Kinetic insights into the role of the reductant in H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are monocopper 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 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 reoxidation (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.

Keywords: CBP21; Serratia marcescens; SmLPMO10A; binding; chitin; copper monooxygenase; enzyme inactivation; enzyme kinetics; hydrogen peroxide; lytic polysaccharide monooxygenase; oxidative degradation; polysaccharide; reductant.