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-activeSmLPMO10A 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 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.