In the present study, the molecular and basic biochemical characterization of endopolygalacturonase E, the fourth Aspergillus niger N400 endopolygalacturonase, is reported. The entire endopolygalacturonase E gene consists of 1293 bp interrupted by three short introns (50, 50, and 59 bp, respectively) as concluded from the cDNA sequence. The deduced amino acid sequence comprises 378 residues that include 39 Nterminal amino acids of the prepropeptide. The calculated M r and pI of the mature protein are 35 584 and 3.6, respectively.Compared with other endopolygalacturonases from A. niger N400, the mature protein endopolygalacturonase E has the highest sequence identity with endopolygalacturonase C (77.6 %) followed by endopolygalacturonase I (57.6 %) and endopolygalacturonase II (54.3 %).For overproduction of endopolygalacturonase E, an A. niger multicopy strain was used that was transformed with a promoter gene fusion construct that directs expression from the glycolytic A. niger pyruvate kinase promoter. The enzyme was purified and characterized as an endopolygalacturonase based on product analysis after polygalacturonate hydrolysis and on bond cleavage frequencies of oligogalacturonates of different degree of polymerisation (n ϭ 2Ϫ7). The pH optimum was 3.8. The K m and V max for polygalacturonate hydrolysis were 2.5Ϯ 0.4 mg · ml Ϫ1 and 1.3Ϯ 0.2 µkat · mg Ϫ1 , respectively. A subsite map was calculated by the combination of the methods of Suganuma et al.

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The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 Å and refined to a crystallographic R factor of 18. Because the R218K PelC-galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the ␤ elimination cleavage of polygalacturonic acid. INTRODUCTIONPectate lyases are depolymerizing enzymes that degrade plant cell walls, causing tissue maceration and death. The enzymes normally are secreted by phytopathogenic organisms and are known to be the primary virulence agents in soft rot diseases caused by Erwinia spp (Collmer and Keen, 1986;Kotoujansky, 1987;Barras et al., 1994). In the latter organisms, the enzymes exist as multiple, independently regulated isozymes that share amino acid sequence identity ranging from 27 to 80%.Pectate lyases share sequence similarities with fungal pectin lyases, plant pollen proteins, and plant style proteins (Henrissat et al., 1996). The three-dimensional structures of five members of the superfamily have been determined and include Erwinia chrysanthemi pectate lyase C (PelC) (Yoder et al., 1993;Yoder and Jurnak, 1995), E. chrysanthemi pectate lyase E (PelE) (Lietzke et al., 1994), Bacillus subtilis pectate lyase ( B. subtilis Pel) (Pickersgill et al., 1994), Aspergillus niger pectin lyase A (PLA) (Mayans et al., 1997), and A. niger pectin lyase B (PLB) (Vitali et al., 1998). All share a similar but an unusual structural motif, termed the parallel ␤ helix, in which the ␤ strands are folded into a large, right-handed coil. The enzyme structures differ in the size and conformation of the loops that protrude from the parallel ␤ helix core. As deduced from sequence similarity and site-directed mutagenesis studies, the protruding loops on one side of the parallel ␤ helix form the pectolytic active site (Kita et al., 1996). The structural differences of the loops are believed to be related to subtle differences in the enzymatic and maceration properties of the proteins.Pectate lyases catalyze the cleavage of pectate, the deesterified product of pectin, which is the major component that maintains the structural integrity of cell walls in higher plants The Plant Cell (Carpita and Gibeaut, 1993). The pectate backbone is composed of blocks of polygalacturonic acid (PGA), which is a helical homopolymer of D -galacturonic acid (Gal p A) units linked by ␣ -(1 → 4) glycosidic bonds. The blocks of PGA are separated by stretches in which (1 → 2)-␣ -L -rhamnose residues alternate with Gal p A (McNaught, 1997). Blocks of PGA may contain as many as 200 Gal p A units and span 100 nm (Thibault et al., 1993). Cations are necessary to neutralize PGA in solution and, as a consequence, influence its struct...

Endopolygalacturonases I, II and C isolated from recombinant Aspergillus niger strains were characterized with respect to pH optimum, activity on polygalacturonic acid and mode of action and kinetics on oligogalacturonates of different chain length (n = 3±7).Apparent V max values using polygalacturonate as a substrate at the pH optimum, pH 4.1, were calculated as 13.8 mkat´mg ±1 , 36.5 mkat´mg ±1 and 415 nkat´mg ±1 for endopolygalacturonases I, II and C, respectively. K m values were , 0.15 mg´mL ±1 for all three enzymes. Product progression analysis using polygalacturonate as a substrate revealed a random cleavage pattern for all three enzymes and suggested processive behavior for endopolygalacturonases I and C. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 5 or 6 for endopolygalacturonase I and endopolygalacturonase C, respectively. The bond-cleavage frequencies obtained for the hydrolysis of the oligogalacturonates were used to assess subsite maps. The maps indicate that the minimum number of subsites is seven for all three enzymes.Using pectins of various degrees of esterification, it was shown that endopolygalacturonase II is the most sensitive to the presence of methyl esters. Like endopolygalacturonase II, endopolygalacturonases I, C and E, which was also included in this part of the study, preferred the non-esterified pectate. Additional differences in substrate specificity were revealed by analysis of the reaction products of hydrolysis of a mixture of pectate lyase-generated D4,5-unsaturated oligogalacturonates of degree of polymerization 4±8. Whereas endopolygalacturonase I showed a strong preference for generating the D4,5-unsaturated dimer, with endopolygalacturonase II the D4,5-unsaturated trimer accumulated, indicating further differences in substrate specificity. For endopolygalacturonases C and E both the D4,5-unsaturated dimer and trimer were observed, although in different ratios.Keywords: Aspergillus niger; endopolygalacturonase; kinetics; processivity; subsites.Saprophytic and plant pathogenic fungi and bacteria produce a vast array of enzymes capable of degrading the complex carbohydrate structures present in the plant cell wall. The most complex carbohydrate, and one of the major constituents of the middle lamella of the plant cell wall, is pectin. Owing to its complex structure, many enzymes are involved in the complete breakdown of pectin. Among these enzymes are pectin methylesterases, pectin and rhamnogalacturonan acetylesterases, pectate, pectin and rhamnogalacturonan lyases, rhamnogalacturonan hydrolases and polygalacturonases. The polygalacturonases [poly(1,4-a-d-galacturonide) glycanohydrolase, EC 3.2.1.15] hydrolyze the a-1,4 glycosidic bonds between adjacent a-dgalacturonic acid residues and are thought to act specifically on the homogalacturonan or`smooth' part of the pectin molecule.At present numerous genes encoding both exo-and endo-acting polygalactu...

Aspergillus niger pectin lyases are encoded by a multigene family. The complete nucleotide sequence of the pectin lyase PLA-encoding gene pelA has been determined. Comparison of the deduced amino acid sequence with the deduced amino acid sequence of the other characterized pectin lyase, PLD, shows that the proteins share 69% amino acid identity. When grown on media with pectin as the sole carbon source, A. niger transformants containing multiple copies of the pelA gene show raised mRNA levels and overexpression of the gene product PLA compared with the wild-type strain. PLA was purified and characterized. In A. nidulans transformants PLA is also produced in medium containing a high concentration of glucose and no pectin.

Several nopaline degrading strains and one octopine degrading strain are shown to loose oncogenicity as well as the ability to utilize these guanidine compounds when they are cured of their TI plasmid. To investigate whether the specific genes involved in the utilization of one or the other compound are located on the plasmid, plasmid-transfer experiments have been performed. The plasmid from a nopaline degrading strain has been transferred to a naturally non oncogenic Agrobacterium namely A. radiobacter. Furthermore, the plasmid from an octopine degrading strain has been transferred to a plasmid-cured strain which originally had the capacity to utilize nopaline. Both kinds of experiments prove that the TI plasmid determines the strain specificity with regard to the utilization of either octopine or nopaline. They also demonstrate that the synthesis of either octopine or nopaline in crown gall cells is also determined by genes located on the TI plasmid harboured by the transforming A. tumefaciens strains.

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 faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin.