Crystal Structures of a Poplar Xyloglucan Endotransglycosylase Reveal Details of Transglycosylation Acceptor Binding

Abstract

Xyloglucan endotransglycosylases (XETs) cleave and religate xyloglucan polymers in plant cell walls via a transglycosylation mechanism. Thus, XET is a key enzyme in all plant processes that require cell wall remodeling. To provide a basis for detailed structure–function studies, the crystal structure of Populus tremula x tremuloides XET16A (PttXET16A), heterologously expressed in Pichia pastoris, has been determined at 1.8-Å resolution. Even though the overall structure of PttXET16A is a curved β-sandwich similar to other enzymes in the glycoside hydrolase family GH16, parts of its substrate binding cleft are more reminiscent of the distantly related family GH7. In addition, XET has a C-terminal extension that packs against the conserved core, providing an additional β-strand and a short α-helix. The structure of XET in complex with a xyloglucan nonasaccharide, XLLG, reveals a very favorable acceptor binding site, which is a necessary but not sufficient prerequisite for transglycosylation. Biochemical data imply that the enzyme requires sugar residues in both acceptor and donor sites to properly orient the glycosidic bond relative to the catalytic residues

A Barley Xyloglucan Xyloglucosyl Transferase Covalently Links Xyloglucan, Cellulosic Substrates, and (1,3;1,4)-β-D-Glucans

“…The models were subsequently supported by the published three-dimensional structure of the Populus tremula ϫ tremuloides XET (36). The plant XTHs and microbial (1,3;1,4)-␤-Dglucan endohydrolases are all classified in the family GH16 group of glycoside hydrolases, although a small number of microbial xyloglucan endohydrolases are also classified within families GH5, GH12, GH44, and GH74 (37).…”

“…The difficulties encountered during the purification procedure of HvXET5 were largely attributable to the presence of numerous hydrophobic patches on the surface of the enzyme, as predicted from the three-dimensional structure of the Populus XET (36). However, after the HvXET5 enzyme was purified, its activity remained more or less constant for at least a year at Ϫ20°C.…”

Group III-AXTHGenes of Arabidopsis Encode Predominant Xyloglucan Endohydrolases That Are Dispensable for Normal Growth

“…1). AtXTH31 and AtXTH32 have 66% and 69% identities with TmNXG1, respectively (82% and 83% similarities, respectively), whereas their identities with the group I representative, Populus tremula 3 Populus tremuloides XET16-34 (Johansson et al, 2004;Kallas et al, 2005), drop to 35% and 37% identities, respectively (50% and 52% similarities, respectively).…”

Structure and Substrate Specificity of a Eukaryotic Fucosidase from Fusarium graminearum

“…FgFCO1 as expressed in P. pastoris or after treatment with peptide N-glycosidase F failed to grow crystals with the same sets of screens, and an overall trend of higher solubility was observed for these highly glycosylated forms of the enzyme compared with Endo H-deglycosylated fucosidase. Endo H-treated The XXFG nonasaccharide structure is drawn in stereo in analogy to the structures of the xyloglucan fragment XLLG in complex with GH16-family endoxyloglucanase (PDB codes 2VH9 and 1UMZ) (77,78). The shorthand nomenclature for xyloglucan oligosaccharides XXFG and XLLG (the latter not shown) is based on McDougall and Fry (76).…”