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Title: A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 ( PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’smore » N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Lastly, our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.« less
Authors:
 [1] ;  [2] ;  [3] ;  [2] ;  [2] ;  [4] ;  [5]
  1. Univ. of Virginia School of Medicine, Charlottesville, VA (United States)
  2. The Pennsylvania State Univ., University Park, PA (United States)
  3. Royal Institute of Technology, Stockholm (Sweden)
  4. Royal Institute of Technology, Stockholm (Sweden); Univ. of Adelaide, Urrbrae (Australia)
  5. Univ. of Virginia School of Medicine, Charlottesville, VA (United States); Univ. of California, Berkeley, CA (United States)
Publication Date:
Grant/Contract Number:
SC0001090
Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 113; Journal Issue: 40; Related Information: CLSF partners with Pennsylvania State University (lead); North Carolina State University; University of Rhode Island; Virginia Tech University; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
Energy Frontier Research Centers (EFRC), Washington, D.C. (United States). Center for Lignocellulose Structure and Formation (CLSF)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; biofuels (including algae and biomass); bio-inspired; membrane; carbon sequestration; materials and chemistry by design; synthesis (self-assembly)
OSTI Identifier:
1325834
Alternate Identifier(s):
OSTI ID: 1388837

Purushotham, Pallinti, Cho, Sung Hyun, Diaz-Moreno, Sara M., Kumar, Manish, Nixon, B. Tracy, Bulone, Vincent, and Zimmer, Jochen. A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro. United States: N. p., Web. doi:10.1073/pnas.1606210113.
Purushotham, Pallinti, Cho, Sung Hyun, Diaz-Moreno, Sara M., Kumar, Manish, Nixon, B. Tracy, Bulone, Vincent, & Zimmer, Jochen. A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro. United States. doi:10.1073/pnas.1606210113.
Purushotham, Pallinti, Cho, Sung Hyun, Diaz-Moreno, Sara M., Kumar, Manish, Nixon, B. Tracy, Bulone, Vincent, and Zimmer, Jochen. 2016. "A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro". United States. doi:10.1073/pnas.1606210113.
@article{osti_1325834,
title = {A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro},
author = {Purushotham, Pallinti and Cho, Sung Hyun and Diaz-Moreno, Sara M. and Kumar, Manish and Nixon, B. Tracy and Bulone, Vincent and Zimmer, Jochen},
abstractNote = {Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Lastly, our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.},
doi = {10.1073/pnas.1606210113},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 40,
volume = 113,
place = {United States},
year = {2016},
month = {9}
}