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

Bastien Bissaro,Agnes Heering,Silja Kuusk,Piret Kuusk,Riin Kont,Priit Väljamäe,Morten Sørlie,Vincent G.H. Eijsink

doi:10.1074/jbc.ra118.006196

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 (KmRapp). Theoretical analyses revealed that KmRapp 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.

Highlights

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides in the presence of an external electron donor
The kinetics of H2O2-driven oxidation of chitin (14C-labeled crystalline ␣-chitin nanowhiskers (CNWs)) by SmLPMO10A with 0.1 mM ascorbic acid (AscA) as reductant has been characterized in detail before [25]
This latter study showed that reduction of N. crassa LPMO9C increased both the binding strength and binding capacity but that the effect was strongest (4.7-fold increase) at the level of the partition coefficient (initial slope of Recent findings showing that H2O2 acts as a cosubstrate of LPMOs [22, 23, 25, 26] call for detailed kinetic studies of H2O2driven degradation of polysaccharides

Summary

Results

The kinetics of H2O2-driven oxidation of chitin (14C-labeled crystalline ␣-chitin nanowhiskers (CNWs)) by SmLPMO10A with 0.1 mM AscA as reductant has been characterized in detail before [25]. The addition of catalase caused more than 10-fold reduction of the background activity (Fig. 1B) These results suggest that, under the conditions used here, the background activity is related to the formation of H2O2 by divalent metal ion-catalyzed oxidation of AscA by O2. Unlike reactions with AscA, within the time scales used, there was no significant background signal when GA and methyl hydroquinone (MHQ) were used as reductants (Fig. S1), regardless of the CNW batch used. This is corroborated by a reported much higher stability of the latter reductants against oxidation (i.e. H2O2 production) compared with AscA [10]. Considering the above, and as a result of precisely tailoring the reaction conditions, in all of the experiments

Time curves with different reductants

Binding to CNWs in the absence of reductant

Methylhydroquinone Gallic acida

Discussion

Substrates and enzymes