The Active Site Topology of Aspergillus nigerEndopolygalacturonase II as Studied by Site-directed Mutagenesis

Abstract

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 ...

Structural requirements of endo polygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

“…A single displacement reaction may be catalyzed where an acidic residue acts as a general acid (Asp212) by donating a proton to the glycosydic oxygen, and residues Asp-191 and Asp-213 activate a water molecule, which performs a nucleophilic attack on the anomeric carbon. The unusual distance between the three Asp residues and between Asp-212 and the water molecule is consistent with the suggestion that the proton donation and the nucleophilic attack are performed at the same side with respect to the ␣-glycosydic bond to be hydrolyzed (40). It is worth noting the relative low presence of positive charges in the cleft (see Fig.…”

Fungal Endopolygalacturonases Are Recognized as Microbe-Associated Molecular Patterns by the Arabidopsis Receptor-Like Protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1

“…The substitutions chosen for generating the catalytically inactive Bcpg3 D353E/D354N protein were designed based on studies of Armand et al (2000) on the catalytic residues of Aspergillus niger pgB. For mutant protein production in P. pastoris, sitedirected mutagenesis was carried out by amplifying two fragments of mutant allele Bcpg3 D353E/D354N from pPIC3.5-Bcpg3 (Kars et al, 2005) with primers AT261/ AT269 and AT270/AT262, which were linked by overlap PCR with primers AT261/AT262.…”

Targeted Modification of Homogalacturonan by Transgenic Expression of a Fungal Polygalacturonase Alters Plant Growth

“…The level of PG expression was higher for lines 1 and 2 and lower for lines 5, 6, and 13. Arabidopsis plants were also transformed with the gene encoding the mutated form of AnPGII (AnPGII D201N) in which the substitution of Asp-201 by Asn causes a complete loss of enzyme activity (Armand et al, 2000;Federici et al, 2001). Western-blot analysis of protein extracts with antibodies against AnPGII showed the presence of a 38-kD band in the plants transformed with either the wild type or the mutated AnPGII gene.…”