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

Tobias M. Hedison, Erik Breslmayr, Muralidharan Shanmugam, Kwankao Karnpakdee, Derren J. Heyes, Anthony P. Green, Roland Ludwig, Nigel S. Scrutton, Daniel Kracher

Research output: Contribution to journalArticlepeer-review

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 2O 2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H 2O 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 2O 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 2O 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 2O 2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H 2O 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 2O 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.

Original languageEnglish
JournalThe FEBS Journal
DOIs
Publication statusPublished - 23 Dec 2020

Research Beacons, Institutes and Platforms

  • Manchester Institute of Biotechnology

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