This research explores the emergence of new enzyme functions from unevolved sequences. Using ultrahigh-throughput droplet microfluidics, a library of over one million de novo designed 4-helix bundle proteins was screened for phosphoesterase activity. The study found that function acquisition involved significant sequence changes, notably truncations removing over 40% of the protein chain. A highly active truncated protein, mini-cAMPase, was characterized as a manganese-dependent metalloenzyme with broad phosphodiesterase activity, particularly towards cAMP, exhibiting rate acceleration comparable to evolved enzymes.

J. David Schnettler, Michael S. Wang, Maximilian Gantz, H. Adrian Bunzel, Christina Karas, Florian Hollfelder, Michael H. Hecht