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Title: Molecular mechanism of the chitinolytic peroxygenase reaction

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H 2 O 2 as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H 2 O 2 -mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H 2 O 2 into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper–oxyl intermediate. The initial cleavage of H 2 O 2 and subsequent hydrogen atom abstraction from chitin by the copper–oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO–Cu(II) to LPMO–Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO–Cu(I) is 2,000-fold faster with H 2 O 2 than with O 2 , the latter being several orders of magnitude slower than rates reported formore » other monooxygenases. The presence of substrate accelerated reoxidation by H 2 O 2 , whereas reoxidation by O 2 became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.« less


Citation Formats

Bissaro, Bastien, Streit, Bennett, Isaksen, Ingvild, Eijsink, Vincent G. H., Beckham, Gregg T., DuBois, Jennifer L., and Røhr, Åsmund K. Molecular mechanism of the chitinolytic peroxygenase reaction. United States: N. p., 2020. Web. doi:10.1073/pnas.1904889117.
Bissaro, Bastien, Streit, Bennett, Isaksen, Ingvild, Eijsink, Vincent G. H., Beckham, Gregg T., DuBois, Jennifer L., & Røhr, Åsmund K. Molecular mechanism of the chitinolytic peroxygenase reaction. United States. doi:10.1073/pnas.1904889117.
Bissaro, Bastien, Streit, Bennett, Isaksen, Ingvild, Eijsink, Vincent G. H., Beckham, Gregg T., DuBois, Jennifer L., and Røhr, Åsmund K. Mon . "Molecular mechanism of the chitinolytic peroxygenase reaction". United States. doi:10.1073/pnas.1904889117.
@article{osti_1581447,
title = {Molecular mechanism of the chitinolytic peroxygenase reaction},
author = {Bissaro, Bastien and Streit, Bennett and Isaksen, Ingvild and Eijsink, Vincent G. H. and Beckham, Gregg T. and DuBois, Jennifer L. and Røhr, Åsmund K.},
abstractNote = {Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H 2 O 2 as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H 2 O 2 -mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H 2 O 2 into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper–oxyl intermediate. The initial cleavage of H 2 O 2 and subsequent hydrogen atom abstraction from chitin by the copper–oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO–Cu(II) to LPMO–Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO–Cu(I) is 2,000-fold faster with H 2 O 2 than with O 2 , the latter being several orders of magnitude slower than rates reported for other monooxygenases. The presence of substrate accelerated reoxidation by H 2 O 2 , whereas reoxidation by O 2 became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.},
doi = {10.1073/pnas.1904889117},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = ,
volume = ,
place = {United States},
year = {2020},
month = {1}
}

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Works referenced in this record:

Fungal Cellulases
journal, January 2015

  • Payne, Christina M.; Knott, Brandon C.; Mayes, Heather B.
  • Chemical Reviews, Vol. 115, Issue 3
  • DOI: 10.1021/cr500351c

On the catalytic mechanisms of lytic polysaccharide monooxygenases
journal, April 2016


Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism
journal, December 2013

  • Kim, S.; Stahlberg, J.; Sandgren, M.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 1
  • DOI: 10.1073/pnas.1316609111

Harnessing the potential of LPMO-containing cellulase cocktails poses new demands on processing conditions
journal, November 2015

  • Müller, Gerdt; Várnai, Anikó; Johansen, Katja Salomon
  • Biotechnology for Biofuels, Vol. 8, Issue 1
  • DOI: 10.1186/s13068-015-0376-y

The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases
journal, February 2016

  • Frandsen, Kristian E. H.; Simmons, Thomas J.; Dupree, Paul
  • Nature Chemical Biology, Vol. 12, Issue 4
  • DOI: 10.1038/nchembio.2029

The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces
journal, January 2012


The Non-catalytic Chitin-binding Protein CBP21 from Serratia marcescens Is Essential for Chitin Degradation
journal, June 2005

  • Vaaje-Kolstad, Gustav; Horn, Svein J.; van Aalten, Daan M. F.
  • Journal of Biological Chemistry, Vol. 280, Issue 31
  • DOI: 10.1074/jbc.M504468200

Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars
journal, November 2017


How a Lytic Polysaccharide Monooxygenase Binds Crystalline Chitin
journal, March 2018


Stimulation of Lignocellulosic Biomass Hydrolysis by Proteins of Glycoside Hydrolase Family 61 Structure and Function of a Large, Enigmatic Family
journal, April 2010

  • Harris, Paul; Welner, Ditte; McFarland, K.
  • Biochemistry, Vol. 49, Issue 15, p. 3305-3316
  • DOI: 10.1021/bi100009p

Oxidoreductases and Reactive Oxygen Species in Conversion of Lignocellulosic Biomass
journal, September 2018

  • Bissaro, Bastien; Várnai, Anikó; Røhr, Åsmund K.
  • Microbiology and Molecular Biology Reviews, Vol. 82, Issue 4
  • DOI: 10.1128/MMBR.00029-18

Kinetic insights into the role of the reductant in H 2 O 2 -driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase
journal, December 2018

  • Kuusk, Silja; Kont, Riin; Kuusk, Piret
  • Journal of Biological Chemistry, Vol. 294, Issue 5
  • DOI: 10.1074/jbc.RA118.006196

An Oxidative Enzyme Boosting the Enzymatic Conversion of Recalcitrant Polysaccharides
journal, October 2010

  • Vaaje-Kolstad, Gustav; Westereng, Bjørge; Horn, Svein J.
  • Science, Vol. 330, Issue 6001, p. 219-222
  • DOI: 10.1126/science.1192231

Structural basis for the enhancement of virulence by viral spindles and their in vivo crystallization
journal, March 2015

  • Chiu, Elaine; Hijnen, Marcel; Bunker, Richard D.
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 13, p. 3973-3978
  • DOI: 10.1073/pnas.1418798112

Crystal and Molecular Structure of Hydrogen Peroxide: A Neutron‐Diffraction Study
journal, May 1965

  • Busing, William R.; Levy, Henri A.
  • The Journal of Chemical Physics, Vol. 42, Issue 9
  • DOI: 10.1063/1.1696379

An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion
journal, February 2018


Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components
journal, August 2011

  • Quinlan, R. J.; Sweeney, M. D.; Lo Leggio, L.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 37, p. 15079-15084
  • DOI: 10.1073/pnas.1105776108

Discovery of the combined oxidative cleavage of plant xylan and cellulose by a new fungal polysaccharide monooxygenase
journal, July 2015

  • Frommhagen, Matthias; Sforza, Stefano; Westphal, Adrie H.
  • Biotechnology for Biofuels, Vol. 8, Issue 1
  • DOI: 10.1186/s13068-015-0284-1

Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure
journal, January 2017

  • Villares, Ana; Moreau, Céline; Bennati-Granier, Chloé
  • Scientific Reports, Vol. 7, Issue 1
  • DOI: 10.1038/srep40262

Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2
journal, August 2017

  • Bissaro, Bastien; Røhr, Åsmund K.; Müller, Gerdt
  • Nature Chemical Biology, Vol. 13, Issue 10
  • DOI: 10.1038/nchembio.2470

Generating Cu II -Oxyl/Cu III -Oxo Species from Cu I -α-Ketocarboxylate Complexes and O 2 : In Silico Studies on Ligand Effects and CH-Activation Reactivity
journal, May 2009

  • Huber, Stefan M.; Ertem, Mehmed Z.; Aquilante, Francesco
  • Chemistry - A European Journal, Vol. 15, Issue 19
  • DOI: 10.1002/chem.200802338

Targeting the reactive intermediate in polysaccharide monooxygenases
journal, July 2017

  • Hedegård, Erik D.; Ryde, Ulf
  • JBIC Journal of Biological Inorganic Chemistry, Vol. 22, Issue 7
  • DOI: 10.1007/s00775-017-1480-1

Lytic xylan oxidases from wood-decay fungi unlock biomass degradation
journal, January 2018

  • Couturier, Marie; Ladevèze, Simon; Sulzenbacher, Gerlind
  • Nature Chemical Biology, Vol. 14, Issue 3
  • DOI: 10.1038/nchembio.2558

Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases
journal, April 2015

  • Vermaas, Josh V.; Crowley, Michael F.; Beckham, Gregg T.
  • The Journal of Physical Chemistry B, Vol. 119, Issue 20
  • DOI: 10.1021/acs.jpcb.5b00778

Kinetic studies of Rhus vernicifera laccase
journal, October 1976

  • Andréasson, Lars-Erik; Reinhammar, Bengt
  • Biochimica et Biophysica Acta (BBA) - Enzymology, Vol. 445, Issue 3
  • DOI: 10.1016/0005-2744(76)90112-1

Expression of an insecticidal fern protein in cotton protects against whitefly
journal, September 2016

  • Shukla, Anoop Kumar; Upadhyay, Santosh Kumar; Mishra, Manisha
  • Nature Biotechnology, Vol. 34, Issue 10
  • DOI: 10.1038/nbt.3665

Extracellular electron transfer systems fuel cellulose oxidative degradation
journal, April 2016


Discovery and industrial applications of lytic polysaccharide mono-oxygenases
journal, February 2016

  • Johansen, Katja S.
  • Biochemical Society Transactions, Vol. 44, Issue 1
  • DOI: 10.1042/BST20150204

Catalytic Mechanism of Fungal Lytic Polysaccharide Monooxygenases Investigated by First-Principles Calculations
journal, December 2017


The impact of hydrogen peroxide supply on LPMO activity and overall saccharification efficiency of a commercial cellulase cocktail
journal, July 2018

  • Müller, Gerdt; Chylenski, Piotr; Bissaro, Bastien
  • Biotechnology for Biofuels, Vol. 11, Issue 1
  • DOI: 10.1186/s13068-018-1199-4

Copper Active Sites in Biology
journal, January 2014

  • Solomon, Edward I.; Heppner, David E.; Johnston, Esther M.
  • Chemical Reviews, Vol. 114, Issue 7
  • DOI: 10.1021/cr400327t

A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase
journal, August 2014


The Role of the Secondary Coordination Sphere in a Fungal Polysaccharide Monooxygenase
journal, March 2017


Oxidative Cleavage of Cellulose by Fungal Copper-Dependent Polysaccharide Monooxygenases
journal, December 2011

  • Beeson, William T.; Phillips, Christopher M.; Cate, Jamie H. D.
  • Journal of the American Chemical Society, Vol. 134, Issue 2
  • DOI: 10.1021/ja210657t

Spectroscopic characterization of the peroxide intermediate in the reduction of dioxygen catalyzed by the multicopper oxidases
journal, October 1991

  • Cole, James L.; Ballou, David P.; Solomon, Edward I.
  • Journal of the American Chemical Society, Vol. 113, Issue 22
  • DOI: 10.1021/ja00022a064

Kinetics of H 2 O 2 -driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase
journal, November 2017

  • Kuusk, Silja; Bissaro, Bastien; Kuusk, Piret
  • Journal of Biological Chemistry, Vol. 293, Issue 2
  • DOI: 10.1074/jbc.M117.817593

Density‐functional thermochemistry. III. The role of exact exchange
journal, April 1993

  • Becke, Axel D.
  • The Journal of Chemical Physics, Vol. 98, Issue 7, p. 5648-5652
  • DOI: 10.1063/1.464913

Single-molecule study of oxidative enzymatic deconstruction of cellulose
journal, October 2017


Cleavage of cellulose by a CBM33 protein
journal, August 2011

  • Forsberg, Zarah; Vaaje-Kolstad, Gustav; Westereng, Bjørge
  • Protein Science, Vol. 20, Issue 9
  • DOI: 10.1002/pro.689

Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content
journal, January 2012

  • Cannella, David; Hsieh, Chia-wen C.; Felby, Claus
  • Biotechnology for Biofuels, Vol. 5, Issue 1
  • DOI: 10.1186/1754-6834-5-26

Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase
journal, December 2016

  • O'Dell, William B.; Agarwal, Pratul K.; Meilleur, Flora
  • Angewandte Chemie International Edition, Vol. 56, Issue 3
  • DOI: 10.1002/anie.201610502

Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability
journal, December 2017

  • Kracher, Daniel; Andlar, Martina; Furtmüller, Paul G.
  • Journal of Biological Chemistry, Vol. 293, Issue 5
  • DOI: 10.1074/jbc.RA117.000109

The Paleozoic Origin of Enzymatic Lignin Decomposition Reconstructed from 31 Fungal Genomes
journal, June 2012


Kinetic Isotope Effects as Probes of the Mechanism of Galactose Oxidase
journal, June 1998

  • Whittaker, Mei M.; Ballou, David P.; Whittaker, James W.
  • Biochemistry, Vol. 37, Issue 23
  • DOI: 10.1021/bi980328t

Molecular mechanism of lytic polysaccharide monooxygenases
journal, January 2018

  • Hedegård, Erik Donovan; Ryde, Ulf
  • Chemical Science, Vol. 9, Issue 15
  • DOI: 10.1039/C8SC00426A

The carbohydrate-active enzymes database (CAZy) in 2013
journal, November 2013

  • Lombard, Vincent; Golaconda Ramulu, Hemalatha; Drula, Elodie
  • Nucleic Acids Research, Vol. 42, Issue D1
  • DOI: 10.1093/nar/gkt1178

The Poulos−Kraut Mechanism of Compound I Formation in Horseradish Peroxidase:  A QM/MM Study
journal, June 2006

  • Derat, Etienne; Shaik, Sason
  • The Journal of Physical Chemistry B, Vol. 110, Issue 21
  • DOI: 10.1021/jp055412e

Oxidoreductive Cellulose Depolymerization by the Enzymes Cellobiose Dehydrogenase and Glycoside Hydrolase 61
journal, August 2011

  • Langston, James A.; Shaghasi, Tarana; Abbate, Eric
  • Applied and Environmental Microbiology, Vol. 77, Issue 19, p. 7007-7015
  • DOI: 10.1128/AEM.05815-11

Correlation of copper valency with product formation in single turnovers of dopamine .beta.-monooxygenase
journal, May 1989

  • Brenner, Mitchell C.; Klinman, Judith P.
  • Biochemistry, Vol. 28, Issue 11
  • DOI: 10.1021/bi00437a023

The equilibrium structure of hydrogen peroxide
journal, January 2018

  • Baraban, Joshua H.; Changala, P. Bryan; Stanton, John F.
  • Journal of Molecular Spectroscopy, Vol. 343
  • DOI: 10.1016/j.jms.2017.09.014

Inner-Sphere Mechanism for Molecular Oxygen Reduction Catalyzed by Copper Amine Oxidases
journal, July 2008

  • Mukherjee, Arnab; Smirnov, Valeriy V.; Lanci, Michael P.
  • Journal of the American Chemical Society, Vol. 130, Issue 29
  • DOI: 10.1021/ja801378f

Climbing the Density Functional Ladder: Nonempirical Meta–Generalized Gradient Approximation Designed for Molecules and Solids
journal, September 2003


Physiological and Molecular Understanding of Bacterial Polysaccharide Monooxygenases
journal, June 2017

  • Agostoni, Marco; Hangasky, John A.; Marletta, Michael A.
  • Microbiology and Molecular Biology Reviews, Vol. 81, Issue 3
  • DOI: 10.1128/MMBR.00015-17

Crystal Structure and Binding Properties of the Serratia marcescens Chitin-binding Protein CBP21
journal, December 2004

  • Vaaje-Kolstad, G.; Houston, D. R.; Riemen, A. H. K.
  • Journal of Biological Chemistry, Vol. 280, Issue 12
  • DOI: 10.1074/jbc.M407175200

A Random-Sequential Kinetic Mechanism for Polysaccharide Monooxygenases
journal, April 2018


Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases
journal, June 2014

  • Kjaergaard, C. H.; Qayyum, M. F.; Wong, S. D.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 24
  • DOI: 10.1073/pnas.1408115111

Multipoint Precision Binding of Substrate Protects Lytic Polysaccharide Monooxygenases from Self-Destructive Off-Pathway Processes
journal, June 2018


Fueling biomass-degrading oxidative enzymes by light-driven water oxidation
journal, January 2016

  • Bissaro, Bastien; Forsberg, Zarah; Ni, Yan
  • Green Chemistry, Vol. 18, Issue 19
  • DOI: 10.1039/C6GC01666A

Reactivity of O 2 versus H 2 O 2 with polysaccharide monooxygenases
journal, April 2018

  • Hangasky, John A.; Iavarone, Anthony T.; Marletta, Michael A.
  • Proceedings of the National Academy of Sciences, Vol. 115, Issue 19
  • DOI: 10.1073/pnas.1801153115

Modulation of the ligand-based fluorescence of 3d metal complexes upon spin state change
journal, January 2015

  • Lochenie, Charles; Wagner, Kristina G.; Karg, Matthias
  • Journal of Materials Chemistry C, Vol. 3, Issue 30
  • DOI: 10.1039/C5TC00837A

Cellulose Degradation by Polysaccharide Monooxygenases
journal, June 2015


Structural diversity of lytic polysaccharide monooxygenases
journal, June 2017

  • Vaaje-Kolstad, Gustav; Forsberg, Zarah; Loose, Jennifer SM
  • Current Opinion in Structural Biology, Vol. 44
  • DOI: 10.1016/j.sbi.2016.12.012

Oxidations catalyzed by fungal peroxygenases
journal, April 2014