title: Bonds broken and formed during the mixed-linkage glucan : xyloglucan endotransglucosylase reaction catalysed byEquisetumhetero-trans-β-glucanase.
abstract: Mixed-linkage glucan∶xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-β-glucanase (HTG), which among land plants is known only fromEquisetumspecies. The biochemical details of the MXE reaction were incompletely understood - details that would promote understanding of MXE's rolein vivoand enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1→3),(1→4)-β-d-glucan (MLG), with radioactive oligosaccharides of xyloglucan as the acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and gel-permeation chromatography (GPC).EquisetumHTG consistently cleaved the MLG at the third consecutive β-(1→4)-bond following (towards the reducing terminus) a β-(1→3)-bond. It then formed a β-(1→4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its xyloglucan endotransglucosylase/hydrolase subfamily membership. Using size-homogeneous barley MLG as the donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative to the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases.
authors.affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, The King's Buildings, Edinburgh EH9 3BF, U.K.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Simmons
authors.affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, The King's Buildings, Edinburgh EH9 3BF, U.K. s.fry@ed.ac.uk.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Fry
mesh_terms: D055162:Biocatalysis; D002240:Carbohydrate Sequence; D002473:Cell Wall / Q000737:chemistry / Q000201:enzymology; D002482:Cellulose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D057075:Enzyme Assays; D020829:Equisetum / Q000737:chemistry / Q000201:enzymology*; D005936:Glucans / Q000737:chemistry* / Q000378:metabolism; D016695:Glycosyltransferases / Q000378:metabolism*; D010936:Plant Extracts / Q000737:chemistry; D010940:Plant Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D013780:Tetroses / Q000737:chemistry* / Q000378:metabolism; D014990:Xylans / Q000737:chemistry* / Q000378:metabolism; D047071:beta-Glucans / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005936:Glucans; D010936:Plant Extracts; D010940:Plant Proteins; D013780:Tetroses; D014990:Xylans; D047071:beta-Glucans; C029353:xyloglucan; C048468:cellotetraose; D002482:Cellulose; D016695:Glycosyltransferases; C473049:xyloglucan - xyloglucosyltransferase
keywords: Equisetum; cell walls; hetero-transglycosylation; mixed-linkage glucan; transglycanases; xyloglucan