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J. Biol. Chem., Vol. 283, Issue 32, 21853-21863, August 8, 2008

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Active-site Mapping of a Populus Xyloglucan endo-Transglycosylase with a Library of Xylogluco-oligosaccharides^*

Marc Saura-Valls, Régis Fauré¹, Harry Brumer^¶2, Tuula T. Teeri^¶, Sylvain Cottaz, Hugues Driguez, and Antoni Planas³

From the Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain, the Centre de Recherche sur les Macromolécules Végétales, CNRS, 38041 Grenoble Cedex 9, France, and the ^¶School of Biotechnology, Royal Institute of Technology, AlbaNova University Centre, SE-106 91 Stockholm, Sweden

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 XXXG moiety (G = Glcβ(14) and X = Xyl(16)Glcβ(14)) 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 = Galβ(14)) 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.

Received for publication, April 22, 2008 , and in revised form, May 28, 2008.

^* This work was supported in part by European Union Contract QLK5-CT-2001-00443, Grant 2005SGR 00883 from the Generalitat de Catalunya, Grant BIO2007-67904 from Ministerio de Educación y Ciencia, Spain, and CNRS. The Centre de Recherche sur les Macromolécules Végétales is affiliated with University Joseph Fourier and is a member of the Institut de Chimie Moléculaire de Grenoble, FR-CNRS 2607. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2, Schemes S1 and S2, and additional references.

¹ Present address: INSA Toulouse, F-31077 Toulouse, France.

² Fellow (Rådsforskare) of the Swedish Research Council.

³ To whom correspondence should be addressed. E-mail: antoni.planas{at}iqs.edu.

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