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Insights into the H 2 O 2 ‐driven catalytic mechanism of fungal lytic polysaccharide monooxygenases
Author(s) -
Hedison Tobias M.,
Breslmayr Erik,
Shanmugam Muralidharan,
Karnpakdee Kwankao,
Heyes Derren J.,
Green Anthony P.,
Ludwig Roland,
Scrutton Nigel S.,
Kracher Daniel
Publication year - 2021
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.15704
Subject(s) - cellobiose dehydrogenase , cellobiose , chemistry , monooxygenase , polysaccharide , cellulose , xyloglucan , substrate (aquarium) , biochemistry , enzyme , cellulase , biology , ecology , cytochrome p450
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 Nc LPMO9C. 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.