Xyloglucan is ubiquitous in the cell walls of land plants and is also an essential storage polymer in seeds of many species. We studied the hydrolysis of the non-reducing end xylosyl residue of xyloglucan oligosaccharides (XGOs) by the Escherichia coli α-xylosidase (YicI). Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS) and ion fragmentation analysis together with high performance anion exchange chromatography with pulsed amperometric detection revealed that YicI preferentially removes the xylosyl residue from the glycosyl residue of non-galactosylated oligosaccharides. The YicI shows decreasing activity against the galactosylated oligosaccharides XXXG>XXLG≥XLXG. Studies of the XGOs interaction with active site residues by molecular dynamics simulations suggested that hydrogen bond interactions between the D49 and galactosylated oligosaccharides play an important role in enzyme-XGO interactions. This was confirmed by site-directed mutagenesis, where the D49A mutant affected catalytic efficiency against galactosylated XGOs. Our findings advance xyloglucan disassembly models and highlight the importance of YicI for biotechnology applications.
Keywords: Binding site; ESI-MS; HPAEC; Molecular dynamics simulation; Xyloglucan; Xylooligosaccharides.
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