title: The backbone of the pectic polysaccharide rhamnogalacturonan I is cleaved by an endohydrolase and an endolyase.
abstract: Rhamnogalacturonan I (RG-I), a major pectic component of the primary walls of plant cells, is believed to play an important role in determining both the structure and functions of the walls. A more detailed structural description of RG-I is likely to lead to a greater understanding of the biological roles of this polysaccharide. Two enzymes secreted by Aspergillus aculeatus that have been cloned and expressed in a fungal system (Kofod et al., J. Biol. Chem., 269, 29182-29189, 1994) cleave the RG-I backbone in an endo fashion and should assist in the further structural characterization of this polysaccharide. We found that both of the available preparations of the cloned enzymes were contaminated with exoglycanases, reducing their utility in structurally characterizing RG-I. We purified the enzymes to apparent homogeneity by ion-exchange 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 1H-NMR spectroscopy, electrospray MS, GC-MS, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and UV spectroscopy. The results of these analyses establish that rhamnogalacturonase A (RGase A) is an endohydrolase that cleaves the -4)-alpha-D-GalpA-(1-2)-alpha-L-Rhap glycosidic linkage. However, the purported rhamnogalacturonase B (RGase B) is, in fact, an endolyase that cleaves the -2)-alpha-L-Rhap-(1-4)-alpha-D-GalpA glycosidic linkage, thereby generating oligosaccharides terminating at the non-reducing end with a hex-4-enopyranosyluronic acid residue.
authors.affiliation: Complex Carbohydrate Research Center, Department of Biochemistry, University of Georgia, Athens, USA.
authors.forename: P
authors.initials: P
authors.lastname: Azadi
authors.forename: M A
authors.initials: MA
authors.lastname: O'Neill
authors.forename: C
authors.initials: C
authors.lastname: Bergmann
authors.forename: A G
authors.initials: AG
authors.lastname: Darvill
authors.forename: P
authors.initials: P
authors.lastname: Albersheim
mesh_terms: D002240:Carbohydrate Sequence; D002473:Cell Wall / Q000737:chemistry*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D006026:Glycoside Hydrolases / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010368:Pectins / Q000737:chemistry* / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014197:Trees / Q000737:chemistry*
chemical_list: C042491:rhamnogalacturonan I; D010368:Pectins; D006026:Glycoside Hydrolases; C067959:rhamnogalacturonase A; D011133:Polysaccharide-Lyases; C098240:rhamnogalacturonase B