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Active-site mapping of a Populus xyloglucan endo-transglycosylase with a library of xylogluco-oligosaccharides
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO), Glycoscience.
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 32, 21853-21863 p.Article in journal (Refereed) Published
Abstract [en]

Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and + 1). Donor binding is dominated by the higher affinity of the XXXGmoiety (G = Glc beta(1 -> 4) and X = Xyl alpha(1 -> 6)Glc beta(1 -> 4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K-m values for the donor substrates showed that a GG-unit on negative and-XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal = Gal beta(1 -> 4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.

Place, publisher, year, edition, pages
2008. Vol. 283, no 32, 21853-21863 p.
Keyword [en]
plant-cell wall, glycosyl-enzyme intermediate, endoxyloglucan, transferase, endotransglycosylase, mechanism, acceptor, binding, seeds, purification, nomenclature
URN: urn:nbn:se:kth:diva-17741DOI: 10.1074/jbc.M803058200ISI: 000258114700004ScopusID: 2-s2.0-52049124583OAI: diva2:335786
QC 20100525Available from: 2010-08-05 Created: 2010-08-05Bibliographically approved

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