Glycosylation Is Vital for Industrial Performance of Hyperactive Cellulases

Chung, Daehwan; Sarai, Nicholas S.; Knott, Brandon C.; Hengge, Neal; Russell, Jordan F.; Yarbrough, John M.; Brunecky, Roman; Young, Jenna; Supekar, Nitin; Vander Wall, Todd; Sammond, Deanne W.; Crowley, Michael F.; Szymanski, Christine M.; Wells, Lance; Azadi, Parastoo; Westpheling, Janet; Himmel, Michael E.; Bomble, Yannick J.

doi:10.1021/acssuschemeng.8b05049

Title: Glycosylation Is Vital for Industrial Performance of Hyperactive Cellulases

Journal Article · 31 January 2019 · ACS Sustainable Chemistry & Engineering

DOI: https://doi.org/10.1021/acssuschemeng.8b05049 · OSTI ID:1497985

Chung, Daehwan ^[1]; Sarai, Nicholas S. ^[2]; Knott, Brandon C. ^[1]; Hengge, Neal ^[1]; Russell, Jordan F. ^[3]; Yarbrough, John M. ^[1]; Brunecky, Roman ^[1]; Young, Jenna ^[3]; Supekar, Nitin ^[3]; Vander Wall, Todd ^[1];

^[1];

^[1]; Szymanski, Christine M. ^[3]; Wells, Lance ^[3]; Azadi, Parastoo ^[3]; Westpheling, Janet ^[3]; Himmel, Michael E. ^[1];

^[1]

National Renewable Energy Lab. (NREL), Golden, CO (United States)
National Renewable Energy Lab. (NREL), Golden, CO (United States); California Inst. of Technology (CalTech), Pasadena, CA (United States)
Univ. of Georgia, Athens, GA (United States)

In the terrestrial biosphere, biomass deconstruction is conducted by microbes employing a variety of complementary strategies, many of which remain to be discovered. Moreover, the biofuels industry seeks more efficient (and less costly) cellulase formulations upon which to launch the nascent sustainable bioenergy economy. The glycan decoration of fungal cellulases has been shown to protect these enzymes from protease action and to enhance binding to cellulose. We show here that thermal tolerant bacterial cellulases are glycosylated as well, although the types and extents of decoration differ from their Eukaryotic counterparts. Our major findings are that glycosylation of CelA is uniform across its three linker peptides and composed of mainly galactose disaccharides (which is unique) and that this glycosylation dramatically impacts the hydrolysis of insoluble substrates, proteolytic and thermal stability, and substrate binding and changes the dynamics of the enzyme. This study suggests that the glycosylation of CelA is crucial for its exceptionally high cellulolytic activity on biomass and provides the robustness needed for this enzyme to function in harsh environments including industrial settings.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)

Grant/Contract Number:: AC36-08GO28308

OSTI ID:: 1497985

Report Number(s):: NREL/JA-2700-73100

Journal Information:: ACS Sustainable Chemistry & Engineering, Journal Name: ACS Sustainable Chemistry & Engineering Journal Issue: 5 Vol. 7; ISSN 2168-0485

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

References (49)

Glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies Kayser, Veysel; Chennamsetty, Naresh; Voynov, Vladimir Biotechnology Journal, Vol. 6, Issue 1 https://doi.org/10.1002/biot.201000091	journal	October 2010
Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris cutinase: Role of glycosylation: Cutinase Thermal Inactivation Shirke, Abhijit N.; Su, An; Jones, J. Andrew Biotechnology and Bioengineering, Vol. 114, Issue 1 https://doi.org/10.1002/bit.26052	journal	September 2016
Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective Guex, Nicolas; Peitsch, Manuel C.; Schwede, Torsten ELECTROPHORESIS, Vol. 30, Issue S1 https://doi.org/10.1002/elps.200900140	journal	June 2009
Scalable molecular dynamics with NAMD Phillips, James C.; Braun, Rosemary; Wang, Wei Journal of Computational Chemistry, Vol. 26, Issue 16, p. 1781-1802 https://doi.org/10.1002/jcc.20289	journal	January 2005
CHARMM: The biomolecular simulation program Brooks, B. R.; Brooks, C. L.; Mackerell, A. D. Journal of Computational Chemistry, Vol. 30, Issue 10 https://doi.org/10.1002/jcc.21287	journal	July 2009
Computational protein design with explicit consideration of surface hydrophobic patches Jacak, Ron; Leaver-Fay, Andrew; Kuhlman, Brian Proteins: Structure, Function, and Bioinformatics, Vol. 80, Issue 3 https://doi.org/10.1002/prot.23241	journal	December 2011
Glycomic and glycoproteomic analysis of glycoproteins—a tutorial Shajahan, Asif; Heiss, Christian; Ishihara, Mayumi Analytical and Bioanalytical Chemistry, Vol. 409, Issue 19 https://doi.org/10.1007/s00216-017-0406-7	journal	June 2017
Probing the role of N-linked glycans in the stability and activity of fungal cellobiohydrolases by mutational analysis Adney, William S.; Jeoh, Tina; Beckham, Gregg T. Cellulose, Vol. 16, Issue 4 https://doi.org/10.1007/s10570-009-9305-1	journal	June 2009
Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains Langsford, M. L.; Gilkes, N. R.; Singh, B. FEBS Letters, Vol. 225, Issue 1-2 https://doi.org/10.1016/0014-5793(87)81150-X	journal	December 1987
Isolation and characterization of plant cell walls and cell wall components York, William S.; Darvill, Alan G.; McNeil, Michael Methods in Enzymology https://doi.org/10.1016/0076-6879(86)18062-1	book	January 1986
[12] High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates Hardy, Mark R.; Reid Townsend, R. Methods in Enzymology, Vol. 230, 208-225 https://doi.org/10.1016/0076-6879(94)30014-3	book	January 1994
The nature of the carbohydrate-peptide linkage region in glycoproteins from the cellulosomes of Clostridium thermocellum and Bacteroides cellulosolvens. Gerwig, G. J.; Kamerling, J. P.; Vliegenthart, J. F. Journal of Biological Chemistry, Vol. 268, Issue 36 https://doi.org/10.1016/S0021-9258(19)74203-3	journal	December 1993
Protein Structure Prediction Using Rosetta Rohl, Carol A.; Strauss, Charlie E. M.; Misura, Kira M. S. Methods in Enzymology https://doi.org/10.1016/S0076-6879(04)83004-0	book	January 2004
Interrelationship of Steric Stabilization and Self-Crowding of a Glycosylated Protein Høiberg-Nielsen, R.; Westh, P.; Skov, L. K. Biophysical Journal, Vol. 97, Issue 5 https://doi.org/10.1016/j.bpj.2009.05.045	journal	September 2009
The O-Glycosylated Linker from the Trichoderma reesei Family 7 Cellulase Is a Flexible, Disordered Protein Beckham, Gregg T.; Bomble, Yannick J.; Matthews, James F. Biophysical Journal, Vol. 99, Issue 11 https://doi.org/10.1016/j.bpj.2010.10.032	journal	December 2010
Lignocellulose deconstruction in the biosphere Bomble, Yannick J.; Lin, Chien-Yuan; Amore, Antonella Current Opinion in Chemical Biology, Vol. 41 https://doi.org/10.1016/j.cbpa.2017.10.013	journal	December 2017
Harnessing glycosylation to improve cellulase activity Beckham, Gregg T.; Dai, Ziyu; Matthews, James F. Current Opinion in Biotechnology, Vol. 23, Issue 3 https://doi.org/10.1016/j.copbio.2011.11.030	journal	June 2012
Tool for Rapid Analysis of Glycopeptide by Permethylation via One-Pot Site Mapping and Glycan Analysis Shajahan, Asif; Supekar, Nitin T.; Heiss, Christian Analytical Chemistry, Vol. 89, Issue 20 https://doi.org/10.1021/acs.analchem.7b01730	journal	October 2017
Sabatier Principle for Interfacial (Heterogeneous) Enzyme Catalysis Kari, Jeppe; Olsen, Johan P.; Jensen, Kenneth ACS Catalysis, Vol. 8, Issue 12 https://doi.org/10.1021/acscatal.8b03547	journal	October 2018
Role of glycosylation on the conformation and chain dimensions of O-linked glycoproteins: light-scattering studies of ovine submaxillary mucin Shogren, Randal; Gerken, Thomas A.; Jentoft, Neil Biochemistry, Vol. 28, Issue 13 https://doi.org/10.1021/bi00439a029	journal	June 1989
Protein glycosylation in bacteria: sweeter than ever Nothaft, Harald; Szymanski, Christine M. Nature Reviews Microbiology, Vol. 8, Issue 11 https://doi.org/10.1038/nrmicro2383	journal	October 2010
The Multi Domain Caldicellulosiruptor bescii CelA Cellulase Excels at the Hydrolysis of Crystalline Cellulose Brunecky, Roman; Donohoe, Bryon S.; Yarbrough, John M. Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-017-08985-w	journal	August 2017
Modeling protein quaternary structure of homo- and hetero-oligomers beyond binary interactions by homology Bertoni, Martino; Kiefer, Florian; Biasini, Marco Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-017-09654-8	journal	September 2017
The impact of O -glycan chemistry on the stability of intrinsically disordered proteins Prates, Erica T.; Guan, Xiaoyang; Li, Yaohao Chemical Science, Vol. 9, Issue 15 https://doi.org/10.1039/C7SC05016J	journal	January 2018
A Colorimetric Assay for Glycoproteins Based on the Periodic Acid/Schiff Stain Mantle, MichÈLe; Allen, Adrian Biochemical Society Transactions, Vol. 6, Issue 3 https://doi.org/10.1042/bst0060607	journal	June 1978
Cloning, expression and characterization of a Family 48 exocellulase, Cel48A, from Thermobifida fusca: Thermobifida fusca exocellulase Cel48A Irwin, Diana C.; Zhang, Sheng; Wilson, David B. European Journal of Biochemistry, Vol. 267, Issue 16 https://doi.org/10.1046/j.1432-1327.2000.01546.x	journal	August 2000
Comparison of simple potential functions for simulating liquid water Jorgensen, William L.; Chandrasekhar, Jayaraman; Madura, Jeffry D. The Journal of Chemical Physics, Vol. 79, Issue 2 https://doi.org/10.1063/1.445869	journal	July 1983
Effect of glycosylation on protein folding: A close look at thermodynamic stabilization Shental-Bechor, D.; Levy, Y. Proceedings of the National Academy of Sciences, Vol. 105, Issue 24 https://doi.org/10.1073/pnas.0801340105	journal	June 2008
Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis Gao, D.; Chundawat, S. P. S.; Sethi, A. Proceedings of the National Academy of Sciences, Vol. 110, Issue 27 https://doi.org/10.1073/pnas.1213426110	journal	June 2013
Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose Payne, Christina M.; Resch, Michael G.; Chen, Liqun Proceedings of the National Academy of Sciences, Vol. 110, Issue 36 https://doi.org/10.1073/pnas.1309106110	journal	August 2013
Specificity of O-glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules Chen, L.; Drake, M. R.; Resch, M. G. Proceedings of the National Academy of Sciences, Vol. 111, Issue 21 https://doi.org/10.1073/pnas.1402518111	journal	May 2014
Distinct roles of N- and O-glycans in cellulase activity and stability Amore, Antonella; Knott, Brandon C.; Supekar, Nitin T. Proceedings of the National Academy of Sciences, Vol. 114, Issue 52 https://doi.org/10.1073/pnas.1714249114	journal	December 2017
Native protein sequences are close to optimal for their structures Kuhlman, B.; Baker, D. Proceedings of the National Academy of Sciences, Vol. 97, Issue 19 https://doi.org/10.1073/pnas.97.19.10383	journal	September 2000
Predicting Enzyme Adsorption to Lignin Films by Calculating Enzyme Surface Hydrophobicity Sammond, Deanne W.; Yarbrough, John M.; Mansfield, Elisabeth Journal of Biological Chemistry, Vol. 289, Issue 30 https://doi.org/10.1074/jbc.M114.573642	journal	May 2014
Toward the estimation of the absolute quality of individual protein structure models Benkert, Pascal; Biasini, Marco; Schwede, Torsten Bioinformatics, Vol. 27, Issue 3 https://doi.org/10.1093/bioinformatics/btq662	journal	December 2010
Biological roles of glycans Varki, Ajit Glycobiology, Vol. 27, Issue 1 https://doi.org/10.1093/glycob/cww086	journal	August 2016
The SWISS-MODEL Repository—new features and functionality Bienert, Stefan; Waterhouse, Andrew; de Beer, Tjaart A. P. Nucleic Acids Research, Vol. 45, Issue D1 https://doi.org/10.1093/nar/gkw1132	journal	November 2016
SWISS-MODEL: homology modelling of protein structures and complexes Waterhouse, Andrew; Bertoni, Martino; Bienert, Stefan Nucleic Acids Research, Vol. 46, Issue W1 https://doi.org/10.1093/nar/gky427	journal	May 2018
Primary structure of O-linked carbohydrate chains in the cellulosome of different Clostridium thermocellum strains Gerwig, Gerrit J.; Kamerling, Johannis P.; Vliegenthart, Johannes F. G. European Journal of Biochemistry, Vol. 196, Issue 1 https://doi.org/10.1111/j.1432-1033.1991.tb15793.x	journal	February 1991
Novel oligosaccharide constituents of the cellulase complex of Bacteroides cellulosolvens Gerwig, Gerrit J.; Kamerling, Johannis P.; Vliegenthart, Johannes F. G. European Journal of Biochemistry, Vol. 205, Issue 2 https://doi.org/10.1111/j.1432-1033.1992.tb16844.x	journal	April 1992
Revealing Nature's Cellulase Diversity The Digestion Mechanism of Caldicellulosiruptor bescii CelA Brunecky, R.; Alahuhta, M.; Xu, Q. Science, Vol. 342, Issue 6165, p. 1513-1516 https://doi.org/10.1126/science.1244273	journal	December 2013
Use of Label-Free Quantitative Proteomics To Distinguish the Secreted Cellulolytic Systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis Lochner, Adriane; Giannone, Richard J.; Rodriguez, Miguel Applied and Environmental Microbiology, Vol. 77, Issue 12 https://doi.org/10.1128/AEM.02811-10	journal	April 2011
Sequence Analysis of Scaffolding Protein CipC and ORFXp, a New Cohesin-Containing Protein inClostridium cellulolyticum: Comparison of Various Cohesin Domains and Subcellular Localization of ORFXp Pagès, Sandrine; Bélaïch, Anne; Fierobe, Henri-Pierre Journal of Bacteriology, Vol. 181, Issue 6 https://doi.org/10.1128/JB.181.6.1801-1810.1999	journal	March 1999
Implications of cellobiohydrolase glycosylation for use in biomass conversion Jeoh, Tina; Michener, William; Himmel, Michael E. Biotechnology for Biofuels, Vol. 1, Issue 1 https://doi.org/10.1186/1754-6834-1-10	journal	January 2008
Deletion of Caldicellulosiruptor besciiCelA reveals its crucial role in the deconstruction of lignocellulosic biomass Young, Jenna; Chung, Daehwan; Bomble, Yannick J. Biotechnology for Biofuels, Vol. 7, Issue 1 https://doi.org/10.1186/s13068-014-0142-6	journal	October 2014
New perspective on glycoside hydrolase binding to lignin from pretreated corn stover Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth Biotechnology for Biofuels, Vol. 8, Issue 1 https://doi.org/10.1186/s13068-015-0397-6	journal	December 2015
High activity CAZyme cassette for improving biomass degradation in thermophiles Brunecky, Roman; Chung, Daehwan; Sarai, Nicholas S. Biotechnology for Biofuels, Vol. 11, Issue 1 https://doi.org/10.1186/s13068-018-1014-2	journal	February 2018
Homologous Expression of the Caldicellulosiruptor bescii CelA Reveals that the Extracellular Protein Is Glycosylated Chung, Daehwan; Young, Jenna; Bomble, Yannick J. PLOS ONE, Vol. 10, Issue 3 https://doi.org/10.1371/journal.pone.0119508	journal	March 2015
Synergism in Binary Mixtures of Thermobifida fusca Cellulases Cel6B, Cel9A, and Cel5A on BMCC and Avicel Watson, Donna L.; Wilson, David B.; Walker, Larry P. Applied Biochemistry and Biotechnology, Vol. 101, Issue 2 https://doi.org/10.1385/ABAB:101:2:097	journal	January 2002

Cited By (2)

Iron incorporation both intra- and extra-cellularly improves the yield and saccharification of switchgrass (Panicum virgatum L.) biomass Lin, Chien-Yuan; Donohoe, Bryon S.; Bomble, Yannick J. Biotechnology for Biofuels, Vol. 14, Issue 1 https://doi.org/10.1186/s13068-021-01891-4	journal	March 2021
Engineering Geobacillus thermoglucosidasius for direct utilisation of holocellulose from wheat straw Bashir, Zeenat; Sheng, Lili; Anil, Annamma Biotechnology for Biofuels, Vol. 12, Issue 1 https://doi.org/10.1186/s13068-019-1540-6	journal	August 2019

Similar Records

Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated

Journal Article · 2015 · PLoS ONE · OSTI ID:1220729

Chung, Daehwan; Young, Jenna; Bomble, Yannick J.; +4 more

Harnessing Glycosylation to Improve Cellulase Activity

Journal Article · 2012 · Current Opinion in Biotechnology · OSTI ID:1045063

Beckham, Gregg T.; Dai, Ziyu; Matthews, James F.; +5 more

Harnessing glycosylation to improve cellulase activity

Journal Article · 2012 · Current Opinion in Biotechnology, 23(3):338-345 · OSTI ID:1048603

Beckham, Gregg T; Dai, Ziyu; Mattews, James F; +5 more

Related Subjects

09 BIOMASS FUELS
CAZymes
Caldicellulosiruptor bescii
biofuels
cellulolytic anaerobes
enzyme stability
galactose
glycosylation

Title: Glycosylation Is Vital for Industrial Performance of Hyperactive Cellulases

Citation Formats

References (49)

Cited By (2)

Similar Records

Related Subjects