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Title: Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases

Abstract

In nature, polysaccharide glycosidic bonds are cleaved by hydrolytic enzymes for a vast array of biological functions. Recently, a new class of enzymes that utilize an oxidative mechanism to cleave glycosidic linkages was discovered; these enzymes are called lytic polysaccharide monooxygenases (LPMO). These oxidative enzymes are synergistic with cocktails of hydrolytic enzymes and are thought to act primarily on crystalline regions, in turn providing new sites of productive attachment and detachment for processive hydrolytic enzymes. In the case of cellulose, the homopolymer of ..beta..-1,4-d-glucose, enzymatic oxidation occurs at either the reducing end or the nonreducing end of glucose, depending on enzymatic specificity, and results in the generation of oxidized chemical substituents at polymer chain ends. LPMO oxidation of cellulose is thought to produce either a lactone at the reducing end of glucose that can spontaneously or enzymatically convert to aldonic acid or 4-keto-aldose at the nonreducing end that may further oxidize to a geminal diol. Here, we use molecular simulation to examine the effect of oxidation on the structure of crystalline cellulose. The simulations highlight variations in behaviors depending on the chemical identity of the oxidized species and its location within the cellulose fibril, as different oxidized species introduce stericmore » effects that disrupt local crystallinity and in some cases reduce the work needed for polymer decrystallization. Reducing-end oxidations are easiest to decrystallize when located at the end of the fibril, whereas nonreducing end oxidations readily decrystallize from internal cleavage sites despite their lower solvent accessibility. The differential in decrystallization free energy suggests a molecular mechanism consistent with experimentally observed LPMO/cellobiohydrolase synergy. Additionally, the soluble oxidized cellobiose products released by hydrolytic cellulases may bind to the active sites of cellulases with different affinities relative to cellobiose itself, which potentially affects hydrolytic turnover through product inhibition. To examine the effect of oxidation on cello-oligomer binding, we use thermodynamic integration to compute the relative change in binding free energy between the hydrolyzed and oxidized products in the active site of Family 7 and Family 6 processive glycoside hydrolases, Trichoderma reesei Cel7A and Cel6A, which are key industrial cellulases and commonly used model systems for fungal cellulases. Our results suggest that the equilibrium between the two reducing end oxidized products, favoring the linear aldonic acid, may increase product inhibition, which would in turn reduce processive substrate turnover. In the case of LMPO action at the nonreducing end, oxidation appears to lower affinity with the nonreducing end specific cellulase, reducing product inhibition and potentially promoting processive cellulose turnover. Overall, this suggests that oxidation of recalcitrant polysaccharides by LPMOs accelerates degradation not only by increasing the concentration of chain termini but also by reducing decrystallization work, and that product inhibition may be somewhat reduced as a result.« less

Authors:
; ; ;
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
OSTI Identifier:
1405914
Report Number(s):
NREL/JA-5100-70390
Journal ID: ISSN 1520-6106
DOE Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article
Journal Name:
Journal of Physical Chemistry. B, Condensed Matter, Materials, Surfaces, Interfaces and Biophysical Chemistry
Additional Journal Information:
Journal Volume: 119; Journal Issue: 20; Journal ID: ISSN 1520-6106
Publisher:
American Chemical Society
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; oxidation; crystalline cellulose; cellulases; structure

Citation Formats

Vermaas, Josh V., Crowley, Michael F., Beckham, Gregg T., and Payne, Christina M. Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases. United States: N. p., 2015. Web. doi:10.1021/acs.jpcb.5b00778.
Vermaas, Josh V., Crowley, Michael F., Beckham, Gregg T., & Payne, Christina M. Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases. United States. doi:10.1021/acs.jpcb.5b00778.
Vermaas, Josh V., Crowley, Michael F., Beckham, Gregg T., and Payne, Christina M. Thu . "Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases". United States. doi:10.1021/acs.jpcb.5b00778.
@article{osti_1405914,
title = {Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases},
author = {Vermaas, Josh V. and Crowley, Michael F. and Beckham, Gregg T. and Payne, Christina M.},
abstractNote = {In nature, polysaccharide glycosidic bonds are cleaved by hydrolytic enzymes for a vast array of biological functions. Recently, a new class of enzymes that utilize an oxidative mechanism to cleave glycosidic linkages was discovered; these enzymes are called lytic polysaccharide monooxygenases (LPMO). These oxidative enzymes are synergistic with cocktails of hydrolytic enzymes and are thought to act primarily on crystalline regions, in turn providing new sites of productive attachment and detachment for processive hydrolytic enzymes. In the case of cellulose, the homopolymer of ..beta..-1,4-d-glucose, enzymatic oxidation occurs at either the reducing end or the nonreducing end of glucose, depending on enzymatic specificity, and results in the generation of oxidized chemical substituents at polymer chain ends. LPMO oxidation of cellulose is thought to produce either a lactone at the reducing end of glucose that can spontaneously or enzymatically convert to aldonic acid or 4-keto-aldose at the nonreducing end that may further oxidize to a geminal diol. Here, we use molecular simulation to examine the effect of oxidation on the structure of crystalline cellulose. The simulations highlight variations in behaviors depending on the chemical identity of the oxidized species and its location within the cellulose fibril, as different oxidized species introduce steric effects that disrupt local crystallinity and in some cases reduce the work needed for polymer decrystallization. Reducing-end oxidations are easiest to decrystallize when located at the end of the fibril, whereas nonreducing end oxidations readily decrystallize from internal cleavage sites despite their lower solvent accessibility. The differential in decrystallization free energy suggests a molecular mechanism consistent with experimentally observed LPMO/cellobiohydrolase synergy. Additionally, the soluble oxidized cellobiose products released by hydrolytic cellulases may bind to the active sites of cellulases with different affinities relative to cellobiose itself, which potentially affects hydrolytic turnover through product inhibition. To examine the effect of oxidation on cello-oligomer binding, we use thermodynamic integration to compute the relative change in binding free energy between the hydrolyzed and oxidized products in the active site of Family 7 and Family 6 processive glycoside hydrolases, Trichoderma reesei Cel7A and Cel6A, which are key industrial cellulases and commonly used model systems for fungal cellulases. Our results suggest that the equilibrium between the two reducing end oxidized products, favoring the linear aldonic acid, may increase product inhibition, which would in turn reduce processive substrate turnover. In the case of LMPO action at the nonreducing end, oxidation appears to lower affinity with the nonreducing end specific cellulase, reducing product inhibition and potentially promoting processive cellulose turnover. Overall, this suggests that oxidation of recalcitrant polysaccharides by LPMOs accelerates degradation not only by increasing the concentration of chain termini but also by reducing decrystallization work, and that product inhibition may be somewhat reduced as a result.},
doi = {10.1021/acs.jpcb.5b00778},
journal = {Journal of Physical Chemistry. B, Condensed Matter, Materials, Surfaces, Interfaces and Biophysical Chemistry},
issn = {1520-6106},
number = 20,
volume = 119,
place = {United States},
year = {2015},
month = {5}
}