| Popis: |
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of polysaccharides, such as cellulose and chitin. LPMO action is key to the efficient varlorization of biomass, but the instability of LPMOs in turnover conditions limits their efficiency. LPMO catalysis requires the presence of a reductant, such as ascorbic acid, and hydrogen peroxide, which can be generatedin situin the presence of molecular oxygen and various electron donors.. While it is known that reduced LPMOs are prone to auto-catalytic 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 insight into how the structure of the chitin-activeSmLPMO10A is affected by oxidative damage, using NMR and CD spectroscopy. Incubation with ascorbic acid, led 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 intactSmLPMO10A reappeared, indicating dissociation of catalytically competent LPMO. Activity assays confirmed thatSmLPMO10A 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 ofSmLPMO10A and demonstrates the protective effect of the substrate. The impact of turnover conditions on aromatic residues in the core of the enzyme suggests a role for these residues in dealing with redox-active species generated in the copper center. |
