title: A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro.
abstract: 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. 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.
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908.
authors.forename: Pallinti
authors.initials: P
authors.lastname: Purushotham
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802.
authors.forename: Sung Hyun
authors.initials: SH
authors.lastname: Cho
authors.affiliation: Division of Glycoscience, Royal Institute of Technology, Stockholm, SE-10691, Sweden.
authors.forename: Sara M
authors.initials: SM
authors.lastname: Díaz-Moreno
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802.
authors.forename: Manish
authors.initials: M
authors.lastname: Kumar
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802.
authors.forename: B Tracy
authors.initials: BT
authors.lastname: Nixon
authors.affiliation: Division of Glycoscience, Royal Institute of Technology, Stockholm, SE-10691, Sweden; Australian Research Council Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Urrbrae, SA, 5064, Australia.
authors.forename: Vincent
authors.initials: V
authors.lastname: Bulone
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908; jochen_zimmer@virginia.edu.
authors.forename: Jochen
authors.initials: J
authors.lastname: Zimmer
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D002480:Cellulase / Q000378:metabolism; D002482:Cellulose / Q000096:biosynthesis* / Q000648:ultrastructure; D003600:Cytosol / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry; D020894:Microfibrils / Q000378:metabolism* / Q000648:ultrastructure; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D032107:Populus / Q000201:enzymology*; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D013997:Time Factors
chemical_list: D006027:Glycosides; D007527:Isoenzymes; D008055:Lipids; D010940:Plant Proteins; D011994:Recombinant Proteins; D002482:Cellulose; D005964:Glucosyltransferases; C478648:cellulose synthase; D002480:Cellulase
keywords: biopolymer; cellulose; glycosyltransferase; membrane transport; plant cell wall