Barley xyloglucan xyloglucosyl transferases bind xyloglucan-derived oligosaccharides in their acceptor-binding regions in multiple conformational states

Abstract

Three barley xyloglucan endotransglycosylases (HvXETs), known as xyloglucan xyloglucosyl transferases (EC 2.4.1.207), were subjected to kinetic and computational docking studies. The k(cat) x K(m)(-1) values with the reduced [3H]-labelled XXXG, XXLG/XLXG and XLLG acceptor substrates were 0.02 x 10(-2), 0.1 x 10(-2) and 3.2 x 10(-2) s(-1) microM(-1), while the K(m) constants were 10.6, 8.6 and 5.3 mM, obtained for HvXET3, HvXET4 and HvXET6, respectively. Docking of XLLG in acceptor-binding regions revealed that at least two conformational states were likely to participate in all isoforms. The assessments of kinetic and computational data indicated that the disposition of aromatic residues at the entrance to the active sites and the flexibility of proximal COOH-terminal loops could orient acceptors more or less favourably during binding, thus leading to tighter or weaker K(m) constants. The data suggested that binding of acceptors in HvXETs is guided by contributions from the conserved residues in the active sites and by the of neighbouring loops.Gustav Vaaje-Kolstad, Vladimír Farkaš, Geoffrey B. Fincher, Maria Hrmov