Abstract: Acknowledgements: We thank O. J. Klein for help with LC-MS measurements. J.D.S. was supported by a Gates Cambridge Scholarship. M.G. was supported by a Trinity College/Benn W. Levy SBS DTP studentship. The work in Cambridge was supported by the BBSRC (BB/W000504/1), the Volkswagen Foundation (98182) and the EU HORIZON 2020 programme via an ERC Advanced Investigator grant (to F.H., 695669). H.A.B. thanks the SNSF for funding (P5R5PB_210999). The work in Princeton was supported by NSF grant MCB-1947720 to M.H.H. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. ; Funder: Gates Cambridge Trust; doi: https://doi.org/10.13039/501100005370 ; Funder: University of Cambridge | Trinity College, University of Cambridge; doi: https://doi.org/10.13039/501100000727 ; The ability of unevolved amino acid sequences to become biological catalysts was key to the emergence of life on Earth. However, billions of years of evolution separate complex modern enzymes from their simpler early ancestors. To probe how unevolved sequences can develop new functions, we use ultrahigh-throughput droplet microfluidics to screen for phosphoesterase activity amidst a library of more than one million sequences based on a de novo designed 4-helix bundle. Characterization of hits revealed that acquisition of function involved a large jump in sequence space enriching for truncations that removed >40% of the protein chain. Biophysical characterization of a catalytically active truncated protein revealed that it dimerizes into an α-helical structure, with the gain of function accompanied by increased structural dynamics. The identified phosphodiesterase is a manganese-dependent metalloenzyme that hydrolyses a range of phosphodiesters. It is most active towards cyclic AMP, with a rate acceleration of ~109 and a catalytic proficiency of >1014 M-1, comparable to larger enzymes shaped by billions of years of evolution.
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