Inversion of configuration during hydrolysis of α‐1,4‐galacturonidic linkage by three Aspergillus polygalacturonases

Abstract

Endopolygalacturonases I and lI (PGI and PGII) ofAspergillus niger and an exopolygalacturonase (ExoPG) of A. tubingensis were investigated to reveal the stereochemistry of their hydrolytic action. Reduced pentagalacturonic acid (pentaGalU-ol) and reduced trigalacturonic acid (triGalU-ol) were used as non-reducing substrates for the enzymes. The configuration of the reducing ends in the products formed in D20 reaction mixtures was followed by I H-NMR spectroscopy. It has been unambiguously established that primary cleavage of pentaGalUol by both PGI and PGII leads to diGalU-ol and the [~-anomer of triGalUA. The primary products of hydrolysis of triGalU-ol by ExoPG were diGalU-ol and the ~anomer of GaIUA. Thus, all three Aspergillus polygalacturonases belong to the so-called inverting glycanases, i.e. they utilize the single displacement mechanism of hydrolysis of the glycosidic linkage.

Reaction Mechanism Based on X-ray Crystallography at Atomic Resolution of Endopolygalacutronase I from Fungus <i>Stereum purpureum</i>

“…EndoPG is an inverting enzyme. 25) As indicated previously, 11,12) the distances between the absolutely conserved three aspartates are 4.4 A (Asp153 Asp173), 5.5 A (Asp153 Asp174), and 4.8 A (Asp173 Asp174), respectively. No conserved acidic residues are found at a distance of about 10 A from each other.…”

AspergillusEnzymes Involved in Degradation of Plant Cell Wall Polysaccharides

“…Recently the activity of an endo-acting xylogalacturonase has been characterized (377) that is specific for a xylose-substituted galacturonic acid backbone. The stereochemical course of hydrolysis of several enzymes involved in the degradation of the main chain of the pectin hairy regions was studied recently (39,297). Enzymes acting on the hairy regions of pectin, exogalacturonase, rhamnogalacturonan hydrolase, rhamnogalacturonan rhamnohydrolase, and ␣-rhamnosidase (297), as well as enzymes acting on the smooth regions, endopolygalacturonase I and II (39), all hydrolyzed the substrate via an inverting mechanism.…”

“…The stereochemical course of hydrolysis of several enzymes involved in the degradation of the main chain of the pectin hairy regions was studied recently (39,297). Enzymes acting on the hairy regions of pectin, exogalacturonase, rhamnogalacturonan hydrolase, rhamnogalacturonan rhamnohydrolase, and ␣-rhamnosidase (297), as well as enzymes acting on the smooth regions, endopolygalacturonase I and II (39), all hydrolyzed the substrate via an inverting mechanism. Sequencing of the corresponding genes grouped all Aspergillus endo-and exopolygalacturonases and rhamnogalacturonases in the same glycosidase family, which has an inverting mechanism of hydrolysis (Table 8).…”

ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1), ADPG2, and QUARTET2 Are Polygalacturonases Required for Cell Separation during Reproductive Development inArabidopsis

“…Homogalacturonan-rich pectin is commonly found in the middle lamella region of the cell wall where two adjacent cells abut and pectin integrity is important for cell adhesion (MacDougall et al, 1996;Ridley et al, 2001). Endopolygalacturonases (endo-PGs) catalyze random hydrolysis of a-1,4-glycosidic linkages in polygalacturonic acid (GalUA), a polymer that constitutes the main chain of the homogalacturonan region of pectin (Biely et al, 1996). Although there is only limited direct genetic evidence for the physiological importance of individual PGs, correlations have been reported between increasing PG activity and cell separation in fruit ripening and in the shedding of leaves, flowers, and fruit (Taylor et al, 1993;Kalaitzis et al, 1995;Brown, 1997;Kalaitzis et al, 1997).…”