Active-site mapping of a Populus xyloglucan endo-transglycosylase with a library of xylogluco-oligosaccharides.
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=Glcbeta(1-->4) and X=Xylalpha(1-->6)Glcbeta(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=Galbeta(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.
Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain.
Marc
M
Saura-Valls
Régis
R
Fauré
Harry
H
Brumer
Tuula T
TT
Teeri
Sylvain
S
Cottaz
Hugues
H
Driguez
Antoni
A
Planas
D001665:Binding Sites; D019075:Electrophoresis, Capillary; D016695:Glycosyltransferases / Q000378:metabolism*; D007700:Kinetics; D009281:Naphthalenes / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D032107:Populus / Q000201:enzymology*; D054852:Small Molecule Libraries / Q000378:metabolism*; D013997:Time Factors
D009281:Naphthalenes; D009844:Oligosaccharides; D054852:Small Molecule Libraries; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid; D016695:Glycosyltransferases; C073693:xyloglucan endotransglycosylase