Evolution of an enzyme from a solute-binding protein

15 Much of the functional diversity observed in modern enzyme superfamilies originates from 16 molecular tinkering with existing enzymes. New enzymes frequently evolve from enzymes 17 with latent, promiscuous activities, and often inherit key features of the ancestral enzyme, 18 retaining conserved catalytic groups and stabilizing analogous intermediates or transition 19 states. While experimental evolutionary biochemistry has yielded considerable insight into 20 the evolution of new enzymes from existing enzymes, the emergence of catalytic activity de 21 novo remains poorly understood. Although certain enzymes are thought to have evolved from 22 non-catalytic proteins, the mechanisms underlying these complete evolutionary transitions 23 have not been described. Here we show how the enzyme cyclohexadienyl dehydratase (CDT) 24 evolved from a cationic amino acid-binding protein belonging to the solute-binding protein 25 (SBP) superfamily. Analysis of the evolutionary trajectory between reconstructed ancestors 26 and extant proteins showed that the emergence and optimization of catalytic activity involved 27 several distinct processes. The emergence of CDT activity was potentiated by the 28 incorporation of a desolvated general acid into the ancestral binding site, which provided an 29 intrinsically reactive catalytic motif, and reshaping of the ancestral binding site, which 30 facilitated enzyme-substrate complementarity. Catalytic activity was subsequently gained via 31 the introduction of hydrogen-bonding networks that positioned the catalytic residue precisely 32 and contributed to transition state stabilization. Finally, catalytic activity was enhanced by 33 remote substitutions that refined the active site structure and reduced sampling of non34 catalytic states. Our work shows that the evolutionary processes that underlie the emergence 35 of enzymes by natural selection in the wild are mirrored by recent examples of computational 36 design and directed evolution of enzymes in the laboratory. 37 . CC-BY-NC 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/157495 doi: bioRxiv preprint first posted online Jun. 30, 2017;