Structure of xylogalacturonan fragments from watermelon cell-wall pectin. Endopolygalacturonase can accommodate a xylosyl residue on the galacturonic acid just following the hydrolysis site Original Research Article

A combination of xylogalacturonan (XGA), homogalacturonan, and rhamnogalacturonan was extracted from watermelon fruit cell walls with 0.1 M NaOH. In contrast to the resistance of xylogalacturonans from most other sources to endopolygalacturonase (EPG), about 50% of the extracted XGA could be converted into oligosaccharides by EPG digestion with a commercial EPG from Megazyme International. The oligosaccharides were fractionated by ion-exchange chromatography, and their structures were investigated by mass spectrometry and NMR spectroscopy. The smallest oligosaccharide was β-d-Xylp-(1→3)-α-d-GalAp-(1→4)-α-d-GalAp-(1→4)-α-d-GalAp-(1→4)-GalAp. The most abundant was β-d-Xylp-(1→3)-α-d-GalAp-(1→4)-α-d-GalAp-(1→4)(β-d-Xylp-(1→3)-α-d-GalAp-(1→4))-α-d-GalAp-(1→4)-α-d-GalAp-(1→4)-GalAp. Given that the nonreducing ends of the oligosaccharides often were xylosylated GalA residues, and that fungal EPG digests homogalacturonans between the third and fourth GalA bound to the enzyme, it appears that EPG can accommodate a xylosylated GalA in the site that binds the fourth GalA. Since all of the oligosaccharides characterized had three unsubstituted GalA residues at their reducing ends, the enzyme appears not to accommodate xylosylated residues in the first three sugar-binding sites. Thus, XGA regions with fewer than three unsubstituted residues between branch points will be resistant to EPG. The EPG-susceptible XGA was not recovered from cell walls prepared using phosphate buffer for the homogenization of the watermelon tissue, probably because it was degraded by endogenous watermelon EPG and lost during isolation of the walls. Use of Tris-buffered phenol during wall isolation to prevent enzyme action caused some amidation of GalA residues with Tris.