THE BACKBONE OF THE PECTIC POLYSACCHARIDE RHAMNOGALACTURONAN-I IS CLEAVED BY AN ENDOHYDROLASE AND AN ENDOLYASE

Rhamnogalacturonan I(RG-I), a major pectic component of the primary wa lls of plant cells, is believed to play an important role in determini ng both the structure and functions of the walls, A more detailed stru ctural description of RG-I is likely to lead to a greater understandin g of the biological roles of this polysaccharide. Two enzymes secreted by Aspergillus aculeatus that have been cloned and expressed in a fun gal system (Kofod et al., J, Biol. Chem., 269, 29182-29189, 1994) clea ve the RG-I backbone in an endo fashion and should assist in the furth er structural characterization of this polysaccharide. We found that b oth of the available preparations of the cloned enzymes were contamina ted with exoglycanases, reducing their utility in structurally charact erizing RG-I, We purified the enzymes to apparent homogeneity by ion-e xchange chromatography and then used the purified enzymes to generate backbone oligosaccharide fragments from partially debranched sycamore RG-I, The backbone oligosaccharides, which were separated from larger pieces of partially debranched RG-I by gel-permeation chromatography: have been structurally characterized by H-1-NMR spectroscopy, electros pray MS, GC-MS, highperformance anion-exchange chromatography with pul sed amperometric detection (HPAEC-PAD) and UV spectroscopy, The result s of these analyses establish that rhamnogalacturonase A (RGase A) is an endohydrolase that cleaves the -4-alpha-D-Galp A-(1-2)-alpha-L-Rhap glycosidic linkage, However, the purported rhamnogalacturonase B (RGa se B) is, in fact, an endolyase that cleaves the -2-alpha-L-Rhap-(1-4) -alpha-D-Galp A glycosidic linkage, thereby generating oligosaccharide s terminating at the non-reducing end with a hex-4-enopyranosyluronic acid residue.