Phylogenetic variation in glycosidases and glycanases acting on plant cell wall polysaccharides, and the detection of transglycosidase and trans-β-xylanase activities

Wall polysaccharide chemistry varies phylogenetically, suggesting a need for variation in wall enzymes. Although plants possess the genes for numerous putative enzymes acting on wall carbohydrates, the activities of the encoded proteins often remain conjectural. To explore phylogenetic differences in demonstrable enzyme activities, we extracted proteins from 57 rapidly growing plant organs with three extractants, and assayed their ability to act on six oligosaccharides 'modelling' selected cell-wall polysaccharides. Based on reaction products, we successfully distinguished exo- and endo-hydrolases and found high taxonomic variation in all hydrolases screened: beta-D-xylosidase, endo-(1 -> 4)-beta-D-xylanase, beta-D-mannosidase, endo-(1 -> 4)-beta-D-mannanase, alpha-D-xylosidase, beta-D-galactosidase, alpha-L-arabinosidase and alpha-L-fucosidase. The results, as GHATAbase, a searchable compendium in Excel format, also provide a compilation for selecting rich sources of enzymes acting on wall carbohydrates. Four of the hydrolases were accompanied, sometimes exceeded, by transglycosylase activities, generating products larger than the substrate. For example, during beta-xylosidase assays on (1 -> 4)-beta-D-xylohexaose (Xyl(6)), Marchantia, Selaginella and Equisetum extracts gave negligible free xylose but approximately equimolar Xyl(5) and Xyl(7), indicating trans-beta-xylosidase activity, also found in onion, cereals, legumes and rape. The yield of Xyl(9) often exceeded that of Xyl(7-8), indicating that beta-xylanase was accompanied by an endotransglycosylase activity, here called trans-beta-xylanase, catalysing the reaction 2Xyl(6) -> Xyl(3) + Xyl(9). Similar evidence also revealed trans-alpha-xylosidase, trans-alpha-arabinosidase and trans-alpha-arabinanase activities acting on xyloglucan oligosaccharides and (1 -> 5)-alpha-L-arabino-oligosaccharides. In conclusion, diverse plants differ dramatically in extractable enzymes acting on wall carbohydrate, reflecting differences in wall polysaccharide composition. Besides glycosidase and glycanase activities, five new transglycosylase activities were detected. We propose that such activities function in the assembly and re-structuring of the wall matrix.