title: Enzymatic Activity of Xyloglucan Xylosyltransferase 5.
abstract: Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a β-(1,4)-glucan backbone decorated with d-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher Km and 9-fold lower kcat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone.
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011.
authors.forename: Alan T
authors.initials: AT
authors.lastname: Culbertson
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011.
authors.forename: Yi-Hsiang
authors.initials: YH
authors.lastname: Chou
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011.
authors.forename: Adrienne L
authors.initials: AL
authors.lastname: Smith
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011.
authors.forename: Zachary T
authors.initials: ZT
authors.lastname: Young
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011.
authors.forename: Alesia A
authors.initials: AA
authors.lastname: Tietze
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Cottaz
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011.
authors.forename: Régis
authors.identifier: 0000-0002-5107-9009
authors.initials: R
authors.lastname: Fauré
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 zabotina@iastate.edu.
authors.forename: Olga A
authors.identifier: 0000-0001-6938-6923
authors.initials: OA
authors.lastname: Zabotina
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D016296:Mutagenesis; D009844:Oligosaccharides / Q000378:metabolism; D010430:Pentosyltransferases / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014540:Uridine Diphosphate Xylose / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; C099423:cellohexaose; D014540:Uridine Diphosphate Xylose; D010430:Pentosyltransferases; C029368:xyloglucan xylosyltransferase