Redox robustness drives LPMO evolution
- Iván Ayuso-Fernández
- Tom Z. Emrich-Mills
- Ole Golten
- Zarah Forsberg
- Kelsi R. Hall
- László G. Nagy
- Morten Sørlie
- Åsmund Kjendseth Røhr
- Vincent G. H. Eijsink
2026-01-13
Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are exceptionally powerful small redox enzymes that master the controlled generation and productive use of potentially damaging hydroxyl radicals in what is essentially a H 2 O 2 -driven peroxygenase reaction. We have used ancestral sequence reconstruction and enzyme resurrection to unravel evolutionary steps leading to this exceptional catalytic ability. Real-time monitoring of copper reoxidation and amino acid radical formation showed evolutionary improvement of both the capacity to avoid futile turnover of H 2 O 2 and the ability to scavenge damaging radicals resulting from such turnover through a hole hopping pathway. Through mutational studies of ancestral LPMOs, we show that adoption of an extant-like conformation of residues in the hole hopping pathway yields improvements in redox robustness to near-extant levels. These results show how selective pressure imposed by the need for generating a highly oxidizing intermediate is a key driver of metalloenzyme evolution, involving large parts of the enzyme, well beyond the catalytic center.