Characterisation of five GH16 glycanase and transglycanase activities and of their hemicellulosic substrates

Abstract

Plant primary cell walls are hydrated extracellular complexes composed largely of polysaccharides: cellulose, hemicellulose and pectin. Cell wall constituents and composition vary in cell-, environment-, and species-dependent manners. For example, within land plant hemicelluloses xyloglucan is ubiquitous while mixedlinkage (1→3),(1→4)-β-D-glucan (MLG) is found only in the Poales and Equisetum. Glycosyl hydrolase 16 (GH16) enzyme family members include numerous enzymes with pertinence to the understanding of the ‘lives’ of cell wall hemicelluloses. However, despite this, the details of the interactions between GH16 enzymes and their substrates have often not been elucidated. Likewise, the true preferences of many of these enzymes and the range of substrates which they can utilise remain to be fully explored. By providing a greater wealth of information for the correlation of enzyme structure with reaction catalysed, such an understanding would enable better predictions of the activities of novel enzymes. Crucially, this would also allow better identification of roles performed by these enzymes in planta as well as of the potential applications of these enzymes. This work sought to further our understanding of the interactions between GH16 enzymes and their substrates by the study of five activities exhibited by GH16 enzymes – xyloglucan endotransglucosylase (XET), xyloglucan endoglucanase/hydrolase (XEG/XEH), mixed-linkage glucan : xyloglucan endotransglucosylase (MXE), lichenase and cellulose : xyloglucan endotransglucosylase (CXE). All of the analysed activities act on xyloglucan and/or MLG. Of particular focus is the novel enzyme MXE from the evolutionarily isolated genus Equisetum (horsetail), which acts on both. Notable findings include: identification of MXE/CXE gene; determination of the substrate specificity of MXE; defining of the sites of attack of lichenase, XEG, XET and MXE; discovery of novel xyloglucan structures and discrepancies between the xyloglucan present in different barley organs.