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

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.

[1]  Natércia F. Brás,et al.  Docking and molecular dynamics studies on the stereoselectivity in the enzymatic synthesis of carbohydrates , 2009 .

[2]  K. Dill,et al.  Binding of small-molecule ligands to proteins: "what you see" is not always "what you get". , 2009, Structure.

[3]  J. Vincken,et al.  Two General Branching Patterns of Xyloglucan, XXXG and XXGG , 1997, Plant physiology.

[4]  Peter J. Reilly,et al.  Specific empirical free energy function for automated docking of carbohydrates to proteins , 2003, J. Comput. Chem..

[5]  Pekka Mark,et al.  Analysis of nasturtium TmNXG1 complexes by crystallography and molecular dynamics provides detailed insight into substrate recognition by family GH16 xyloglucan endo‐transglycosylases and endo‐hydrolases , 2009, Proteins.

[6]  David C. Cantu,et al.  Theory and computation show that Asp463 is the catalytic proton donor in human endoplasmic reticulum alpha-(1-->2)-mannosidase I. , 2008, Carbohydrate research.

[7]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[8]  Karl Nicholas Kirschner,et al.  GLYCAM06: A generalizable biomolecular force field. Carbohydrates , 2008, J. Comput. Chem..

[9]  S. Fry,et al.  Pre-formed xyloglucans and xylans increase in molecular weight in three distinct compartments of a maize cell-suspension culture , 2003, Planta.

[10]  K. Nishitani,et al.  The XTH family of enzymes involved in xyloglucan endotransglucosylation and endohydrolysis: current perspectives and a new unifying nomenclature. , 2002, Plant & cell physiology.

[11]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[12]  Brandi L. Cantarel,et al.  The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics , 2008, Nucleic Acids Res..

[13]  G. Fincher,et al.  A Barley Xyloglucan Xyloglucosyl Transferase Covalently Links Xyloglucan, Cellulosic Substrates, and (1,3;1,4)-β-D-Glucans* , 2007, Journal of Biological Chemistry.

[14]  S. Fry,et al.  Purification of xyloglucan endotransglycosylases (XETs): a generally applicable and simple method based on reversible formation of an enzyme-substrate complex. , 1999, The Biochemical journal.

[15]  T. Teeri,et al.  Substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase HvXET6 from barley (Hordeum vulgare L.) , 2009, The FEBS journal.

[16]  Anthony D. Hill,et al.  Automated docking to explore subsite binding by glycoside hydrolase family 6 cellobiohydrolases and endoglucanases. , 2007, Biopolymers.

[17]  R. Baran,et al.  Ping-pong character of nasturtium-seed xyloglucan endotransglycosylase (XET) reaction. , 2000, General physiology and biophysics.

[18]  Modelling of xyloglucan, pectins and pectic side chains binding onto cellulose microfibrils , 2008 .

[19]  K. Nishitani,et al.  Endo-xyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecule to another xyloglucan molecule. , 1992, The Journal of biological chemistry.

[20]  R. Newman,et al.  Solid-state 13C NMR study of a composite of tobacco xyloglucan and Gluconacetobacter xylinus cellulose: molecular interactions between the component polysaccharides. , 2009, Biomacromolecules.

[21]  P. H. Schünmann,et al.  Expression of XET‐related genes and its relation to elongation in leaves of barley (Hordeum vulgare L.) , 1997 .

[22]  P. Reilly,et al.  The fate of β-d-mannopyranose after its formation by endoplasmic reticulum α-(1→2)-mannosidase I catalysis , 2007 .

[23]  K. Mazeau,et al.  The xyloglucan–cellulose assembly at the atomic scale , 2006, Biopolymers.

[24]  M. Sternberg,et al.  Protein structure prediction on the Web: a case study using the Phyre server , 2009, Nature Protocols.

[25]  Chandrika Mulakala,et al.  Docking studies on glycoside hydrolase Family 47 endoplasmic reticulum alpha-(1-->2)-mannosidase I to elucidate the pathway to the substrate transition state. , 2006, Carbohydrate research.

[26]  M. Sippl.  Recognition of errors in three‐dimensional structures of proteins , 1993, Proteins.

[27]  R. Baran,et al.  Divergent modes of action on xyloglucan of two isoenzymes of xyloglucan endo-transglycosylase from Tropaeolum majus , 2003 .

[28]  Z. Sulová,et al.  Cleavage of xyloglucan by nasturtium seed xyloglucanase and transglycosylation to xyloglucan subunit oligosaccharides. , 1992, Archives of biochemistry and biophysics.

[29]  David S. Goodsell,et al.  Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function , 1998 .

[30]  David S. Goodsell,et al.  A semiempirical free energy force field with charge‐based desolvation , 2007, J. Comput. Chem..

[31]  Andrew G. Glen,et al.  APPL , 2001 .

[32]  E. Baydoun,et al.  Biosynthesis and cell-wall deposition of a pectin–xyloglucan complex in pea , 2005, Planta.

[33]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[34]  H. Brumer,et al.  Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremulaxtremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris. , 2005, The Biochemical journal.

[35]  M S Waterman,et al.  Identification of common molecular subsequences. , 1981, Journal of molecular biology.

[36]  Daniel J. Cosgrove,et al.  Loosening of plant cell walls by expansins , 2000, Nature.

[37]  H. Brumer,et al.  Structural Evidence for the Evolution of Xyloglucanase Activity from Xyloglucan Endo-Transglycosylases: Biological Implications for Cell Wall Metabolism[W] , 2007, The Plant Cell Online.

[38]  Peter J. Reilly,et al.  A Gibbs free energy correlation for automated docking of carbohydrates , 2008, J. Comput. Chem..

[39]  H. Brumer,et al.  Crystal Structures of a Poplar Xyloglucan Endotransglycosylase Reveal Details of Transglycosylation Acceptor Binding , 2004, The Plant Cell Online.

[40]  S. Fry,et al.  Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants. , 1992, The Biochemical journal.

[41]  S. Fry,et al.  The Arabidopsis TCH4 Xyloglucan Endotransglycosylase (Substrate Specificity, pH Optimum, and Cold Tolerance) , 1997, Plant physiology.

[42]  K. Keegstra,et al.  The Structure of Plant Cell Walls: III. A Model of the Walls of Suspension-cultured Sycamore Cells Based on the Interconnections of the Macromolecular Components. , 1973, Plant physiology.

[43]  J. Botto,et al.  The plant cell , 2007, Plant Molecular Biology Reporter.

[44]  P. Campbell,et al.  In vitro activities of four xyloglucan endotransglycosylases from Arabidopsis. , 1999, The Plant journal : for cell and molecular biology.