Activation of bacterial lytic polysaccharide monooxygenases with cellobiose dehydrogenase

Abstract

Lytic polysaccharide monooxygenases (LPMOs) represent a recent addition to the carbohydrate-active enzymes and are classified as auxiliary activity (AA) families 9, 10, 11, and 13. LPMOs are crucial for effective degradation of recalcitrant polysaccharides like cellulose or chitin. These enzymes are copper-dependent and utilize a redox mechanism to cleave glycosidic bonds that is dependent on molecular oxygen and an external electron donor. The electrons can be provided by various sources, such as chemical compounds (e.g., ascorbate) or by enzymes (e.g., cellobiose dehydrogenases, CDHs, from fungi). Here, we demonstrate that a fungal CDH from Myriococcum thermophilum (MtCDH), can act as an electron donor for bacterial family AA10 LPMOs. We show that employing an enzyme as electron donor is advantageous since this enables a kinetically controlled supply of electrons to the LPMO. The rate of chitin oxidation by CBP21 was equal to that of cosubstrate (lactose) oxidation by MtCDH, verifying the usage of two electrons in the LPMO catalytic mechanism. Furthermore, since lactose oxidation correlates directly with the rate of LPMO catalysis, a method for indirect determination of LPMO activity is implicated. Finally, the one electron reduction of the CBP21 active site copper by MtCDH was determined to be substantially faster than chitin oxidation by the LPMO. Overall, MtCDH seems to be a universal Statement for Broader Audience: Lytic polysaccharide monooxygenases (LPMOs) are redox-active enzymes that cleave the glycosidic bonds of recalcitrant polysaccharides. The present study shows that fungal cellobiose dehydrogenases can act as a universal electron donor for these enzymes and that appropriate dosing of electrons to the LPMO is important for stable activity. Finally, our results verify that two electrons are consumed by each LPMO reaction.Abbreviations: AA, auxiliary activity; CAZy, carbohydrate-active enzyme database; CAZymes, carbohydrate-active enzymes; CDH, cellobiose dehydrogenase; CYT, cytochrome; DH, dehydrogenase; DP, degree of polymerization; FAD, flavin adenine dinucleotide; LPMO, lytic polysaccharide monooxygenase; ox, oxidized electron donor for both bacterial and fungal LPMOs, indicating that their electron transfer mechanisms are similar.

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

“…Prereduction of CDH with cellobiose was performed under atmospheric conditions with or without catalase to investigate the effect of the by‐product H 2 O 2 on the observed rate. The presence of H 2 O 2 had no effect on the LPMO reduction rate and in both cases, the apparent bimolecular rate constant for the CDH oxy+ /LPMO interaction was 1.27 × 10 6 m −1 ·s −1 , which is similar to the reported value for Ch CDH/LPMO (0.63 × 10 6 m −1 ·s −1 ; ).…”

“…While Ch CDH and CDH oxy+ transfer electrons to the LPMO with similar efficiencies, they produce different amounts of H 2 O 2 , which translates into different LPMO activities. In accordance with the correlation between H 2 O 2 production by the CDH and LPMO activity, it has been noted earlier that the rate of Ch CDH‐driven LPMO activity is similar to the rate of H 2 O 2 production by Ch CDH in reactions with O 2 as the only electron acceptor . It has been suggested that H 2 O 2 ‐driven LPMO activity implies that, when once reduced, an LPMO could carry out multiple oxidative cleavages .…”

Methylation of the N‐terminal histidine protects a lytic polysaccharide monooxygenase from auto‐oxidative inactivation

“…In this respect, it is interesting to note the relatively stable kinetics obtained with CDH (Fig. A), which is known to form H 2 O 2 in a controlled manner at rates comparable to observed catalytic rates of LPMOs . In line with the recent claims by Bissaro et al, it is tempting to speculate that in these reactions the produced H 2 O 2 is consumed immediately by the LPMO, avoiding H 2 O 2 accumulation and thus oxidative inactivation.…”

“…Reactions with various electron‐donating systems (ascorbic acid, CDH, or natural compounds in steam‐exploded birch) did not reveal differences in terms of the LPMO's ability to recruit electrons from varying sources. While progress curves with CDH showed that product formation increased during most of the monitored incubation period, the reactions with ascorbic acid, expectedly, terminated before the monitoring period was over (Fig. ).…”

Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars

“…93,110,119,130 More recently, Loose et al demonstrated activation of a bacterial AA10 LPMO by a CDH from the fungus Myriococcum thermophilum . 158 The interactions of CDH with LPMOs are discussed further in sections 4 and 5.…”