The emergence of enzymes through the neofunctionalization of noncatalytic proteins is ultimately responsible for the extraordinary range of biological catalysts observed in nature. Although the evolution of some enzymes from binding proteins can be inferred by homology, we have a limited understanding of the nature of the biochemical and biophysical adaptations along these evolutionary trajectories and the sequence in which they occurred. Here we reconstructed and characterized evolutionary intermediate states linking an ancestral solute-binding protein to the extant enzyme cyclohexadienyl dehydratase. We show how the intrinsic reactivity of a desolvated general acid was harnessed by a series of mutations radiating from the active site, which optimized enzyme–substrate complementarity and transition-state stabilization and minimized sampling of noncatalytic conformations. Our work reveals the molecular evolutionary processes that underlie the emergence of enzymes de novo, which are notably mirrored by recent examples of computational enzyme design and directed evolution.
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B.E.C. and J.A.K. were supported by Australian Postgraduate Awards. B.E.C. was also supported by a Rod Rickards PhD scholarship and an Alan Sargeson scholarship. This research was undertaken with the assistance of resources, services, and staff from the Australian National Computational Infrastructure (NCI), the Australian Synchrotron, and the CSIRO Collaborative Crystallisation Centre, and funding from the Australian Research Council Discovery Project scheme (C.J.J.). We thank A. Saeed, P. Yates, L. Tan and S. Warring for additional technical contributions. We thank H. Janovjak (IST Austria) for gifting us the pDOTS7 plasmid.
The authors declare no competing interests
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Clifton, B.E., Kaczmarski, J.A., Carr, P.D. et al. Evolution of cyclohexadienyl dehydratase from an ancestral solute-binding protein. Nat Chem Biol 14, 542–547 (2018). https://doi.org/10.1038/s41589-018-0043-2
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