Polygalacturonases specifically hydrolyze polygalacturonate, a major constituent of plant cell wall pectin. To understand the catalytic mechanism and substrate and product specificity of these enzymes, we have solved the x-ray structure of endopolygalacturonase II of Aspergillus niger and we have carried out site-directed mutagenesis studies. The enzyme folds into a right-handed parallel -helix with 10 complete turns. The -helix is composed of four parallel -sheets, and has one very small ␣-helix near the N terminus, which shields the enzyme's hydrophobic core. Loop regions form a cleft on the exterior of the -helix. The plant cell wall consists of a network of complex carbohydrates like cellulose, hemicellulose, and pectin. The latter is the most complex of these carbohydrates. It contains so-called "smooth regions" and "hairy regions." The smooth regions, also known as homogalacturonan, consist of ␣(1,4)-linked D-galacturonic acid residues, whereas the hairy regions, or rhamnogalacturonan I, are characterized by stretches of alternating ␣(1,2)-linked D-galacturonic acid and L-rhamnose (1). The rhamnose residues can be substituted at their O4 atoms by arabinose or galactose (2). Throughout the pectin molecule, the galacturonic acid residues can be methylated at O6 and/or acetylated at O2 and/or O3 (3). Due to its complex structure, modification of pectin by plants or complete breakdown by microorganisms requires many different enzymes.In microorganisms several classes of pectinases have been identified. These classes comprise pectate-, pectin-, and rhamnogalacturonan lyases, rhamnogalacturonan hydrolases, and polygalacturonases, which all depolymerize the main chain; and pectin methylesterases and pectin-and rhamnogalacturonan acetylesterases, which act on the substituents of the main chain. Crystal structures are known of members of several classes of main chain depolymerizing pectinases. These include pectate lyases from Erwinia chrysanthemi and Bacillus subtilis (4 -6), pectin lyases from Aspergillus niger (7,8), and rhamnogalacturonase A from Aspergillus aculeatus (9). Recently, the crystal structure of endopolygalacturonase from the bacterium Erwinia carotovora was solved (10). The lyases cleave the substrate by -elimination, whereas rhamnogalacturonases and polygalacturonases use acid/base-catalyzed hydrolysis (11,12). Despite their completely different reaction mechanisms, and their groupings in different sequence homology families, the x-ray structures of pectate lyase, pectin lyase, and rhamnogalacturonase reveal a similar unique right-handed parallel -helix topology (13,14).
Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K m and V max ) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta-and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.Pectic polysaccharides are among the most complex plant cell wall polysaccharides. In the homogalacturonan part, the so-called smooth regions, the 1,4-␣-D-galacturonic acid backbone is partly esterified. These smooth regions are interspersed by the rhamnogalacturonan parts consisting of repeating stretches of 1,2-␣-L-rhamnose-1,4-␣-D-galacturonic acid dimers. Other sugar residues can be attached to the rhamnose residues (1). Because of this complexity, a wide range of enzymes, the so-called pectinases, is necessary for the complete degradation of pectic substances. Two main classes of depolymerizing enzymes act on these polysaccharides: the hydrolases (endopolygalacturonases and rhamnogalacturonases) and the lyases (pectin lyase, pectate lyase, and rhamnogalacturonan lyase).Endopolygalacturonases (PGs; EC 3.2.1.15) 1 catalyze the random hydrolysis of 1,4-␣-D-galactosiduronic linkages in pectates. They have been isolated from a variety of organisms (eukaryotae and prokaryotae). Furthermore, over 40 genes encoding PGs have been cloned and sequenced. The corresponding enzymes have been grouped in family 28 of the general classification of glycosyl hydrolases based on amino acid sequence similarities (2, 3).The gene encoding the endopolygalacturonase II (PGII) from Aspergillus niger has been previously cloned, sequenced, and expressed in A. niger (4). The enzyme hydrolyses the glycosidic linkages with inversion of configuration (5). Recently, PGII was extensively characterized with respect to activity on polygalacturonic acid, mode of action, and kinetics on oligogalacturonates (6).Two different mechanisms have been identified for glycosyl hydrolases: one resulting in retention and the other in inversion of the configuration at the anomeric carbon of the scissile bond (7,8). Despite this difference, in most glycosidases two residues ...
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