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 ...

The cDNA encoding vanillyl-alcohol oxidase (EC 1.1.3.7) was selected from a cDNA library constructed from mRNA isolated from Penicillium simplicissimum CBS 170.90 grown on veratryl alcohol by immunochemical screening. The vaoA-cDNA nucleotide sequence revealed an open reading frame of 1680 base pairs encoding a 560-amino acid protein with a deduced mass of 62,915 Da excluding the covalently bound FAD. The deduced primary structure shares 31% sequence identity with the 8␣-(O-tyrosyl)-FAD containing subunit of the bacterial flavocytochrome p-cresol methyl hydroxylase.The vaoA gene was isolated from a P. simplicissimum genomic library constructed in EMBL3 using the vaoA-cDNA as a probe. Comparison of the nucleotide sequence of the vaoA gene with the cDNA nucleotide sequence demonstrated that the gene is interrupted by five short introns.Aspergillus niger NW156 prtF pyrA leuA cspA transformed with the pyrA containing plasmid and a plasmid harboring the complete vaoA gene including the promoter and terminator was able to produce vaoA mRNA and active vanillyl-alcohol oxidase when grown on veratryl alcohol and anisyl alcohol. A similar induction of the vaoA gene was found for P. simplicissimum, indicating that similar regulatory systems are involved in the induction of the vaoA gene in these fungi.Introduction of a consensus ribosome binding site, AGAAGGAG, in the vaoA-cDNA resulted in elevated expression levels of active vanillyl-alcohol oxidase from the lac promoter in Escherichia coli TG2. The catalytic and spectral properties of the purified recombinant enzyme were indistinguishable from the native enzyme.Vanillyl-alcohol oxidase (EC 1.1.3.7) is a novel type of flavoprotein oxidase that was first isolated from Penicillium simplicissimum CBS 170.90 grown on veratryl alcohol (1). The enzyme is a homo-octamer with each 65-kDa subunit harboring an 8␣-(N 3 -histidyl)-FAD (2). Vanillyl-alcohol oxidase has a broad substrate specificity. In addition to the conversion of vanillyl alcohol to vanillin (Equation 1), the enzyme catalyzes the conversion of a wide range of phenolic compounds bearing side chains of variable size at the para-position of the aromatic ring (3, 4). Due to its broad substrate spectrum, vanillyl-alcohol oxidase may be applied in the fine chemical industry (5). Based on induction experiments, 4-(methoxymethyl)phenol has been proposed to represent the physiological substrate (6). Recently, from rapid reaction kinetics conclusive spectral evidence was obtained that the vanillyl-alcohol oxidase-mediated oxidative demethylation of 4-(methoxymethyl)phenol proceeds through the formation of a quinone-methide product intermediate (4). In the absence of oxygen, this intermediate is stabilized in the active site of the reduced enzyme. Upon flavin reoxidation, the quinone methide of 4-(methoxymethyl)phenol readily reacts with water, yielding 4-hydroxybenzaldehyde, methanol, and hydrogen peroxide as final products.Recently, the three-dimensional structure of vanillyl-alcohol oxidase was elucidated (7). The crystallo...