Title: Distinct roles of N- and O-glycans in cellulase activity and stability

Abstract

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. In this paper, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalytic domain (CD) - a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans,more » namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict a-helix formation and decreased cellulose interaction for the nonglycosylated linker. In conclusion, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.« less

Authors:

Amore, Antonella ^[1]; Knott, Brandon C. ^[2]; Supekar, Nitin T.  ^[3];

• Search DOE PAGES for author "Supekar, Nitin T."
• Search DOE PAGES for ORCID "0000-0002-7006-7177"

Shajahan, Asif ^[3]; Azadi, Parastoo ^[3]; Zhao, Peng ^[3]; Wells, Lance ^[3]; Linger, Jeffrey G. ^[2]; Hobdey, Sarah E. ^[1]; Vander Wall, Todd A. ^[1]; Shollenberger, Todd ^[1]; Yarbrough, John M. ^[1]; Tan, Zhongping  ^[4];

• Search DOE PAGES for author "Tan, Zhongping"
• Search DOE PAGES for ORCID "0000-0002-9302-150X"

Crowley, Michael F. ^[1]; Himmel, Michael E. ^[1]; Decker, Stephen R.  ^[1];

• Search DOE PAGES for author "Decker, Stephen R."
• Search DOE PAGES for ORCID "0000-0001-7002-9034"

Beckham, Gregg T.  ^[2];

• Search DOE PAGES for author "Beckham, Gregg T."
• Search DOE PAGES for ORCID "0000-0002-3480-212X"

Taylor, II, Larry E. ^[1]

---

+ Show Author Affiliations

1. Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401,
2. National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401,
3. Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602,
4. Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80303,, BioFrontiers Institute, University of Colorado, Boulder, CO 80303

Publication Date:

Mon Dec 11 00:00:00 EST 2017

Research Org.:

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

Sponsoring Org.:

USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office (BETO); USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences, and Biosciences Division

OSTI Identifier:

1518529

Alternate Identifier(s):

OSTI ID: 1417800

Report Number(s):

NREL/JA-5100-70178
Journal ID: ISSN 0027-8424

Grant/Contract Number:  

SC0015662; AC36-08GO28308

Resource Type:

Published Article

Journal Name:

Proceedings of the National Academy of Sciences of the United States of America

Additional Journal Information:

Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 114 Journal Issue: 52; Journal ID: ISSN 0027-8424

Publisher:

Proceedings of the National Academy of Sciences

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; 54 ENVIRONMENTAL SCIENCES; 09 BIOMASS FUELS; glycoside hydrolase; glycosylation; linker; intrinsically disordered protein; mannosylation; cellulase