Department of Biochemistry, Biophysics and Molecular Biology - Iowa State University
Host: Olga Zabotina
"Structure and Function of the Xyloglucan Xylosyltransferases"
The plant cell wall is a complex network composed mainly of polysaccharides, the most abundant biopolymers on earth and a rich source of biorenewable materials. Biosynthesis of these plant polysaccharides is poorly understood, largely due to difficulties in the structural characterization of glycosyltransferases and lack of suitable substrates for in vitro analysis. Xyloglucan Xylosyltransferases (XXTs) initiate side-chain extensions from a linear glucan polymer by transferring the xylosyl group from UDP-xylose during xyloglucan biosynthesis.
We optimized protein expression and enzymatic activity conditions of XXTs through numerous N- and C-terminal truncations, various E. coli strains, solubility tags, and storage conditions. This procedure was used for protein expression of three XXTs (XXT1, XXT2, and XXT5) and we show that XXT5 is catalytically active in vitro, though at a significantly slower rate compared to XXT1 or XXT2. As no structural information was available for any of the XXTs, we built a homology model of XXT2. This model was used to predict amino acids involved in UDP-xylose binding that were verified through mutagenesis. The crystal structure of XXT1 demonstrates that XXT1 alone cannot produce xylosylation patterns observed for native xyloglucans because of steric constraints imposed within the acceptor binding cleft. Homology modeling of XXT2 and XXT5, using the crystal structure of XXT1 as template, reveals a structurally altered cleft in XXT5 that could accommodate a partially xylosylated glucan chain produced by XXT1 and/or XXT2. This suggests that XXT1 and XXT2 xylosylate a growing glucan chain to produce the GXXG repeat, which is then utilized by XXT5 to produce the biologically observed XXXG repeat of native xyloglucan present in most of the plants.
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