Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides in the presence of an external electron donor (reductant). In the classical O₂-driven monooxygenase reaction, the reductant is needed in stoichiometric amounts. In a recently discovered, more efficient H₂O₂-driven reaction, the reductant would be needed only for the initial reduction (priming) of the LPMO to its catalytically active Cu(I) form. However, the influence of the reductant on reducing the LPMO or on H₂O₂ production in the reaction remains undefined. Here, we conducted a detailed kinetic characterization to investigate how the reductant affects H₂O₂-driven degradation of ¹⁴C-labeled chitin by a bacterial LPMO, SmLPMO10A (formerly CBP21). Sensitive detection of ¹⁴C-labeled products and careful experimental set-ups enabled discrimination between the effects of the reductant on LPMO priming and other effects, in particular enzyme-independent production of H₂O₂ through reactions with O₂. When supplied with H₂O₂, SmLPMO10A catalyzed 18 oxidative cleavages per one molecule of ascorbic acid, suggesting a “priming reduction” reaction. The dependence of initial rates of chitin degradation on reductant concentration followed hyperbolic saturation kinetics, and differences between the reductants were manifested in large variations in their half-saturating concentrations (K_mR^app). Theoretical analyses revealed that K_mR^app decreases with a decreasing rate of polysaccharide-independent LPMO re-oxidation (by either O₂ or H₂O₂).We conclude that the efficiency of LPMO priming depends on the relative contributions of reductant reactivity, on the LPMO’s polysaccharide monooxygenase/peroxygenase and reductant oxidase/peroxidase activities, and on reaction conditions such as O₂, H₂O₂, and polysaccharide concentrations.

Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are recognized as important contributors to aerobic enzymatic degradation of recalcitrant polysaccharides such as chitin and cellulose. LPMOs are remarkably abundant in nature, with some fungal species possessing more than 50 LPMO genes, and the biological implications of this diversity remain enigmatic. For example, chitin-active LPMOs have been encountered in biological niches where chitin conversion does not seem to take place. We have carried out an in-depth kinetic characterization of a putatively chitin-active LPMO from Aspergillus fumigatus (AfAA11B), which, as we show here, has multiple unusual properties, such as a low redox potential and high oxidase activity. Furthermore, AfAA11B is hardly active on chitin, while being very active on soluble oligomers of N-acetylglucosamine. In the presence of chitotetraose, the enzyme can withstand considerable amounts of H₂O₂, which it uses to efficiently and stoichiometrically convert this substrate. The unique properties of AfAA11B allowed experiments showing that it is a strict peroxygenase and does not catalyze a monooxygenase reaction. This study shows that nature uses LPMOs for breaking glycosidic bonds in non-polymeric substrates in reactions that depend on H₂O₂. The quest for the true substrates of these enzymes, possibly carbohydrates in the cell wall of the fungus or its competitors, will be of major interest.

Significance: Lytic polysaccharide monooxygenases (LPMOs) have unique catalytic centers, at which a single copper catalyzes the oxidative cleavage of a glycosidic bond. The mechanism by which LPMOs activate molecular oxygen is key to understanding copper (bio)catalysis but remains poorly understood, largely because the insoluble and heterogeneous nature of LPMO substrates precludes the use of usual laboratory techniques. Using an integrated NMR/EPR approach, we have unraveled structural and electronic details of the interactions of an LPMO fromBacillus licheniformisand β-chitin. EPR spectroscopy on uniformly isotope¹⁵N-labeled⁶³Cu(II)-LPMO provided insight into substrate-driven rearrangement of the copper coordination sphere that predisposes the enzyme for O₂activation.