Insights into the H₂O₂‐driven catalytic mechanism of fungal lytic polysaccharide monooxygenases

Abstract

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H 2 O 2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H 2 O 2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H 2 O 2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single 'priming' electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H 2 O 2 -driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped-flow spectroscopy, alongside electron paramagnetic resonance and UV-Vis spectroscopy, we reveal how H 2 O 2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H 2 O 2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu + state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H 2 O 2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu + state of LPMOs, which may prevent the formation of uncoupled side reactions.

Kinetic Characterization of a Putatively Chitin-Active LPMO Reveals a Preference for Soluble Substrates and Absence of Monooxygenase Activity

“… 10 The general picture, emerging from studies on multiple bacterial and fungal LPMOs, is that these enzymes are effective peroxygenases. 16 , 28 , 30 It remains, however, difficult to fully exclude a monooxygenase reaction because it is difficult to create “monooxygenase conditions” that do not lead to in situ generation of H 2 O 2 and because LPMOs may have varying catalytic properties. As to the latter, next to demonstrating a novel LPMO functionality, efficient cleavage of soluble chito-oligomers, our data show that, despite the conserved copper histidine brace, LPMOs show considerable variation in redox potential.…”

“… 12 Central to LPMO action is a unique mononuclear copper-active site made up of two histidines, where the N-terminal histidine coordinates with both the imidazole ring and the N-terminal amine. 8 , 13 When reduced to Cu(I), LPMOs can activate O 2 3 , 14 or H 2 O 2 11 , 15 , 16 to create a reactive oxygen-containing intermediate that catalyzes the oxidation of glycosidic bonds in chitin, 3 cellulose, 17 and other plant-based polysaccharides 18 − 20 (EC 1.14.99.53–1.14.99.56).…”

Quantifying Oxidation of Cellulose-Associated Glucuronoxylan by Two Lytic Polysaccharide Monooxygenases from Neurospora crassa

“…LPMOs are mono-copper enzymes that oxidize chitin or cellulose by hydroxylating either the C-1 or C-4 position of the scissile glycosidic bond, which leads to spontaneous bond cleavage ( 9 – 13 ). LPMOs were originally considered monooxygenases, using O 2 as a cosubstrate ( 9 , 14 ), but recent work indicates that LPMOs are efficient peroxygenases, using H 2 O 2 as a cosubstrate ( 15 – 19 ).…”

Characterization of a lytic polysaccharide monooxygenase from Aspergillus fumigatus shows functional variation among family AA11 fungal LPMOs

“…In the case of an O 2 -driven reaction, the reaction requires two externally delivered electrons per catalytic cycle ( 2 ). In the case of H 2 O 2 -driven reaction, which tends to be much faster ( 16 , 18 , 19 , 20 ), a reduced LPMO can carry out multiple reactions without the need for additional externally delivered electrons ( 16 , 21 , 22 ). Cellulose-active LPMOs oxidize either the C1 or the C4 carbon of the scissile glycosidic bond; some LPMOs exclusively act on C1 or C4, whereas others are less specific and produce a mixture of C1-oxidized and C4-oxidized products ( 23 , 24 ).…”