Abstract
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of polysaccharides, such as cellulose and chitin. LPMO catalysis requires a reductant, such as ascorbic acid, and hydrogen peroxide, which can be generated in situ in the presence of molecular oxygen and various electron donors. While it is known that reduced LPMOs are prone to autocatalytic oxidative damage 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 the chitin-active SmLPMO10A is affected by oxidative damage using NMR and circular dichroism spectroscopy. Incubation with ascorbic acid could lead to rearrangements of aromatic residues, followed by more profound structural changes near the copper-active site and loss of activity. Longer incubation times induced changes in larger parts of the structure, indicative of progressing oxidative damage. Incubation with ascorbic acid in the presence of chitin led to similar changes in the observable (i.e., not substrate-bound) fraction of the enzyme. Upon subsequent addition of H2O2, which drastically speeds up chitin hydrolysis, NMR signals corresponding to seemingly intact SmLPMO10A reappeared, indicating dissociation of catalytically competent LPMO. Activity assays confirmed that SmLPMO10A retained catalytic activity when pre-incubated with chitin before being subjected to conditions that induce oxidative damage. Overall, this study provides structural insights into the process of oxidative damage of SmLPMO10A and demonstrates the protective effect of the substrate.