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Design and evolution of chimeric streptavidin for protein-enabled dual gold catalysis


Artificial metalloenzymes result from anchoring an organometallic catalyst within an evolvable protein scaffold. Thanks to its dimer of dimers quaternary structure, streptavidin allows the precise positioning of two metal cofactors to activate a single substrate, thus expanding the reaction scope accessible to artificial metalloenzymes. To validate this concept, we report herein on our efforts to engineer and evolve an artificial hydroaminase based on dual gold activation of alkynes. Guided by modelling, we designed a chimeric streptavidin equipped with a hydrophobic lid shielding its active site, which enforces the advantageous positioning of two synergistic biotinylated gold cofactors. Three rounds of directed evolution using Escherichia coli cell-free extracts led to the identification of mutants favouring either the anti-Markovnikov product (an indole carboxamide with 96% regioselectivity, 51 turnover numbers), resulting from a dual gold σ,π-activation of an ethynylphenylurea substrate, or the Markovnikov product (a phenyl-dihydroquinazolinone with 99% regioselectivity, 333 turnover numbers), resulting from the π-activation of the alkyne by gold.

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Fig. 1: Engineering and evolving an HAMase based on dual gold activation of alkynes.
Fig. 2: Chemo-genetic optimization of HAMase activity.
Fig. 3: Design and structural characterization of the chimeric ArM.
Fig. 4: Analysis of the transition state structure and close-lying amino acid residues in chimeric Sav.
Fig. 5: Directed evolution of a HAMase based on Sav-SOD.

Data availability

Data relating to the materials and methods, detailed substrate and cofactor synthesis, optimization studies, catalytic experiments, protein expression, MD and DFT calculations, selected UPLC-MS chromatograms, high-resolution MS spectra and NMR studies are available in the Supplementary Information. Crystallographic data for biot-Au 2·Sav-SOD K121A is available free of charge from the PDB under reference number 7ALX. All other data are available from the authors upon request.


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T.R.W. thanks the European Research Council (ERC) advanced grant (the Directed Evolution of Artificial Metalloenzymes (DrEAM), grant agreement 694424), the Swiss National Science Foundation (grant SNF 200020_182046) and the National Centre of Competence in Research (NCCR) Molecular Systems Engineering for generous support. We thank the Analytical Team of the Chemistry Department of the University of Basel, in particular M. Pfeffer and S. Mittelheisser, for high-resolution MS analysis and D. Häussinger for assistance with the two-dimensional NMR experiments. We thank J. Klehr and A. Santos Kron for their assistance with protein expression and protein purification as well as J.G. Rebelein for assistance with the protein crystallography. L.T.-S., A.L. and J.-D.M. thank the Spanish Ministerio de Economía, Industria y Competitividad MINECO (grant CTQ2017-87889-P) and the Generalitat de Catalunya (2017SGR1323) for the financial support. L.T.-S. thanks the Spanish Ministerio de Ciencia, Innovación y Universidades (grant FPU18/05895) for the financial support. We thank G. Sciortino and J.E. Sánchez Aparicio for assistance with the molecular modelling set-up and analysis.

Author information




T.R.W., R.L.P. and F.C. conceived and designed the study. F.C., M.M.P. and B.L. contributed to the synthesis of the substrates, products and complexes. N.V.I., D.C.S., R.L.P. and F.C. contributed to mutagenesis, protein expression, protein purification and protein characterization. N.V.I. performed the crystallization, X-ray structure determinations and native MS experiments. F.C. performed the catalytic, preparative and deuterium-labelling experiments, designed the screening protocol and recorded the data. T.R.W., F.C. and N.V.I. analysed the data. J.D.M., A.L. and L.T.S. contributed to the molecular modelling experiments. T.R.W., F.C. and N.V.I. wrote the manuscript, which was further supplemented through contributions from R.L.P. and J.-D.M. All authors have given approval to the final version of the manuscript.

Corresponding authors

Correspondence to Jean-Didier Maréchal, Ryan L. Peterson or Thomas R. Ward.

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The authors declare no competing interests.

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Peer review information Nature Catalysis thanks the anonymous reviewers for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Notes 1–11, Figs. 1–40 and Tables 1–17.

Reporting Summary

Supplementary Data 1

DFT-optimized structure of transition states.

Supplementary Data 2

Coordinates from MD simulations.

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Christoffel, F., Igareta, N.V., Pellizzoni, M.M. et al. Design and evolution of chimeric streptavidin for protein-enabled dual gold catalysis. Nat Catal 4, 643–653 (2021).

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