Functional Characterization of a Lytic Polysaccharide Monooxygenase from Schizophyllum Commune That Degrades Non-crystalline Substrates

Overview

Journal Sci Rep

Specialty Science

Date 2023 Oct 13

PMID 37833388

Authors

Heidi Ostby

Idd A Christensen

Karen Hennum

Aniko Varnai

Edith Buchinger

Siri Grandal

Gaston Courtade

Olav A Hegnar

Finn L Aachmann

Vincent G H Eijsink

Affiliations

Soon will be listed here.

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that use O or HO to oxidatively cleave glycosidic bonds. LPMOs are prevalent in nature, and the functional variation among these enzymes is a topic of great interest. We present the functional characterization of one of the 22 putative AA9-type LPMOs from the fungus Schizophyllum commune, ScLPMO9A. The enzyme, expressed in Escherichia coli, showed C4-oxidative cleavage of amorphous cellulose and soluble cello-oligosaccharides. Activity on xyloglucan, mixed-linkage β-glucan, and glucomannan was also observed, and product profiles differed compared to the well-studied C4-oxidizing NcLPMO9C from Neurospora crassa. While NcLPMO9C is also active on more crystalline forms of cellulose, ScLPMO9A is not. Differences between the two enzymes were also revealed by nuclear magnetic resonance (NMR) titration studies showing that, in contrast to NcLPMO9C, ScLPMO9A has higher affinity for linear substrates compared to branched substrates. Studies of HO-fueled degradation of amorphous cellulose showed that ScLPMO9A catalyzes a fast and specific peroxygenase reaction that is at least two orders of magnitude faster than the apparent monooxygenase reaction. Together, these results show that ScLPMO9A is an efficient LPMO with a broad substrate range, which, rather than acting on cellulose, has evolved to act on amorphous and soluble glucans.

Citing Articles

Functional characterization of two AA10 lytic polysaccharide monooxygenases from Cellulomonas gelida.

Turunen R, Tuveng T, Forsberg Z, Schiml V, Eijsink V, Arntzen M Protein Sci. 2025; 34(3):e70060.

PMID: 39969139 PMC: 11837042. DOI: 10.1002/pro.70060.

References

Hegnar O, Ostby H, Petrovic D, Olsson L, Varnai A, Eijsink V . Quantifying Oxidation of Cellulose-Associated Glucuronoxylan by Two Lytic Polysaccharide Monooxygenases from Neurospora crassa. Appl Environ Microbiol. 2021; 87(24):e0165221. PMC: 8612270. DOI: 10.1128/AEM.01652-21. View

Stepnov A, Eijsink V, Forsberg Z . Enhanced in situ HO production explains synergy between an LPMO with a cellulose-binding domain and a single-domain LPMO. Sci Rep. 2022; 12(1):6129. PMC: 9005612. DOI: 10.1038/s41598-022-10096-0. View

Sorlie M, Seefeldt L, Parker V . Use of stopped-flow spectrophotometry to establish midpoint potentials for redox proteins. Anal Biochem. 2000; 287(1):118-25. DOI: 10.1006/abio.2000.4826. View

Beeson W, Vu V, Span E, Phillips C, Marletta M . Cellulose degradation by polysaccharide monooxygenases. Annu Rev Biochem. 2015; 84:923-46. DOI: 10.1146/annurev-biochem-060614-034439. View

Armougom F, Moretti S, Poirot O, Audic S, Dumas P, Schaeli B . Expresso: automatic incorporation of structural information in multiple sequence alignments using 3D-Coffee. Nucleic Acids Res. 2006; 34(Web Server issue):W604-8. PMC: 1538866. DOI: 10.1093/nar/gkl092. View

Levasseur A, Drula E, Lombard V, Coutinho P, Henrissat B . Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes. Biotechnol Biofuels. 2013; 6(1):41. PMC: 3620520. DOI: 10.1186/1754-6834-6-41. View

Frandsen K, Haon M, Grisel S, Henrissat B, Lo Leggio L, Berrin J . Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs). J Biol Chem. 2020; 296:100086. PMC: 7949027. DOI: 10.1074/jbc.RA120.015545. View

Kuusk S, Bissaro B, Kuusk P, Forsberg Z, Eijsink V, Sorlie M . Kinetics of HO-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase. J Biol Chem. 2017; 293(2):523-531. PMC: 5767858. DOI: 10.1074/jbc.M117.817593. View

Kuusk S, Kont R, Kuusk P, Heering A, Sorlie M, Bissaro B . Kinetic insights into the role of the reductant in HO-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase. J Biol Chem. 2018; 294(5):1516-1528. PMC: 6364757. DOI: 10.1074/jbc.RA118.006196. View

10.

Frommhagen M, Westphal A, van Berkel W, Kabel M . Distinct Substrate Specificities and Electron-Donating Systems of Fungal Lytic Polysaccharide Monooxygenases. Front Microbiol. 2018; 9:1080. PMC: 5987398. DOI: 10.3389/fmicb.2018.01080. View

11.

Jones S, Transue W, Meier K, Kelemen B, Solomon E . Kinetic analysis of amino acid radicals formed in HO-driven Cu LPMO reoxidation implicates dominant homolytic reactivity. Proc Natl Acad Sci U S A. 2020; 117(22):11916-11922. PMC: 7275769. DOI: 10.1073/pnas.1922499117. View

12.

Westereng B, Arntzen M, Aachmann F, Varnai A, Eijsink V, Agger J . Simultaneous analysis of C1 and C4 oxidized oligosaccharides, the products of lytic polysaccharide monooxygenases acting on cellulose. J Chromatogr A. 2016; 1445:46-54. DOI: 10.1016/j.chroma.2016.03.064. View

13.

Quinlan R, Sweeney M, Lo Leggio L, Otten H, Poulsen J, Johansen K . Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc Natl Acad Sci U S A. 2011; 108(37):15079-84. PMC: 3174640. DOI: 10.1073/pnas.1105776108. View

14.

Liu Y, Seefeldt L, Parker V . Entropies of redox reactions between proteins and mediators: the temperature dependence of reversible electrode potentials in aqueous buffers. Anal Biochem. 1997; 250(2):196-202. DOI: 10.1006/abio.1997.2222. View

15.

Isaksen T, Westereng B, Aachmann F, Agger J, Kracher D, Kittl R . A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides. J Biol Chem. 2013; 289(5):2632-42. PMC: 3908397. DOI: 10.1074/jbc.M113.530196. View

16.

Hangasky J, Iavarone A, Marletta M . Reactivity of O versus HO with polysaccharide monooxygenases. Proc Natl Acad Sci U S A. 2018; 115(19):4915-4920. PMC: 5949000. DOI: 10.1073/pnas.1801153115. View

17.

Frommhagen M, Sforza S, Westphal A, Visser J, Hinz S, Koetsier M . Discovery of the combined oxidative cleavage of plant xylan and cellulose by a new fungal polysaccharide monooxygenase. Biotechnol Biofuels. 2015; 8:101. PMC: 4504452. DOI: 10.1186/s13068-015-0284-1. View

18.

Kelley L, Mezulis S, Yates C, Wass M, Sternberg M . The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015; 10(6):845-58. PMC: 5298202. DOI: 10.1038/nprot.2015.053. View

19.

Petrovic D, Varnai A, Dimarogona M, Mathiesen G, Sandgren M, Westereng B . Comparison of three seemingly similar lytic polysaccharide monooxygenases from suggests different roles in plant biomass degradation. J Biol Chem. 2019; 294(41):15068-15081. PMC: 6791328. DOI: 10.1074/jbc.RA119.008196. View

20.

Cannella D, Hsieh C, Felby C, Jorgensen H . Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content. Biotechnol Biofuels. 2012; 5(1):26. PMC: 3458932. DOI: 10.1186/1754-6834-5-26. View