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

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 HO, instead of O, 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 HO as cosubstrate. The use of C-labeled chitin provided convenient and sensitive detection of the released soluble products, which enabled detailed kinetic measurements. The for chitin oxidation found here (5.6 s) is more than an order of magnitude higher than previously reported (apparent) rate constants for reactions containing O but no added HO The / for HO-driven degradation of chitin was on the order of 10 m s, indicating that LPMOs have catalytic efficiencies similar to those of peroxygenases. Of note, HO 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 HO levels, we conclude that controlled generation of HO seems most optimal for fueling LPMO-catalyzed oxidation of polysaccharides.

Characterization and synergistic action of a tetra‐modular lytic polysaccharide monooxygenase from Bacillus cereus

“…Bissaro et al further showed that too high concentrations of H 2 O 2 lead to oxidative damage of the LPMO active site and, thus, enzyme inactivation. In a subsequent kinetic study, Kuusk et al [10] showed that the inactivation reaction is about 1000 times slower than substrate oxidation. The ability of LPMOs to exploit H 2 O 2 has been confirmed by others [11], but the question whether H 2 O 2 or O 2 is the true natural cosubstrate remains a matter of debate [6,11].…”

Polysaccharide oxidation by lytic polysaccharide monooxygenase is enhanced by engineered cellobiose dehydrogenase

“…In contrast, a single reduction step to yield Cu(I) could be followed by binding of H 2 O 2 to directly form an oxyl-or a hydroxyl species [15]. The reported turnover numbers of LPMO with H 2 O 2 as cosubstrate are one to two orders of magnitude higher than those reported with O 2 depending on the substrate and reaction conditions [15,16,18]. However, exceeding H 2 O 2 concentrations also lead to enzyme inactivation via autooxidation of, primarily, the Cu-coordinating histidine residues [15].…”

“…The detailed catalytic mechanism of LPMOs and the nature of their cosubstrate are currently debated. Both O 2 [12][13][14] and H 2 O 2 [15,16] are reported to interact with reduced LPMO, but with different implications on the reaction pathway. The monooxygenase reaction using O 2 requires two sequential electron transfer steps and is likely to proceed via an intermittent dioxo [9,12] or oxyl [17] species.…”

“…However, exceeding H 2 O 2 concentrations also lead to enzyme inactivation via autooxidation of, primarily, the Cu-coordinating histidine residues [15]. This makes a controlled supply of H 2 O 2 essential to maintain LPMO stability during catalysis [16,19].…”

“…200 times lower turnover number for O 2 than for flavin-dependent 1,4-benzoquinone reduction [26]. Still, under physiological conditions, CDH might provide catalytically relevant amounts of H 2 O 2 for LPMOs, which have a µM affinity for this cosubstrate [16,27]. To scrutinize the significance of H 2 O 2 in CDH-driven LPMO activity, we enhanced the H 2 O 2 production rate of ChCDH by means of genetic engineering.…”