Golten, O. | bioRxiv Science

bioRxiv2025

Redox robustness drives LPMO evolution

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 H2O2-driven peroxygenase reaction. We have used ancestral sequence reconstruction and enzyme resurrection to unravel evolutionary steps leading to this unprecedented catalytic power. Real-time monitoring of copper re-oxidation and amino acid radical formation showed evolutionary improvement of both the capacity to avoid futile turnover of H2O2 and the ability to scavenge damaging radicals resulting from such turnover through a hole hopping pathway. These results show how selective pressure imposed by the need for generating a highly oxidizing intermediate shapes metalloenzymes, involving large parts of the enzyme, well beyond the catalytic center.

evolutionary biology↗

bioRxiv2025

Discovery and characterization of a copper-binding carbohydrate-binding module (CBM) regulating the activity of lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that hydroxylate recalcitrant polysaccharides such as cellulose. Like other redox enzymes, LPMOs face challenges in handling the reactive oxygen species generated at their active site, which must be controlled to prevent off-pathway reactions that lead to enzyme inactivation. In the case of LPMOs, oxidative damage may be self-reinforcing because free copper released from damaged catalytic centers will promote abiotic redox reactions that generate reactive oxygen species. Here we show that some members of a widely spread family of carbohydrate-binding modules (CBM2s) have evolved the ability to bind copper and that this ability is exclusively found in CBM2s that are appended to LPMOs. We show that the copper site in these CBM2s protects the LPMO from inactivation both by scavenging free copper, preferably Cu(I), and by interacting directly with the reduced catalytic copper site of the LPMO, thus preventing the enzyme from engaging in off-pathway reactions. These effects are demonstrated by studies on the redox stability of a series of engineered LPMO variants as well as AlphaFold3 models of the CBM2-containing enzymes. Interestingly, the copper site and the cellulose-binding surface are located on different sides of these CBM2s, enabling a mode of action in which the CBM inhibits potentially damaging LPMO activity in the absence of substrate, while such inhibition would be relieved upon binding of the CBM2 to cellulose. These findings show that CBMs have biologically relevant functions beyond carbohydrate-binding and reveal a mechanism for substrate-dependent regulation of LPMO reactivity.

biochemistry↗