Following the Fate of Lytic Polysaccharide Monooxygenases under Oxidative Conditions by NMR Spectroscopy

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependentenzymes that catalyze oxidative cleavage of polysaccharides, suchas cellulose and chitin. LPMO catalysis requires a reductant, suchas ascorbic acid, and hydrogen peroxide, which can be generated insitu in the presence of molecular oxygen and various electron donors.While it is known that reduced LPMOs are prone to autocatalytic oxidativedamage due to off-pathway reactions with the oxygen co-substrate,little is known about the structural consequences of such damage.Here, we present atomic-level insights into how the structure of thechitin-active SmLPMO10A is affected by oxidativedamage using NMR and circular dichroism spectroscopy. Incubation withascorbic acid could lead to rearrangements of aromatic residues, followedby more profound structural changes near the copper-active site andloss of activity. Longer incubation times induced changes in largerparts of the structure, indicative of progressing oxidative damage.Incubation with ascorbic acid in the presence of chitin led to similarchanges in the observable (i.e., not substrate-bound) fraction ofthe enzyme. Upon subsequent addition of H2O2, which drastically speeds up chitin hydrolysis, NMR signals correspondingto seemingly intact SmLPMO10A reappeared, indicatingdissociation of catalytically competent LPMO. Activity assays confirmedthat SmLPMO10A retained catalytic activity when pre-incubatedwith chitin before being subjected to conditions that induce oxidativedamage. Overall, this study provides structural insights into theprocess of oxidative damage of SmLPMO10A and demonstratesthe protective effect of the substrate.