catalysed conversion of substrate OMP into product UMP. Crystals of human OMPDC variant Lys314AcLys were soaked with substrate OMP for different reaction times as indicated ranging from 2–30 min at 6 °C. The refined structural models of bound substrate and interacting residue Asp312 are shown for both active sites of the homodimer. The structural models are superposed with the corresponding 2mFo-DFc electron density maps at a contour level of 1σ (in blue). The refined occupancies of substrate OMP are shown. The difference electron density relative to the dataset obtained at 2 min is shown for all later datasets at a contour level of 3σ in red colour indicating the progressive decarboxylation of substrate OMP over time. Refinements were carried out in space group P21 (containing a functional homodimer in the asymmetric unit) for all datasets. Crystallographic statistics are provided in Supplementary Table 1. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate; UMP, uridine-5’-monophosphate.

Extended Data Fig. 6 Detection of cooperativity in human OMPDC catalysis.

Detection of cooperativity in human OMPDC catalysis. (a) Steady-state kinetic analysis of OMPDC-catalysed conversion of OMP into UMP by isothermal titration calorimetry showing the raw data thermogram after a single injection of 0.2 mM OMP to a solution containing 1 µM OMPDC in 20 mM HEPES/NaOH, pH 7.4 at 25 °C and the therefrom calculated v_S plot. The data were fitted with the Hill equation (fit shown in red). Note the estimated Hill coefficient of nH = 2.0 that indicates positive cooperativity between the two active sites in the homodimeric enzyme. All measurements were carried out in triplicate and are shown as mean ± s.d. (b) Thermodynamic analysis of binding of product UMP to human OMPDC using isothermal titration calorimetry showing the raw data thermogram and the integrated heats. The data were fit with a two-site binding model as detailed in the methods section (fit shown in red). The observation of two binding sites suggests a negative cooperativity between the two active sites in the homodimeric enzyme. All experiments were carried out as triplicates with almost identical results. The fitted kinetic and thermodynamic constants along with the associated calculated standard deviation are shown for a representative experiment. (c) Putative communication wires in human OMPCase that link the two remote active sites of the homodimer. Shown is the structure of human OMPDC variant Lys314AcLys in complex with substrate OMP highlighting the two active sites with the bound substrate molecules and two potential signaling pathways involving Asp317*-W1-W2-W3-W4-W5-Asp317 and/or His283-Glu311-Asp285 from both subunits and several water molecules. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate; UMP, uridine-5’-monophosphate.

Extended Data Fig. 7 Structure of human OMPDC in complex with transition-state analog BMP.

Structure of human OMPDC in complex with transition-state analog BMP. Close-up of the active site showing the local interactions of residue Asp317’. The structural model is superposed with the corresponding 2mFo-DFc electron density map (blue, contour level 5.3σ). Peaks in the H-omit mFo-DFc difference electron density map (magenta, contour level 3σ) indicate the positions of hydrogen atoms of the analog and interacting protein groups. The structural data suggest that the side chain Asp317’ is ionized and interacts with Lys314 (-NH3+), the backbone amide of Ile318’ and the 2’-OH group of BMP. Crystallographic statistics are provided in Supplementary Table 1. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; BMP, 6-hydroxy-UMP.

Extended Data Fig. 8 Structure of human OMPDC in complex with transition-state analog 6-aza-UMP.

Structure of human OMPDC in complex with transition-state analog 6-aza-UMP. (a) Close-up of the active site showing the bound analog, interacting protein groups and water molecules (W). Hydrogen bonds and the bond lengths of the C2-O2 and C4-O4 bonds are indicated. Note the syn-conformation of the base that places the 2-oxo group into the vicinity of the catalytic tetrad rather than Gln430 as observed for all other ligands, which bind in the anti-conformation (see panel b). The structural models are superposed with the corresponding 2mFo-DFc electron density maps at a contour level of 5.3σ (in blue). Crystallographic statistics are provided in Supplementary Table 1. (b) Structural superposition of OMPDC in complex with 6-aza-UMP (in yellow) and UMP (in grey) showing the active site including the bound ligand and selected active site residues. Note the different orientation of the the 2-oxo function for both ligands. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; UMP, uridine-5’-monophosphate.

Extended Data Fig. 9 Functional and structural analysis of human OMPDC variant Asp312Asn.

Functional and structural analysis of human OMPDC variant Asp312Asn. The variant exhibits no measurable enzymatic activity for conversion of OMP. (a) Thermodynamic analysis of binding of product UMP to variant Asp312Asn using isothermal titration calorimetry showing the raw data thermogram and the integrated heats (inset). The data were fit with a 1:1 binding model and yielded a dissociation constant of KDapp = 49 ± 6 µM and a stoichiometry N of 0.59 ± 0.01 indicative for half-of-the-sites reactivity. (b) Thermodynamic analysis of binding of substrate OMP to variant Asp312Asn using isothermal titration calorimetry showing the raw date thermogram and the integrated heats (inset). The data were fit with a two-sites binding model and yielded dissociation constants of KD1 = 0.58 ± 0.56 µM KD2 = 0.19 ± 0.09 µM indicative for positive cooperativity. (c) Structure of variant Asp312Asn in complex with substrate showing the active site with bound OMP and interacting residues. The structural model of OMP is superposed with the 2mFo-DFc electron density map (in blue) at a contour level of 2σ. Hydogen-bond interactions of OMP with active-site residues and relative occupancies for residues with alternative conformations are indicated. Note that the carboxylate portion of OMP is interacting with both Lys281 as well as Lys314 forming a stable (anticatalytic) enzyme: substrate complex as shown in the accompanying scheme. All experiments were carried out as triplicates with almost identical results. The fitted kinetic and thermodynamic constants along with the associated calculated standard deviation are shown for a representative experiment. Crystallographic statistics are provided in Supplementary Table 1. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate; UMP, uridine-5’-monophosphate.

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Rindfleisch, S., Krull, M., Uranga, J. et al. Ground-state destabilization by electrostatic repulsion is not a driving force in orotidine-5′-monophosphate decarboxylase catalysis. Nat Catal 5, 332–341 (2022). https://doi.org/10.1038/s41929-022-00771-w

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