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Redox-switchable siderophore anchor enables reversible artificial metalloenzyme assembly

Nature Catalysis volume 1, pages 680–688 (2018)Cite this article

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Abstract

Artificial metalloenzymes that contain protein-anchored synthetic catalysts are attracting increasing interest. An exciting, but still unrealized advantage of non-covalent anchoring is its potential for reversibility and thus component recycling. Here we present a siderophore–protein combination that enables strong but redox-reversible catalyst anchoring, as exemplified by an artificial transfer hydrogenase (ATHase). By linking the iron(iii)-binding siderophore azotochelin to an iridium-containing imine-reduction catalyst that produces racemic product in the absence of the protein CeuE, but a reproducible enantiomeric excess if protein bound, the assembly and reductively triggered disassembly of the ATHase was achieved. The crystal structure of the ATHase identified the residues involved in high-affinity binding and enantioselectivity. While in the presence of iron(iii), the azotochelin-based anchor binds CeuE with high affinity, and the reduction of the coordinated iron(iii) to iron(ii) triggers its dissociation from the protein. Thus, the assembly of the artificial enzyme can be controlled via the iron oxidation state.

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Fig. 1: Design considerations.
Fig. 2: Redox-switchable siderophore anchoring.
Fig. 3: Catalyst development and conjugate synthesis.
Fig. 4: Characterization of [Feiii(AZOTO)]2−⊂CeuE and [Feiii(11)Cp*Iriii]⊂CeuE.
Fig. 5: Catalytic activity of [Feiii(11)Cp*Iriii]⊂CeuE and control compounds.
Fig. 6: Redox-switchable anchoring enables CeuE recycling.

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References

  1. Hoarau, M., Hureau, C., Gras, E. & Faller, P. Coordination complexes and biomolecules: a wise wedding for catalysis upgrade. Coord. Chem. Rev. 308, 445–459 (2016).

    Article  CAS  Google Scholar 

  2. Yu, F. et al. Protein design: toward functional metalloenzymes. Chem. Rev. 114, 3495–3578 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lewis, J. C. Artificial metalloenzymes and metallopeptide catalysts for organic synthesis. ACS Catal. 3, 2954–2975 (2013).

    Article  CAS  Google Scholar 

  5. Bos, J. & Roelfes, G. Artificial metalloenzymes for enantioselective catalysis. Curr. Opin. Chem. Biol. 19, 135–143 (2014).

    Article  CAS  PubMed  Google Scholar 

  6. Matsuo, T. & Hirota, S. Artificial enzymes with protein scaffolds: structural design and modification. Biorg. Med. Chem. 22, 5638–5656 (2014).

    Article  CAS  Google Scholar 

  7. Pàmies, O., Diéguez, M. & Bäckvall, J.-E. Artificial metalloenzymes in asymmetric catalysis: key developments and future directions. Adv. Synth. Catal. 357, 1567–1586 (2015).

    Article  CAS  Google Scholar 

  8. Heinisch, T. & Ward, T. R. Artificial metalloenzymes based on the biotin–streptavidin technology: challenges and opportunities. Acc. Chem. Res. 49, 1711–1721 (2016).

    Article  CAS  PubMed  Google Scholar 

  9. Sauer, D. F. et al. A highly active biohybrid catalyst for olefin metathesis in water: impact of a hydrophobic cavity in a β-barrel protein. ACS Catal. 5, 7519–7522 (2015).

    Article  CAS  Google Scholar 

  10. Cotelle, Y., Lebrun, V., Sakai, N., Ward, T. R. & Matile, S. Anion-π enzymes. ACS Cent. Sci. 2, 388–393 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Schwizer, F. et al. Artificial metalloenzymes: reaction scope and optimization strategies. Chem. Rev. 118, 142–231 (2018).

    Article  CAS  PubMed  Google Scholar 

  12. Jeschek, M., Panke, S. & Ward, T. R. Artificial metalloenzymes on the verge of new-to-nature metabolism. Trends Biotechnol. 36, 60–72 (2018).

    Article  CAS  PubMed  Google Scholar 

  13. Wilson, M. E. & Whitesides, G. M. Conversion of a protein to a homogeneous asymmetric hydrogenation catalyst by site-specific modification with a diphosphinerhodium(i) moiety. J. Am. Chem. Soc. 100, 306–307 (1978).

    Article  CAS  Google Scholar 

  14. Jeschek, M. et al. Directed evolution of artificial metalloenzymes for in vivo metathesis. Nature 537, 661–665 (2016).

    Article  CAS  PubMed  Google Scholar 

  15. Hyster, T. K., Knörr, L., Ward, T. R. & Rovis, T. Biotinylated Rh(iii) complexes in engineered streptavidin for accelerated asymmetric C–H activation. Science 338, 500–503 (2012).

    Article  CAS  PubMed  Google Scholar 

  16. Liu, Z. et al. Upregulation of an artificial zymogen by proteolysis. Angew. Chem. Int. Ed. 55, 11587–11590 (2016).

    Article  CAS  Google Scholar 

  17. Monnard, F. W., Nogueira, E. S., Heinisch, T., Schirmer, T. & Ward, T. R. Human carbonic anhydrase II as host protein for the creation of artificial metalloenzymes: the asymmetric transfer hydrogenation of imines. Chem. Sci. 4, 3269–3274 (2013).

    Article  CAS  Google Scholar 

  18. Matsuo, T. et al. Creation of an artificial metalloprotein with a Hoveyda–Grubbs catalyst moiety through the intrinsic inhibition mechanism of α-chymotrypsin. Chem. Commun. 48, 1662–1664 (2012).

    Article  CAS  Google Scholar 

  19. Key, H. M., Dydio, P., Clark, D. S. & Hartwig, J. F. Abiological catalysis by artificial haem proteins containing noble metals in place of iron. Nature 534, 534–537 (2016).

    Article  CAS  PubMed  Google Scholar 

  20. Ying, L.-Q. & Branchaud, B. P. Design of a reversible biotin analog and applications in protein labeling, detection, and isolation. Chem. Commun. 47, 8593–8595 (2011).

    Article  CAS  Google Scholar 

  21. Terai, T. et al. Artificial ligands of streptavidin (ALiS): discovery, characterization, and application for reversible control of intracellular protein transport. J. Am. Chem. Soc. 137, 10464–10467 (2015).

    Article  CAS  PubMed  Google Scholar 

  22. Pollheimer, P. et al. Reversible biofunctionalization of surfaces with a switchable mutant of avidin. Bioconjug. Chem. 24, 1656–1668 (2013).

    Article  CAS  PubMed  Google Scholar 

  23. Dwyer, M. A. & Hellinga, H. W. Periplasmic binding proteins: a versatile superfamily for protein engineering. Curr. Opin. Struct. Biol. 14, 495–504 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Miethke, M. & Marahiel, M. A. Siderophore-based iron acquisition and pathogen control. Microbiol. Mol. Biol. Rev. 71, 413–451 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hider, R. C. & Kong, X. Chemistry and biology of siderophores. Nat. Prod. Rep. 27, 637–657 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Schalk, I. J. & Guillon, L. Fate of ferrisiderophores after import across bacterial outer membranes: different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways. Amino Acids 44, 1267–1277 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Wilson, B. R., Bogdan, A. R., Miyazawa, M., Hashimoto, K. & Tsuji, Y. Siderophores in iron metabolism: from mechanism to therapy potential. Trends Mol. Med. 22, 1077–1090 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ganne, G. et al. Iron release from the siderophore pyoverdine in Pseudomonas aeruginosa involves three new actors: FpvC, FpvG, and FpvH. ACS Chem. Biol. 12, 1056–1065 (2017).

    Article  CAS  PubMed  Google Scholar 

  29. Raines, D. J. et al. Bacteria in an intense competition for iron: key component of the Campylobacter jejuni iron uptake system scavenges enterobactin hydrolysis product. Proc. Natl Acad. Sci. USA 113, 5850–5855 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Raines, D. J., Moroz, O. V., Wilson, K. S. & Duhme-Klair, A.-K. Interactions of a periplasmic binding protein with a tetradentate siderophore mimic. Angew. Chem. Int. Ed. 52, 4595–4598 (2013).

    Article  CAS  Google Scholar 

  31. Zimbron, J. M. et al. A dual anchoring strategy for the localization and activation of artificial metalloenzymes based on the biotin–streptavidin technology. J. Am. Chem. Soc. 135, 5384–5388 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Dürrenberger, M. et al. Artificial transfer hydrogenases for the enantioselective reduction of cyclic imines. Angew. Chem. Int. Ed. 50, 3026–3029 (2011).

    Article  CAS  Google Scholar 

  33. Madern, N., Talbi, B. & Salmain, M. Aqueous phase transfer hydrogenation of aryl ketones catalysed by achiral ruthenium(ii) and rhodium(iii) complexes and their papain conjugates. Appl. Organomet. Chem. 27, 6–12 (2013).

    Article  CAS  Google Scholar 

  34. Chimiak, A. & Neilands, J. B. Lysine analogues of siderophores. Struct. Bond. 58, 89–96 (1984).

    Article  CAS  Google Scholar 

  35. Lo, C., Ringenberg, M. R., Gnandt, D., Wilson, Y. & Ward, T. R. Artificial metalloenzymes for olefin metathesis based on the biotin-(strept)avidin technology. Chem. Commun. 47, 12065–12067 (2011).

    Article  CAS  Google Scholar 

  36. Stookey, L. L. Ferrozine—a new spectrophotometric reagent for iron. Anal. Chem. 42, 779–781 (1970).

    Article  CAS  Google Scholar 

  37. Ruff, A., Kirby, C., Chan, B. C. & O’Connor, A. R. Base-free transfer hydrogenation of ketones using Cp*Ir(pyridinesulfonamide)Cl precatalysts. Organometallics 35, 327–335 (2016).

    Article  CAS  Google Scholar 

  38. Townsend, T. M., Kirby, C., Ruff, A. & O’Connor, A. R. Transfer hydrogenation of aromatic and linear aldehydes catalyzed using Cp*Ir(pyridinesulfonamide)Cl complexes under base-free conditions. J. Organomet. Chem. 843, 7–13 (2017).

    Article  CAS  Google Scholar 

  39. Fu, Y. et al. The contrasting catalytic efficiency and cancer cell antiproliferative activity of stereoselective organoruthenium transfer hydrogenation catalysts. Dalton Trans. 45, 8367–8378 (2016).

    Article  CAS  PubMed  Google Scholar 

  40. Soni, R. et al. The importance of the N–H bond in Ru/TsDPEN complexes for asymmetric transfer hydrogenation of ketones and imines. Org. Biomol. Chem. 9, 3290–3294 (2011).

    Article  CAS  PubMed  Google Scholar 

  41. Václavík, J. et al. Experimental and theoretical perspectives of the Noyori–Ikariya asymmetric transfer hydrogenation of imines. Molecules 19, 6987–7007 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Müller, A., Wilkinson, A. J., Wilson, K. S. & Duhme-Klair, A.-K. An [{Fe(mecam)}2]6− bridge in the crystal structure of a ferric enterobactin binding protein. Angew. Chem. Int. Ed. 45, 5132–5136 (2006).

    Article  CAS  Google Scholar 

  43. Wilde, E. J. et al. Interactions of the periplasmic binding protein CeuE with Fe(iii) n-LICAM4− siderophore analogues of varied linker length. Sci. Rep. 7, 45941 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Scarrow, R. C., Ecker, D. J., Ng, C., Liu, S. & Raymond, K. N. Iron(iii) coordination chemistry of linear dihydroxyserine compounds derived from enterobactin. Inorg. Chem. 30, 900–906 (1991).

    Article  CAS  Google Scholar 

  45. Okamoto, Y., Köhler, V. & Ward, T. R. An NAD(P)H-dependent artificial transfer hydrogenase for multienzymatic cascades. J. Am. Chem. Soc. 138, 5781–5784 (2016).

    Article  CAS  PubMed  Google Scholar 

  46. Chimiak, A. & Neilands, J. B. Lysine analogues of siderophores. Struct. Bond. 58, 89–96 (1984).

    Article  CAS  Google Scholar 

  47. White, C., Yates, A., Maitlis, P. M. & Heinekey, D. M. (η5‐Pentamethylcyclopentadienyl) rhodium and-iridium compounds. Inorg. Synth. 29, 228–234 (2007).

    Google Scholar 

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Acknowledgements

We thank the Engineering and Physical Sciences Research Council (EPSRC, grant EP/L024829/1) and Biotechnology and Biological Sciences Research Council (BBSRC) for financial support, the Diamond Light Source for access to beamlines I03 and I04 (proposal; mx-13587) and J. Turkenburg and S. Hart for assistance with data collection. In addition, experimental support by K. Heaton (mass spectrometry), A. Leech (CD spectroscopy and mass spectrometry), A. Dixon (HPLC), J. H. Peng (catalyst preparation) and A. C. Whitwood (small-molecule crystallography) is gratefully acknowledged.

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Authors and Affiliations

  1. Department of Chemistry, University of York, York, UK

    Daniel J. Raines, Justin E. Clarke & Anne-K. Duhme-Klair

  2. Structural Biology Laboratory, Department of Chemistry, University of York, York, UK

    Justin E. Clarke, Elena V. Blagova, Eleanor J. Dodson & Keith S. Wilson

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Contributions

The experiments were performed by D.J.R. (chemical synthesis, development of catalysts and screening protocols, binding constant determination, redox reversibility tests), J.E.C. (artificial enzyme production and characterization, catalytic screening, redox reversibility tests), A.-K.D.-K. (redox reversibility tests), E.V.B. (protein production and protein crystal growth), E.J.D (protein crystal structure refinement) and K.S.W. (protein crystal structure refinement). K.S.W. (structural biology) and A.-K.D.-K. (chemistry) supervised and directed the project. The manuscript was produced by D.J.R and A.-K.D.-K. based on contributions by all authors. All authors have given approval to the final version of the manuscript.

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Correspondence to Keith S. Wilson or Anne-K. Duhme-Klair.

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Raines, D.J., Clarke, J.E., Blagova, E.V. et al. Redox-switchable siderophore anchor enables reversible artificial metalloenzyme assembly. Nat Catal 1, 680–688 (2018). https://doi.org/10.1038/s41929-018-0124-3

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