Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Hydrolytic catalysis and structural stabilization in a designed metalloprotein

Nature Chemistry volume 4, pages 118–123 (2012)Cite this article

Subjects

Abstract

Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions—a Zn(II) ion, which is important for catalytic activity, and a Hg(II) ion, which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate (pNPA) hydrolysis with an efficiency only ~100-fold less than that of human carbonic anhydrase (CA)II and at least 550-fold better than comparable synthetic complexes. Similarly, CO2 hydration occurs with an efficiency within ~500-fold of CAII. Although histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO2 hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme reveals necessary design features for future metalloenzymes containing one or more metals.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Ribbon diagrams of the [Hg(II)]S[Zn(II)(H2O/OH−)]N(CSL9PenL23H)3n+ parallel 3SCC (one of two different three-helix bundles present in the asymmetric unit) at pH 8.5.
Figure 2: Overlay of the Zn(II)N3O site in [Hg(II)]S[Zn(II)(H2O/OH−)]N(CSL9PenL23H)3n+ with the active site of human CAII.
Figure 3: pH-dependency of pNPA hydrolysis by [Hg(II)]S[Zn(II)(H2O/OH−)]N(TRIL9CL23H)3n+.

Similar content being viewed by others

References

  1. Holm, R. H., Kennepohl, P. & Solomon, E. I. Structural and functional aspects of metal sites in biology. Chem. Rev. 96, 2239–2314 (1996).

    Article  CAS  Google Scholar 

  2. Lu, Y., Yeung, N., Sieracki, N. & Marshall, N. M. Design of functional metalloproteins. Nature 460, 855–862 (2009).

    Article  CAS  Google Scholar 

  3. Nanda, V. & Koder, R. Designing artificial enzymes by intuition and computation. Nature Chem. 2, 15–24 (2010).

    Article  CAS  Google Scholar 

  4. Kaplan, J. & DeGrado, W. F. De novo design of catalytic proteins. Proc. Natl Acad. Sci. USA 101, 11566–11570 (2004).

    Article  CAS  Google Scholar 

  5. Faiella, M. et al. An artificial di-iron oxo-protein with phenol oxidase activity. Nature Chem. Biol. 5, 882–884 (2009).

    Article  CAS  Google Scholar 

  6. Dieckmann, G. R. et al. The role of protonation and metal chelation preferences in defining the properties of mercury-binding coiled coils. J. Mol. Biol. 280, 897–912 (1998).

    Article  CAS  Google Scholar 

  7. Farrer, B. T., Harris, N. P., Balchus, K. E. & Pecoraro, V. L. Thermodynamic model for the stabilization of trigonal thiolato mercury(II) in designed three-stranded coiled coils. Biochemistry 40, 14696–14705 (2001).

    Article  CAS  Google Scholar 

  8. DeGrado, W. F., Summa, C. M., Pavone, V., Nastri, F. & Lombardi, A. De novo design and structural characterization of proteins and metalloproteins. Annu. Rev. Biochem. 68, 779–819 (1999).

    Article  CAS  Google Scholar 

  9. Peacock, A. F. A., Stuckey, J. A. & Pecoraro, V. L. Chirally switching metal coordination environments in designed peptides. Angew. Chem. Int. Ed. 48, 7371–7374 (2009).

    Article  CAS  Google Scholar 

  10. Handel, T. & DeGrado, W. F. De novo design of a Zn2+-binding protein. J. Am. Chem. Soc. 112, 6710–6711 (1990).

    Article  CAS  Google Scholar 

  11. Pessi, A. et al. A designed metal-binding protein with a novel fold. Nature 362, 367–369 (1993).

    Article  CAS  Google Scholar 

  12. Kiyokawa, T. et al. Binding of Cu(II) or Zn(II) in a de novo designed triple-stranded α-helical coiled-coil toward a prototype for a metalloenzyme. J. Pept. Res. 63, 347–353 (2004).

    Article  CAS  Google Scholar 

  13. Müller, H. N. & Skerra, A. Grafting of a high-affinity Zn(II)-binding site on the β-barrel of retinol-binding protein results in enhanced folding stability and enables simplified purification. Biochemistry 33, 14126–14135 (1994).

    Article  Google Scholar 

  14. Vita, C., Roumestand, C., Toma, F. & Ménez, A. Scorpion toxins as natural scaffolds for protein engineering. Proc. Natl Acad. Sci. USA 92, 6404–6408 (1995).

    Article  CAS  Google Scholar 

  15. Farrer, B. T., McClure, C. P., Penner-Hahn, J. E. & Pecoraro, V. L. Arsenic(III)–cysteine interactions stabilize three-helix bundles in aqueous solution. Inorg. Chem. 39, 5422–5423 (2000).

    Article  CAS  Google Scholar 

  16. Matzapetakis, M., Ghosh, D., Weng, T.-C., Penner-Hahn, J. E. & Pecoraro, V. L. Peptidic models for the binding of Pb(II), Bi(III) and Cd(II) to mononuclear thiolate binding sites. J. Biol. Inorg. Chem. 11, 876–890 (2006).

    Article  CAS  Google Scholar 

  17. Dieckmann, G. R. et al. De novo design of mercury-binding two- and three-helical bundles. J. Am. Chem. Soc. 11, 876–890 (1997).

    Google Scholar 

  18. Pecoraro, V. L., Peacock, A. F. A., Iranzo, O. & Luczkowski, M. Understanding the biological chemistry of mercury using a de novo protein design strategy in Bioinorg. Chem. ACS Sympos. Ser. 183–197 (2009).

  19. Touw, D. S., Nordman, C. E., Stuckey, J. A. & Pecoraro, V. L. Identifying important structural characteristics of arsenic resistance proteins by using designed three-stranded coiled coils. Proc. Natl Acad. Sci. USA 104, 11969–11974 (2007).

    Article  CAS  Google Scholar 

  20. Verpoorte, J. A., Mehta, S. & Edsall, J. T. Esterase activities of human carbonic anhydrases B and C. J. Biol. Chem. 242, 4221–4229 (1967).

    CAS  PubMed  Google Scholar 

  21. Gould, S. M. & Tawfik, D. S. Directed evolution of the promiscuous esterase activity of carbonic anhydrase II. Biochemistry 44, 5444–5452 (2005).

    Article  CAS  Google Scholar 

  22. Kimura, E., Shiota, T., Koike, T., Shiro, M. & Kodama, M. A Zinc(II) complex of 1,5,9-triazacyclododecane ([12]aneN3) as a model for carbonic anhydrase. J. Am. Chem. Soc. 112, 5805–5811 (1990).

    Article  CAS  Google Scholar 

  23. Olmo, C. P., Bohmerle, K. & Vahrenkamp, H. Zinc enzyme modeling with zinc complexes of polar pyrazolylborate ligands. Inorg. Chim. Acta 360, 1510–1516 (2007).

    Article  Google Scholar 

  24. Koerner, T. B. & Brown, R. S. The hydrolysis of an activated ester by a tris(4,5-di-n-propyl-2-imidazolyl)phosphine–Zn2+ complex in neutral micellar medium as a model for carbonic anhydrase. Can. J. Chem. 80, 183–191 (2002).

    Article  CAS  Google Scholar 

  25. Bazzicalupi, C. et al. Carboxy and phosphate esters cleavage with mono- and dinuclear zinc(II) macrocyclic complexes in aqueous solution. Crystal structure of [Zn2L1(μ-PP)2(MeOH)2](ClO4)2 (L1=[30]aneN6O4, PP−=diphenyl phosphate). Inorg. Chem. 36, 2784–2790 (1997).

    Article  CAS  Google Scholar 

  26. Sprigings, T. G. & Hall, D. C. A simple carbonic anhydrase model which achieves catalytic hydrolysis by the formation of an ‘enzyme-substrate’-like complex. J. Chem. Soc. Perkin Trans. 2, 2063–2067 (2001).

  27. Jairam, R., Potvin, P. G. & Balsky, S. Zn2+ inclusion complexes of endodentate tripodands as carbonic anhydrase-inspired artificial esterases. Part 2. Micellar systems. J. Chem. Soc. Perkin Trans. 2, 363–367 (1999).

  28. Koike, T., Takamura, M. & Kimura, E. Role of zinc(II) in β-lactamase II: a model study with a zinc(II)-macrocyclic tetraamine (1,4,7,10-tetraazacyclododecane, cyclen) complex. J. Am. Chem. Soc. 116, 8443–8449 (1994).

    Article  CAS  Google Scholar 

  29. Kimura, E., Hashimoto, H. & Koike, T. Hydrolysis of lipophilic esters catalyzed by a zinc(II) complex of a long alkyl-pendant macrocyclic tetraamine in micellar solution. J. Am. Chem. Soc. 118, 10963–10970 (1996).

    Article  CAS  Google Scholar 

  30. Broo, K., Brive, L., Ahlberg, P. & Baltzer, L. Catalysis of hydrolysis and transesterification reactions of p-nitrophenyl esters by a designed helix-loop-helix dimer. J. Am. Chem. Soc. 119, 11362–11372 (1997).

    Article  CAS  Google Scholar 

  31. Nilsson, J. & Baltzer, L. Reactive-site design in folded-polypeptide catalysts—the leaving group pKa of reactive esters sets the stage for cooperativity in nucleophilic and general-acid catalysis. Chem. Eur. J. 6, 2214–2220 (2000).

    Article  CAS  Google Scholar 

  32. Bolon, D. N. & Mayo, S. L. Enzyme-like proteins by computational design. Proc. Natl Acad. Sci. USA 98, 14274–14279 (2001).

    Article  CAS  Google Scholar 

  33. Innocenti, A. et al. Investigations of the esterase, phosphatase, and sulfatase activities of the cytosolic mammalian carbonic anhydrase isoforms I, II, and XIII with 4-nitrophenyl esters as substrates. Bioorg. Med. Chem. Lett. 18, 2267–2271 (2008).

    Article  CAS  Google Scholar 

  34. Liang, Z., Xue, Y., Behravan, G., Jonsson, B.-H. & Lindskog, S. Importance of the conserved active-site residues Tyr7, Glu106, Thr199 for the catalytic function of human carbonic anhydrase II. Eur. J. Biochem. 211, 821–827 (1993).

    Article  CAS  Google Scholar 

  35. Krebs, J. F., Ippolito, J. A., Christianson, D. W. & Fierke, C. A. Structural and functional importance of a conserved hydrogen bond network in human carbonic anhydrase II. J. Biol. Chem. 268, 27458–27466 (1993).

    CAS  PubMed  Google Scholar 

  36. Woolley, P. Models for metal ion function in carbonic anhydrase. Nature 258, 677–682 (1975).

    Article  CAS  Google Scholar 

  37. Huguet, J. & Brown, R. S. Catalytically active models for the active site in carbonic anhydrase. J. Am. Chem. Soc. 102, 7571–7572 (1980).

    Article  CAS  Google Scholar 

  38. Brown, R. S., Curtis, N. J. & Huguet, J. Tris(4,5-diisopropylimidazol-2-yl)phosphine:zinc(2+). A catalytically active model for carbonic anhydrase. J. Am. Chem. Soc. 103, 6953–6959 (1981).

    Article  CAS  Google Scholar 

  39. Brown, R. S., Salmon, D., Curtis, N. J. & Kusuma, S. Carbonic anhydrase models. 4. [Tris[(4,5-dimethyl-2-imidazolyl)methyl]phosphineoxide]cobalt(2+); a small-molecule mimic of the spectroscopic properties of Co(II) carbonic anhydrase. J. Am. Chem. Soc. 104, 3188–3194 (1982).

    Article  CAS  Google Scholar 

  40. Slebocka-Tilk, H., Cocho, J. L., Frakman, Z. & Brown, R. S. Carbonic anhydrase models. 5. Tris(4,5-di-n-propyl-2-imidazolyl)phosphine-zinc(2+) and bis(4,5-diisopropyl-2-imidazolyl)-2-imidazolylphosphine-zinc(2+). Catalysts facilitating HCO3− ↔ CO2 interconversion. J. Am. Chem. Soc. 106, 2421–2431 (1984).

    Article  CAS  Google Scholar 

  41. Zhang, X., van Eldik, R., Koike, T. & Kimura, E. Kinetics and mechanism of the hydration of CO2 and dehydration of HCO3− catalyzed by a Zn(II) complex of 1,5,9-triazacyclododecane as a model for carbonic anhydrase. Inorg. Chem. 32, 5749–5755 (1993).

    Article  CAS  Google Scholar 

  42. Zhang, X. & van Eldik, R. A functional model for carbonic anhydrase: thermodynamic and kinetic study of a tetraazacyclododecane complex of zinc(II). Inorg. Chem. 34, 5606–5614 (1995).

    Article  CAS  Google Scholar 

  43. Nakata, K. et al. Kinetic study of catalytic CO2 hydration by water-soluble model compound of carbonic anhydrase and anion inhibition effect on CO2 hydration. J. Inorg. Biochem. 89, 255–266 (2002).

    Article  CAS  Google Scholar 

  44. Jackman, J. E., Merz, K. M. Jr & Fierke, C. A. Disruption of the active site solvent network in carbonic anhydrase II decreases the efficiency of proton transfer. Biochemistry 35, 16421–16428 (1996).

    Article  CAS  Google Scholar 

  45. Iranzo, O., Cabello, C. & Pecoraro, V. L. Heterochromia in designed metallopeptides: geometry-selective binding of CdII in a de novo peptide. Angew. Chem. Int. Ed. 46, 6688–6691 (2007).

    Article  CAS  Google Scholar 

  46. Peacock, A. F. A., Hemmingsen, L. & Pecoraro, V. L. Using diastereopeptides to control metal ion coordination in proteins. Proc. Natl Acad. Sci. USA 105, 16566–16571 (2008).

    Article  CAS  Google Scholar 

  47. Wright, J. G., Tsang, H.-T., Penner-Hahn, J. E. & O'Halloran, T. V. Coordination chemistry of the Hg–MerR metalloregulatory protein: evidence for a novel tridentate Hg–cysteine receptor site. J. Am. Chem. Soc. 112, 2434–2435 (1990).

    Article  CAS  Google Scholar 

  48. Gomis-Rüth, F.-X., Kress, L. F. & Bode, W. First structure of a snake venom metalloproteinase: a prototype for matrix metalloproteinases/collagenases. EMBO J. 12, 4151–4157 (1993).

    Article  Google Scholar 

Download references

Acknowledgements

V.L.P. acknowledges support from the National Institutes of Health (grant no. R01 ES0 12236), and M.L.Z. from the National Institutes of Health Chemistry–Biology Interface Training Program. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences (contract no. DE-AC02-06CH11357). Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant no. 085P1000817). J.A.S. is supported by the University of Michigan Center for Structural Biology.

Author information

Author notes

  1. Anna F. A. Peacock

    Present address: Present address: School of Chemistry, University of Birmingham, Edgbaston B15 2TT, UK,

Authors and Affiliations

  1. Department of Chemistry, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Melissa L. Zastrow, Anna F. A. Peacock & Vincent L. Pecoraro

  2. Life Sciences Institute, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Jeanne A. Stuckey

Authors
  1. Melissa L. Zastrow
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anna F. A. Peacock
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeanne A. Stuckey
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vincent L. Pecoraro
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.L.Z. carried out the experimental work and completed crystal structure refinement. A.F.A.P. obtained and solved the crystal structure. M.L.Z., A.F.A.P., J.A.S. and V.L.P. contributed to the design of the experiments and analysis of the data. All authors contributed to writing the paper.

Corresponding author

Correspondence to Vincent L. Pecoraro.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 1140 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zastrow, M., Peacock, A., Stuckey, J. et al. Hydrolytic catalysis and structural stabilization in a designed metalloprotein. Nature Chem 4, 118–123 (2012). https://doi.org/10.1038/nchem.1201

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.1201

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing