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Abstract 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 ~10⁹and a catalytic proficiency of >10¹⁴ M⁻¹, comparable to larger enzymes shaped by billions of years of evolution. 

Protein sequence space is vast; nature uses only an infinitesimal fraction of possible sequences to sustain life. Are there solutions to biological problems other than those provided by nature? Can we create artificial proteins that sustain life? To investigate these questions, we have created combinatorial collections, or libraries, of novel sequences with no homology to those found in living organisms. Previously designed libraries contained numerous functional proteins. However, they often formed dynamic, rather than well-ordered structures, which complicated structural and mechanistic characterization. To address this challenge, we describe the development of new libraries based on the de novo protein S-824, a 4-helix bundle with a very stable 3-dimensional structure. Distinct from previous libraries, we targeted variability to a specific region of the protein, seeking to create potential functional sites. By characterizing variant proteins from this library, we demonstrate that the S-824 scaffold tolerates diverse amino acid substitutions in a putative cavity, including buried polar residues suitable for catalysis. We designed and created a DNA library encoding 1.7 × 106 unique protein sequences. This new library of stable de novo α-helical proteins is well suited for screens and selections for a range of functional activities in vitro and in vivo.