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.
Keywords: Serratia marcescens; copper monooxygenase; enzyme inactivation; enzyme kinetics; hydrogen peroxide; polysaccharide.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.