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Evolution of chalcone isomerase from a noncatalytic ancestor

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Abstract

The emergence of catalysis in a noncatalytic protein scaffold is a rare, unexplored event. Chalcone isomerase (CHI), a key enzyme in plant flavonoid biosynthesis, is presumed to have evolved from a nonenzymatic ancestor related to the widely distributed fatty-acid binding proteins (FAPs) and a plant protein family with no isomerase activity (CHILs). Ancestral inference supported the evolution of CHI from a protein lacking isomerase activity. Further, we identified four alternative founder mutations, i.e., mutations that individually instated activity, including a mutation that is not phylogenetically traceable. Despite strong epistasis in other cases of protein evolution, CHI’s laboratory reconstructed mutational trajectory shows weak epistasis. Thus, enantioselective CHI activity could readily emerge despite a catalytically inactive starting point. Accordingly, X-ray crystallography, NMR, and molecular dynamics simulations reveal reshaping of the active site toward a productive substrate-binding mode and repositioning of the catalytic arginine that was inherited from the ancestral fatty-acid binding proteins.

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Change history

  • 14 May 2018

    In the version of this article originally published, the number for the equal contributions footnote was missing for Miriam Kaltenbach and Jason R. Burke in the author list. The error has been corrected in the PDF and print versions of this article.

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Acknowledgements

We thank K.-P. Cherukuri for help with the synthesis of chalconaringenin, B. Duggan and X. Huang for assistance with NMR, G. Louie for assistance with protein X-ray data collection and processing, and G. Cortina for help with analyzing the simulations. This work was funded by the Israel Science Foundation Grant 980/14 and the Sasson & Marjorie Peress Philanthropic Fund (D.S.T.); the United States National Science Foundation grant EEC-0813570 (J.P.N.); the Knut and Alice Wallenberg Foundation, Wenner-Gren Foundations and the European Research Council (S.C.L.K.). Computer time was provided by the Swedish National Infrastructure for Computing. J.P.N. is the Arthur and Julie Woodrow Chair and a Howard Hughes Medical Institute investigator. D.S.T. is the Nella and Leon Benoziyo Professor of Biochemistry.

Author information

M.K. performed ancestral inference with assistance from A.R. M.K. performed directed evolution. M.K., M.D., and J.R.B. performed mutagenesis, protein expression, stable isotope labeling, and biochemical characterization of the proteins. J.R.B., M.K., D.S.T. and J.P.N. performed and analyzed protein X-ray crystallography and NMR. A.P. and S.C.L.K. performed and analyzed MD simulations with assistance from F.S.M. D.S.T. and J.P.N. planned and directed the project, and, together with M.K., J.R.B., A.P., and S.C.L.K., designed the experiments. M.K., J.R.B., J.P.N. and D.S.T. wrote and edited the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Joseph P. Noel or Dan S. Tawfik.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Tables 1–12, Supplementary Figures 1– 20 and Supplementary Notes 1– 3

  2. Reporting Summary

  3. Supplementary Dataset 1

    >AUGV-6826_P_4dokB

  4. Supplementary Dataset 2

    Posterior probabilities P(amino acid) in ancestral reconstruction including indels.

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Further reading

Fig. 1: The CHI protein family.
Fig. 2: Isomerase activity of the inferred ancestors and evolutionary intermediates.
Fig. 3: Additivity versus epistasis along CHI’s evolution.
Fig. 4: The alternative random mutagenesis trajectory.
Fig. 5: Structural changes over the evolution.
Fig. 6: The conformational ensemble of the catalytic arginine changes over the evolution.