Selection of a promiscuous minimalist cAMP phosphodiesterase from a library of de novo designed proteins

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

Research output: Contribution to journalArticlepeer-review

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

Original languageEnglish (US)
Pages (from-to)1200-1208
Number of pages9
JournalNature chemistry
Volume16
Issue number7
DOIs
StatePublished - Jul 2024

All Science Journal Classification (ASJC) codes

  • General Chemistry
  • General Chemical Engineering

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