Kinetics of H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase

Vincent G.H Eijsink,Priit Väljamäe,Piret Kuusk,Zarah Forsberg,Bastien Bissaro,Morten Sørlie,Silja Kuusk

doi:10.1074/jbc.m117.817593

Abstract

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, and are of interest in biotechnological utilization of these abundant biomaterials. It has recently been shown that LPMOs can use H2O2, instead of O2, as a cosubstrate. This peroxygenase-like reaction by a monocopper enzyme is unprecedented in nature and opens new avenues in chemistry and enzymology. Here, we provide the first detailed kinetic characterization of chitin degradation by the bacterial LPMO chitin-binding protein CBP21 using H2O2 as cosubstrate. The use of 14C-labeled chitin provided convenient and sensitive detection of the released soluble products, which enabled detailed kinetic measurements. The kcat for chitin oxidation found here (5.6 s-1) is more than an order of magnitude higher than previously reported (apparent) rate constants for reactions containing O2 but no added H2O2 The kcat/Km for H2O2-driven degradation of chitin was on the order of 106 m-1 s-1, indicating that LPMOs have catalytic efficiencies similar to those of peroxygenases. Of note, H2O2 also inactivated CBP21, but the second-order rate constant for inactivation was about 3 orders of magnitude lower than that for catalysis. In light of the observed CBP21 inactivation at higher H2O2 levels, we conclude that controlled generation of H2O2in situ seems most optimal for fueling LPMO-catalyzed oxidation of polysaccharides.

Highlights

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, and are of interest in biotechnological utilization of these abundant biomaterials
In light of the observed CBP21 inactivation at higher H2O2 levels, we conclude that controlled generation of H2O2 in situ seems most optimal for fueling LPMO-catalyzed oxidation of polysaccharides
Kinetics of the H2O2-driven degradation of chitin by CBP21 The use of 14C-labeled chitin nanowhiskers (CNWs) made it possible to perform detailed kinetic measurements by providing convenient and sensitive detection of the released 14C-labeled soluble products, the amount of which is expressed in N-acetylglucosamine equivalents (NAGeq) as explained under “Experimental procedures.”

Summary

To whom correspondence should be addressed

LPMOs can act on glycan chains that are embedded in a crystalline lattice, cleaving glycosidic bonds by hydroxylation of the C1 or C4 carbon. This ability is conferred by their unusual active-site architecture, a single copper atom coordinated by two conserved histidines, displayed on a solvent-exposed, flat substrate-binding surface [9, 10]. Kinetic studies on LPMOs are scarce, mostly showing very low rates [11]. Such kinetic studies are challenging because of the multisubstrate nature of the reaction, the insolubility of one of the substrates, and oxidative enzyme inactivation [1]. We set out to provide the first detailed kinetic insights into H2O2 utilization by LPMOs using the bacterial C1-oxidizing, chitin-active CBP21 [5] as a model enzyme and 14C-labeled chitin nanowhiskers (CNWs) [12] as substrate

Results

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Discussion

Experimental procedures