Changing a single amino acid residue switches processive and non-processive behavior of Aspergillus niger endopolygalacturonase I and II.

Processivity, also known as multiple attack on a single chain, is a feature commonly encountered only in enzymes in which the substrate binds in a tunnel. However, of the seven Aspergillus niger endopolygalacturonases, which have an open substrate binding cleft, four enzymes show processive behavior, whereas the other endopolygalacturonases are randomly acting enzymes. In a previous study (Benen, J.A.E., Kester, H.C.M., and Visser, J. (1999) Eur. J. Biochem. 259, 577-585) we proposed that the high affinity for the substrate of subsite -5 of processive endopolygalacturonase I constitutes the origin of the multiple attack behavior. Based on primary sequence alignments of A. niger endopolygalacturonases and three-dimensional structure analysis of endopolygalacturonase II, an arginine residue was identified in the processive enzymes at a position commensurate with subsite -5, whereas a serine residue was present at this position in the non-processive enzymes. In endopolygalacturonase I mutation R95S was introduced, and in endopolygalacturonase II mutation S91R was introduced. Product progression analysis on polymer substrate and bond cleavage frequency studies using oligogalacturonides of defined chain length for the mutant enzymes revealed that processive/non-processive behavior is indeed interchangeable by one single amino acid substitution at subsite -5, Arg-->Ser or Ser-->Arg.

Characterization of Aspergillus niger pectate lyase A.

The Aspergillus niger plyA gene encoding pectate lyase A (EC 4.2.99. 3) was cloned from a chromosomal lambda(EMBL4) library using the Aspergillus nidulans pectate lyase encoding gene [Dean, R. A., and Timberlake, W. E. (1989) Plant Cell 1, 275-284] as a probe. The plyA gene was overexpressed using a promoter fusion with the A. niger pyruvate kinase promoter. Purification of the recombinant pectate lyase A resulted in the identification of two enzyme forms of which one appeared to be N-glycosylated and the other appeared to be free of N-glycosylation. The two enzyme forms showed identical specific activities. The N-glycosylation free pectate lyase A was further characterized with respect to product formation on polygalacturonic acid (alpha-1,4 linked D-galacturonic acid) and mode of action on oligogalacturonides of degree of polymerization 2-8. The bond cleavage frequencies for tetra-, penta-, and hexagalacturonides were studied as a function of [CaCl(2)]. The bond cleavage frequencies changed in a [CaCl(2)]-dependent way for penta- and hexagalacturonide. Kinetic studies using tetra- and hexagalacturonide revealed a strong sigmoidal [CaCl(2)]-dependent relation. The role of Ca(2+) ions in substrate binding is discussed.

Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides.

Synergy in the degradation of two plant cell wall polysaccharides, water insoluble pentosan from wheat flour (an arabinoxylan) and sugar beet pectin, was studied using several main-chain cleaving and accessory enzymes. Synergy was observed between most enzymes tested, although not always to the same extent. Degradation of the xylan backbone by endo-xylanase and beta-xylosidase was influenced most strongly by the action of alpha-L-arabinofuranosidase and arabinoxylan arabinofuranohydrolase resulting in a 2.5-fold and twofold increase in release of xylose, respectively. Ferulic acid release by feruloyl esterase A and 4-O-methyl glucuronic acid release by alpha-glucuronidase depended largely on the degradation of the xylan backbone by endo-xylanase but were also influenced by other enzymes. Degradation of the backbone of the pectin hairy regions resulted in a twofold increase in the release of galactose by beta-galactosidase and endo-galactanase but did not significantly influence the arabinose release by arabinofuranosidase and endo-arabinase. Ferulic acid release from sugar beet pectin by feruloyl esterase A was affected most strongly by the presence of other accessory enzymes.

Structure and function of pectic enzymes: virulence factors of plant pathogens.

The structure and function of Erwinia chrysanthemi pectate lysase C, a plant virulence factor, is reviewed to illustrate one mechanism of pathogenesis at the molecular level. Current investigative topics are discussed in this paper.

Subsite mapping of Aspergillus niger endopolygalacturonase II by site-directed mutagenesis.

To assess the subsites involved in substrate binding in Aspergillus niger endopolygalacturonase II, residues located in the potential substrate binding cleft stretching along the enzyme from the N to the C terminus were subjected to site-directed mutagenesis. Mutant enzymes were characterized with respect to their kinetic parameters using polygalacturonate as a substrate and with respect to their mode of action using oligogalacturonates of defined length (n = 3-6). In addition, the effect of the mutations on the hydrolysis of pectins with various degrees of esterification was studied. Based on the results obtained with enzymes N186E and D282K it was established that the substrate binds with the nonreducing end toward the N terminus of the enzyme. Asn(186) is located at subsite -4, and Asp(282) is located at subsite +2. The mutations D183N and M150Q, both located at subsite -2, affected catalysis, probably mediated via the sugar residue bound at subsite -1. Tyr(291), located at subsite +1 and strictly conserved among endopolygalacturonases appeared indispensable for effective catalysis. The mutations E252A and Q288E, both located at subsite +2, showed only slight effects on catalysis and mode of action. Tyr(326) is probably located at the imaginary subsite +3. The mutation Y326L affected the stability of the enzyme. For mutant E252A, an increased affinity for partially methylesterified substrates was recorded. Enzyme N186E displayed the opposite behavior; the specificity for completely demethylesterified regions of substrate, already high for the native enzyme, was increased. The origin of the effects of the mutations is discussed.

Tandem mass spectrometric analysis of aspergillus niger pectin methylesterase: mode of action on fully methyl-esterified oligogalacturonates.

The substrate specificity and the mode of action of Aspergillus niger pectin methylesterase (PME) was determined using both fully methyl-esterified oligogalacturonates with degrees of polymerization (DP) 2-6 and chemically synthesized monomethyl trigalacturonates. The enzymic activity on the different substrates and a preliminary characterization of the reaction products were performed by using high-performance anion-exchange chromatography at neutral pH. Electrospray ionization tandem MS (ESI-MS/MS) was used to localize the methyl esters on the (18)O-labelled reaction products during the course of the enzymic reaction. A. niger PME is able to hydrolyse the methyl esters of fully methyl-esterified oligogalacturonates with DP 2, and preferentially hydrolyses the methyl esters located on the internal galacturonate residues, followed by hydrolysis of the methyl esters towards the reducing end. This PME is unable to hydrolyse the methyl ester of the galacturonate moiety at the non-reducing end.

Characterization of a novel endopolygalacturonase from Aspergillus niger with unique kinetic properties.

We isolated and characterized a new type of endopolygalacturonase (PG)-encoding gene, pgaD, from Aspergillus niger. The primary structure of PGD differs from that of other A. niger PGs by a 136 amino acid residues long N-terminal extension. Biochemical analysis demonstrated extreme processive behavior of the enzyme on oligomers longer than five galacturonate units. Furthermore, PGD is the only A. niger PG capable of hydrolyzing di-galacturonate. It is tentatively concluded that the enzyme is composed of four subsites. The physiological role of PGD is discussed.

pgaA and pgaB encode two constitutively expressed endopolygalacturonases of Aspergillus niger.

The nucleotide sequence data for pgaA and pgaB have been deposited with the EMBL, GenBank and DDBJ Databases under accession numbers Y18804 and Y18805 respectively. pgaA and pgaB, two genes encoding endopolygalacturonases (PGs, EC 3.2.1.15) A and B, were isolated from a phage genomic library of Aspergillus niger N400. The 1167 bp protein coding region of the pgaA gene is interrupted by one intron, whereas the 1234 bp coding region of the pgaB gene contains two introns. The corresponding proteins, PGA and PGB, consist of 370 and 362 amino acid residues respectively. Northern-blot analysis revealed that pgaA- and pgaB-specific mRNA accumulate in mycelia grown on sucrose. mRNAs are also present upon transfer to media containing D-galacturonic acid and pectin. Recombinant PGA and PGB 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). At their pH optimum the specific activities in a standard assay for PGA (pH 4.2) and PGB (pH 5.0) were 16.5 mu+kat.mg(-1) and 8.3 mu+kat.mg(-1) respectively. Product progression analysis, using polygalacturonate as a substrate, revealed a random cleavage pattern for both enzymes and indicated processive behaviour for PGA. 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 6. Using pectins of various degrees of methyl esterification, it was shown that PGA and PGB both preferred partially methylated substrates.

The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis.

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.

Performance of selected microbial pectinases on synthetic monomethyl-esterified di- and trigalacturonates.

Two monomethyl esters of alpha-(1-4)-linked D-galacturonic dimers and three monomethyl esters of alpha-(1-4)-linked D-galacturonic acid trimers were synthesized chemically and further used as substrates in order to establish the substrate specificity of six different endopolygalacturonases from Aspergillus niger, one exopolygalacturonase from Aspergillus tubingensis, and four selected Erwinia chrysanthemi pectinases; exopolygalacturonan hydrolase X (PehX), exopolygalacturonate lyase X (PelX), exopectate lyase W (PelW), and oligogalacturonan lyase (Ogl). All A. niger endopolygalacturonases (PGs) were unable to hydrolyze the two monomethyldigalacturonates and 2-methyltrigalacturonate, whereas 1-methyltrigalacturonate was only cleaved by PGI, PGII, and PGB albeit at an extremely low rate. The hydrolysis of 3-methyltrigalacturonate into 2-methyldigalacturonate and galacturonate by all endopolygalacturonases demonstrates that these enzymes can accommodate a methylgalacturonate at subsite -2. The A. tubingensis exopolygalacturonase hydrolyzed the monomethyl-esterified digalacturonates and trigalacturonates although at lower rates than for the corresponding oligogalacturonates. 1-Methyltrigalacturonate was hydrolyzed at the same rate as trigalacturonate which demonstrates that the presence of a methyl ester at the third galacturonic acid from the nonreducing end does not have any effect on the performance of exopolygalacturonase. Of the four E. chrysanthemi pectinases, Ogl was the only enzyme able to cleave digalacturonate, whereas all four enzymes cleaved trigalacturonate. Ogl does not cleave monomethyl-esterified digalacturonate and trigalacturonate in case the second galacturonic acid residue from the reducing end is methyl-esterified. PehX did not hydrolyze any of the monomethyl-esterified trigalacturonates. The two lyases, PelX and PelW, were both only able to cleave 1-methyltrigalacturonate into Delta4,5-unsaturated 1-methyldigalacturonate and galacturonate.

1.68-A crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis.

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 beta-helix with 10 complete turns. The beta-helix is composed of four parallel beta-sheets, and has one very small alpha-helix near the N terminus, which shields the enzyme's hydrophobic core. Loop regions form a cleft on the exterior of the beta-helix. Site-directed mutagenesis of Asp(180), Asp(201), Asp(202), His(223), Arg(256), and Lys(258), which are located in this cleft, results in a severe reduction of activity, demonstrating that these residues are important for substrate binding and/or catalysis. The juxtaposition of the catalytic residues differs from that normally encountered in inverting glycosyl hydrolases. A comparison of the endopolygalacturonase II active site with that of the P22 tailspike rhamnosidase suggests that Asp(180) and Asp(202) activate the attacking nucleophilic water molecule, while Asp(201) protonates the glycosidic oxygen of the scissile bond.

The exopolygalacturonase from Aspergillus tubingensis is also active on xylogalacturonan.

Apple-pectin hairy regions were prepared from apple pectin by combined action of the recombinant Aspergillus niger enzymes endopolygalacturonase II and pectin methylesterase and the A. tubigensis exopolygalacturonase. Using this enzymically prepared pectin fraction, an additional activity of the A. tubigensis exopolygalacturonase was discovered only when the substrate was chemically saponified and when D-galacturonate, a potent inhibitor of the enzyme, was removed from the incubation mixture. The new reaction product was purified and could be hydrolysed by A. niger beta-xylosidase into D-galacturonate and beta-D-xylose in a 1:1 ratio, which identified it as xylogalacturonate. The results demonstrate that exopolygalacturonase is not only active on galacturonan but also on xylogalacturonan. The enzyme thus accomodates a substrate in which the terminal galacturonic acid residue carries a single xylose substitution. The well-defined substrate specificity of exopolygalacturonase opens the possibility for use of this enzyme in biotechnological applications, such as preparing pectins that are methylated at the non-reducing end, and for studying the fine structure of xylogalacturonan in pectin.

Structure of a plant cell wall fragment complexed to pectate lyase C.

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 A and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, alpha-D-GalpA-([1-->4]-alpha-D-GalpA)3-(1-->4)-D-GalpA , is composed of five galacturonopyranose units (D-GalpA) linked by alpha-(1-->4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein-oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. 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 beta elimination cleavage of polygalacturonic acid.

Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C.

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 Vmax values using polygalacturonate as a substrate at the pH optimum, pH 4.1, were calculated as 13.8 mukat.mg-1, 36.5 mukat.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 delta 4,5-unsaturated oligogalacturonates of degree of polymerization 4-8. Whereas endopolygalacturonase I showed a strong preference for generating the delta 4,5-unsaturated dimer, with endopolygalacturonase II the delta 4,5-unsaturated trimer accumulated, indicating further differences in substrate specificity. For endopolygalacturonases C and E both the delta 4,5-unsaturated dimer and trimer were observed, although in different ratios.

Characterization of the exopolygalacturonate lyase PelX of Erwinia chrysanthemi 3937.

Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-alpha-D-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains. Using a genomic bank of a 3937 mutant with the major pel genes deleted, we cloned a pectinase gene identified as pelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of another E. chrysanthemi strain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL of E. chrysanthemi but has a N-terminal extension of 324 residues. The transcription of pelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation of pelX expression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved in pelX expression. The pelX mutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers or Saintpaulia ionantha plants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites -2 to +2. PelX and PehX were shown to be localized in the periplasm of E. chrysanthemi 3937. PelX catalyzed the formation of unsaturated digalacturonates by attack from the reducing end of the substrate, while PehX released digalacturonates by attack from the nonreducing end of the substrate. Thus, the two types of exo-degrading enzymes appeared complementary in the degradation of pectic polymers, since they act on both extremities of the polymeric chain.

Molecular cloning, sequencing, and heterologous expression of the vaoA gene from Penicillium simplicissimum CBS 170.90 encoding vanillyl-alcohol oxidase.

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 8alpha-(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 lambdaEMBL3 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.

pgaE encodes a fourth member of the endopolygalacturonase gene family from Aspergillus niger.

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 N-terminal amino acids of the prepropeptide. The calculated Mr 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 = 2-7). The pH optimum was 3.8. The Km and Vmax for polygalacturonate hydrolysis were 2.5 +/- 0.4 mg x ml(-1) and 1.3 +/- 0.2 microkat x mg(-1), respectively. A subsite map was calculated by the combination of the methods of Suganuma et al. [Suganuma, T., Matsuno, R., Ohnishi, M. & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 293-316] and Nitta et al. [Nitta, Y., Mizushima, M., Hiromi, K. & Ono, S. (1971) J. Biochem. (Tokyo) 69, 567-576]. This indicated that the enzyme was composed of at least five subsites.

Cloning of the maoA gene that encodes aromatic amine oxidase of Escherichia coli W3350 and characterization of the overexpressed enzyme.

The mao operon of Escherichia coli W3350, which comprises the genes maoC and maoA, was cloned and appeared to be similar to that of Klebsiella aerogenes [Sugino, H., Sasaki, M., Azakami, H., Yamashita, M. & Murooka, Y. (1992) J. Bacteriol. 174, 2485-2492]. The gene that encodes aromatic amine oxidase (maoA) was isolated, sequenced, and expressed in E. coli TG2. The purified enzyme exhibited properties characteristic of a copper/topaquinone(TPQ)-containing amine oxidase with respect to the optical absorption and EPR spectra, the size of the subunits, and the optical absorption spectra obtained upon derivatization with hydrazines. However, high-resolution anion-exchange chromatography revealed that the preparation was heterogeneous. The enzyme preparation appeared to consist of at least four enzyme species with different specific activities, A474nm/A340nm ratios and TPQ/subunit ratios. Since the overall properties of the overexpressed enzyme and the authentic enzyme were similar and the separated enzyme species had identical N-terminal amino acid sequences, the heterogeneity does not seem to be caused by improper expression of the gene in the recombinant strain but by factors that interfere with the processing of the specific tyrosine in the precursor enzyme to functional TPQ. Although other causes cannot be excluded, the spectral data and TPQ/subunit ratios reported in the literature for other amine oxidases suggest that suboptimal synthesis of functional TPQ also occurs in other organisms.