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PubMed2004

Immunocytochemical localization of polygalacturonase during tracheary element differentiation in Zinnia elegans.

Polygalacturonase (PG) is a cell wall-associated protein that degrades pectin. A ZePG1 cDNA encoding a putative PG was isolated from Zinnia elegens L. and a rabbit antibody specific to the ZePG1 protein was generated. The level of the ZePG1 protein was up-regulated when tracheary element differentiation was initiated. Using gold-labeled secondary antibodies for light and electron microscopy, ZePG1 protein was localized in cultured Zinnia cells. This protein was preferentially distributed on tracheary elements (TEs). At the subcellular level, the protein was localized on secondary wall thickenings, primary walls, Golgi bodies and vesicles. Thus, the putative role of the ZePG1 protein might be the degradation of pectic substances before lignification. Some non-TE cells also accumulated ZePG1 protein on primary walls, Golgi bodies and vesicles. The accumulation of ZePG1 protein on primary walls seems to be at the elongating tips of non-TE cells. In plants, ZePG1 protein was localized on the secondary wall thickenings of differentiating TEs and phloem regions. These results suggest that the expression of the ZePG1 protein is highly regulated both spatially and temporally during in vitro and in situ TE differentiation.

Asteraceae↗
PubMed2006

The characterization of the soybean polygalacturonase-inhibiting proteins (Pgip) gene family reveals that a single member is responsible for the activity detected in soybean tissues.

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins that inhibit fungal endopolygalacturonases (PGs). They are encoded by multigene families whose members show functional redundancy and subfunctionalization for recognition of fungal PGs. In order to expand the information on the structure and functional features of legume PGIP, we have isolated and characterized four members of the soybean Pgip gene family and determined the properties of the encoded protein products. Sequence analysis showed that these genes form two clusters: one cluster of about 5 kbp containing Gmpgip1 and Gmpgip2, and the other containing Gmpgip3 and Gmpgip4 within a 60 kb fragment of a separate BAC clone. Sequence diversification of the four members resides mainly in the xxLxLxx region that includes residues forming the beta-sheet B1. When compared with other legume Pgip genes, Gmpgip3 groups with the bean genes Pvpgip1 and Pvpgip2, suggesting that these genes are closer to the ancestral gene. At the protein level, only GmPGIP3 shows the capability to inhibit fungal PGs. The spectrum of inhibition of GmPGIP3 against eight different fungal PGs mirrors that of the PGIP purified from soybean tissues and is similar to that of the bean PvPGIP2, one of the most efficient inhibitors so far characterized. We also report that the four Gmpgip genes are differentially regulated after wounding or during infection with the fungal pathogen Sclerotinia sclerotiorum. Following fungal infection Gmpgip3 is up regulated promptly, while Gmpgip2 is delayed.

Amino Acid Sequence↗
PubMed1998

The promoter of a gene encoding a polygalacturonase-inhibiting protein of Phaseolus vulgaris L. is activated by wounding but not by elicitors or pathogen infection.

Polygalacturonase-inhibiting proteins (PGIPs), leucine-rich repeat (LRR) proteins evolutionarily related to several plant resistance genes, bind to and regulate the action of fungal endopolygalacturonases. In Phaseolus vulgaris L., PGIPs are encoded by a gene family comprising at least five members. As a start for a systematic analysis of the regulation of the pgip family, we have analysed the ability of the promoter of the bean gene pgip-1 to direct expression of beta-glucuronidase (GUS) in transfected tobacco protoplasts, microbombarded bean and tobacco leaves, and transgenic tobacco plants. In protoplasts, the pgip-1 gene region from nucleotide (nt) -2004 to nt +27 directed a level of expression that was as high as that directed by the cauliflower mosaic virus (CaMV) 35S promoter and could not be further induced by elicitor treatment; alteration of the region immediately following the TATAA sequence at nt -29 abolished expression. Upon stable integration into tobacco plants of the pgip-1 promoter-GUS construct, as well as of a -394 deletion, expression was detected for both constructs mainly in the stigma and, to a lesser extent, in the anthers and in the conductive vascular tissue. The promoter responded to wounding but not to oligogalacturonides, fungal glucan, salicylic acid, cryptogein, or pathogen infection. This expression pattern does not mirror that of the whole pgip gene family.

Artificial Gene Fusion↗
PubMed1998

Genomic organization of six tomato polygalacturonases and 5' upstream sequence identity with tap1 and win2 genes.

Recently, three polygalacturonase (PG) cDNAs (TAPG1, TAPG2, and TAPG4) were identified in a library prepared from tomato (Lycopersicon esculentum cv. Rutgers) leaf abscission zones. Genomic clones encoding these three cDNAs have been identified. Moreover, the genomic clones include three additional PG genes, TPG3, TAPG5 and TPG6, which have not been previously reported. A transcript for TAPG5 was detected in the RNA from leaf and flower abscission zones; however, transcripts for TPG3 and TPG6 were not. DNA sequence analysis revealed that TAPG1, TAPG2, and TPG3 are linked in a close tandem array. TAPG4, TAPG5 and TPG6 are also closely linked to each other but in divergent and inverted orientations and are not closely linked to TAPG1, TAPG2, or TPG3. TAPG4, TAPG5 and TPG6 map to the middle of chromosome 12. TPG6 contains two introns. The other five PG genes include four exons and three introns. The relative positions of introns 1 and 2 are shared by all six PG genes. The position of intron 3 is conserved in the other five. The structure of the tomato fruit PG gene, which contains 8 introns, is compared with that of the six PG genes described above. Of interest is an approximately 300 bp inverted repeat found in TAPG1, TAPG2 and TAPG4 that shares significant sequence identity with sequence in the first intron of the tomato anionic peroxidase gene, tap1. RNA blot analysis indicates that the transcript for an anionic peroxidase increases during abscission. In addition, a 250 bp sequence found in TPG3 shares high sequence identity with a 5' upstream region in a wound-induced win2 gene from potato. Potential sites of transcriptional regulation in these genes are discussed.

Base Sequence↗
PubMed2005

Influence of pectin and glucose on growth and polygalacturonase production by Aspergillus niger in solid-state cultivation.

The solid-state production of endo- and exo-polygalacturonases (PG) by Aspergillus niger was studied in a media containing wheat bran, salts, and different citric pectin and/or glucose concentrations. Kinetic analysis of the process indicated that the formation of PG and the growth of A. niger are associated processes. By increasing citric pectin from 0 to 16% (w/w), the maximum A. niger concentration (X (m)) was raised from 94 to 121 mg/g dry medium suggesting that pectin can be used by A. niger as a growth substrate besides its role as an inducer. With 16% (w/w) pectin, 281 U exo-PG/gdm and 152 U endo-PG/gdm were obtained. Otherwise, pectin concentrations from 20 to 30% (w/w) hindered both production and growth. A. niger concentrations of 108-113 mg/gdm were achieved in runs with glucose from 5 to 12% (w/w), whereas at 16 and 20% (w/w) glucose, lower X (m) values (ca. 100 mg/gdm) were measured. The addition of glucose to the wheat bran medium, up to 10% (w/w) led to maximum endo-PG titers slightly lower than those found in the absence of glucose. Nevertheless, exo-PG formation in these media was strongly increased and activities over 370 U/gdm were achieved. The results suggest that in experiments with pectin concentrations until 16% (w/w), exo-PG production was repressed by pectin-degradation products although these same substances had favored biomass growth. When glucose concentrations over 10% (w/w) were added to the media, the maximum activities of both enzymes decreased drastically, suggesting that glucose at high concentrations also exerts a repressive effect on PG production.

Aspergillus niger↗
PubMed1976

Purification and characterisation of polygalacturonases from a commercial Aspergillus niger preparation.

The polygalacturonase (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) activity of Pectinol is resolved into two fractions (E1 and E2) of about equal total activity on DEAE-cellulose. These fractions are purified from other pectinolytic enzyme activity by Sephadex G-75 chromatography. Both E1 and E2 reduce the viscosity of polygalacturonate by 50% after 7% of the glycosidic bonds are hydrolysed. Their activities are not affected by iodoacetate (1 mM) or EDTA (10 mM). E1 and E2 have different molecular weights (35 000 and 85 000, respectively) and different electrophoretic mobilities on sodium dodecyl sulphate polyacrylamide gels. Their pH (4.1 and 3.8 respectively) and ionic strength optima and specific activities also differ. Both enzymes are inhibited at similar rates by diethyl pyrocarbonate at pH 6 but only E2 is protected from this inhibition by 2% (w/v) polygalacturonate. The rate of change of protein absorbance at 250 nm accompanying this inhibition, and the residues are essential for the activities of both E1 and E2. About 2 molecules of carbethoxyhistidine per subunit of E2 and 0.6 molecules per subunit of E1 are present in the completely inhibited enzymes.20

Aspergillus↗
PubMed1996

An essential tyrosine residue of Aspergillus polygalacturonase.

Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine-reactive agents tested, acetic anhydride, N-acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3-9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms.

Acetic Anhydrides↗
PubMed1996

Inversion of configuration during hydrolysis of alpha-1,4-galacturonidic linkage by three Aspergillus polygalacturonases.

Endopolygalacturonases I and II (PGI and PGII) of Aspergillus niger and an exopolygalacturonase (ExoPG) of A. tubingensis were investigated to reveal the stereochemistry of their hydrolytic action. Reduced pentagalacturonic acid (pentaGalU-ol) and reduced trigalacturonic acid (triGalU-ol) were used as non-reducing substrates for the enzymes. The configuration of the reducing ends in the products formed in D2O reaction mixtures was followed by 1H-NMR spectroscopy. It has been unambiguously established that primary cleavage of pentaGalU-ol by both PGI and PGII leads to diGalU-ol and the beta-anomer of triGalUA. The primary products of hydrolysis of triGalUA-ol by ExoPG were diGal-ol and the beta-anomer of GalUA. Thus, all three Aspergillus polygalacturonases belong to the so-called inverting glycanases, i.e. they utilize the single displacement mechanism of hydrolysis of the glycosidic linkage.

Aspergillus↗
PubMed1996

Polygalacturonase inhibitors in bean pods.

The amount of polygalacturonase-inhibiting protein (PGIP) was 14 times higher in bean pods than in etiolated hypocotyls. The PGIP was extracted from bean pods and partially purified by chromatography on columns of S-Sepharose. DEAE-Sephadex A-50, and Sephadex G-75. Further purification by ion-exchange chromatography on a Mono Q column separated two isoforms of the inhibitor. The two PGIPs were similar in most properties but differed slightly in pI values. They also differed in one residue of the N-terminal amino acid sequences. Both bean pod PGIPs differed in two and possibly three residues of the deduced N-terminal amino acid sequence for hypocotyl PGIP. Small alterations in the structure of PGIP may represent a strategy in bean plants for resistance to a variety of pathogens.

Amino Acid Sequence↗
PubMed1992

Characterization of the Erwinia carotovora peh gene and its product polygalacturonase.

The peh gene, encoding polygalacturonase (Peh), was identified in Erwinia carotovora strain EC and cloned in Escherichia coli. Recombinant Peh (re-Peh) was purified from E. coli strain 706 containing peh on a recombinant plasmid. The activity of the re-Peh protein is optimal at pH 5.5. The N-terminal and internal amino acid (aa) sequences of re-Peh were determined and compared to the aa sequence deduced from the nucleotide (nt) sequence of the cloned peh. The re-Peh has no similarity, based on either the nt sequences or the deduced aa sequences, to pectate lyases from the same Er. carotovora strain or other organisms.

Amino Acid Sequence↗
PubMed1995

Cloning and characterization of a novel polygalacturonase-encoding gene from Aspergillus parasiticus.

Pectinases produced by Aspergillus flavus and A. parasiticus are believed to play a significant role in the ability of these fungi to spread in cotton bolls and other crops. Utilizing a DNA probe, generated by PCR, of the Aspergillus niger pgaII gene, we have isolated a novel, constitutively expressed polygalacturonase (PG)-encoding gene (pecA) from an A. parasiticus cDNA library. DNA sequence analysis and the deduced amino acid (aa) sequence of pecA demonstrated significant identity at the nucleotide and aa levels with other PG of fungal origin. Northern blot analysis of RNA isolated from A. parasiticus grown on either glucose or pectin as the sole carbon source showed that pecA was expressed during growth in both media.

Amino Acid Sequence↗
PubMed2006

On the simulation of enzymatic digest patterns: the fragmentation of oligomeric and polymeric galacturonides by endo-polygalacturonase II.

A simulation methodology for predicting the time-course of enzymatic digestions is described. The model is based solely on the enzyme's subsite architecture and concomitant binding energies. This allows subsite binding energies to be used to predict the evolution of the relative amounts of different products during the digestion of arbitrary mixtures of oligomeric or polymeric substrates. The methodology has been specifically demonstrated by studying the fragmentation of a population of oligogalacturonides of varying degrees of polymerization, when digested by endo-polygalacturonase II (endo-PG II) from Aspergillus niger.

Aspergillus niger↗
PubMed2004

Production, characterization and application of a thermostable polygalacturonase of a thermophilic mould Sporotrichum thermophile Apinis.

The production of polygalacturonase (PGase) by Sporotrichum thermophile Apinis in stirred submerged fermentation (SmF) was high in comparison with that in static conditions. Yeast extract (0.25%) and citrus pectin (2%) at pH 7.0 and 45 degrees C supported a high enzyme production in flasks agitated at 200 rpm. An overall 1.75-fold enhancement in PGase production was achieved as a result of optimization. The enzyme was optimally active at pH 7.0 and 55 degrees C, and exhibited t(1/2) of 4 h at 65 degrees C. The Km and Vmax values of the enzyme (for pectin) were 0.416 mg ml(-1) and 0.52 micromol mg(-1)min(-1), respectively. The PGase activity was stimulated by Mn(2+) and Fe(2+), but inhibited strongly by Mg(2+), and slightly by Tween 80 and Triton X-100. Among the additives tested, beta-mercaptoethanol exerted a strong inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. An increase in the yield of banana, grape and apple juices was recorded due to the treatment of fruit pulps with the mixture of enzymes (pectinase, xylanases and cellulase) of S. thermophile as compared to that with only pectinase. The yield of fruit juices did not increase with enhanced titre of cellulase in the enzyme mixture.

Beverages↗
PubMed2006

Glycan analysis of recombinant Aspergillus niger endo-polygalacturonase A.

The enzyme endo-polygalacturonase A, or PGA, is produced by the fungus, Aspergillus niger, and appears to play a critical role during invasion of plant cell walls. The enzyme has been homologously overexpressed in order to provide sufficient quantities of purified enzyme for structural and biological studies. We have characterized this enzyme in terms of its post-translational modifications (PTMs) and found it to be both N- and O-glycosylated. Additionally, we have characterized the glycosyl moieties using MALDI-TOF and LC-ESI mass spectrometry. The characterization of all PTMs on PGA, along with molecular modeling, allows us to reveal potential roles played by the glycans in modulating the interaction of the enzyme with other macromolecules.

Amino Acid Sequence↗
PubMed2006

Isolation of tomato pectin methylesterase and polygalacturonase on monolithic columns.

An improved cation-exchange chromatographic procedure on Convective Interaction Media (CIM, BIA Separations, Ljubljana, Slovenia) short monolithic methacrylate disk columns was used for the isolation of salt-independent pectin methylesterase (PME; EC 3.1.1.11) isoform and endo-polygalacturonase PG1 (PG, EC 3.2.1.15) from ripe tomato fruit extract after studying the chromatographic conditions including type of disk, binding buffer, pH, eluent composition and different gradients. Between 10 and 20 microg of proteins gave reliable chromatograms. Both carboxymethyl (CM) and sulfonyl (SO3) disks were equally suitable for the fractionation of tomato extract using the new gradient, but only CM disk was appropriate for further purification of the PME and PG fractions, and provided fast and sharp separation of proteins. The isolation of pure PG1 could be achieved only by addition of 20% of acetonitrile to the mobile phase. About 200 microg of proteins were loaded at one chromatographic run at the fractionation and purification. Determination of the molecular weights of the separated proteins showed that dimer of salt-independent PME isoform was formed in concentrated solutions of the enzyme but dissociated upon dilution of the solution. From 6 kg of fresh tomato flesh, 28 mg of purified salt-independent PME, 12.5mg of purified and active PG1 and 4 mg of PG2 fraction contaminated with salt-dependent PME isoform were obtained by means of semi-preparative chromatography on CIM disks.

Carboxylic Ester Hydrolases↗
PubMed2004

Evaluation of polygalacturonase activity in Saccharomyces cerevisiae wine strains.

A total of 61 S. cerevisiae strains, 60 of them isolated from wine ecosystems, were evaluated for the presence of the gene encoding endopolygalacturonase (PGU1) and for polygalacturonase (PG) activity. Nine strains lack the gene PGU1 and did not exhibit PG activity on plate assays. Of the 52 strains showing an amplified band corresponding to the size of PGU1 gene, only 36 degraded polygalacturonic acid (PGA) and 17 did not degrade it at any of the pH values used. The coding region of the PGU1 gene (ORF YJR153w) was not present in some PG activity negative strains. The S. cerevisiae UCLMS-39 strain was selected for its specific activity at different pHs, temperatures and oenological parameters. The temperature and pH optima were 50 degrees C and 3.5-5.5 respectively and it was only affected by ethanol. The PGU1 gene was cloned and sequenced. The production of a biologically functional endoPG in S. cerevisiae UCLMS-39 brings us a step closer to improving the qualities of outstanding enological yeasts naturally lacking PG activity.

Amino Acid Sequence↗
PubMed2005

Endo-polygalacturonase in Saccharomyces wine yeasts: effect of carbon source on enzyme production.

Eight wine yeast strains of Saccharomyces sp. were tested for polygalacturonase (PGase) activity, after cultivation on various carbon sources. No strain showed any activity when grown on glucose, while five strains produced PGase in the presence of galactose and polygalacturonate. These data suggest that the PGase of wine strains is repressed by glucose and induced by galactose and polygalacturonate. The existence of the PGase gene in the wine strains and its similarity with that of the laboratory strains was proved by Southern hybridization and PCR amplification. The promoter region of the PGase gene in the wine strains was slightly different from that of the laboratory strains. This possibly explains the different pattern of gene expression in wine and laboratory strains. The PGase of wine strains produced di- or tri-galacturonic acid from polygalacturonic acid, different from the fungal PGase.

Carbon↗
PubMed2004

Interaction of Sclerotinia sclerotiorum with Brassica napus: cloning and characterization of endo- and exo-polygalacturonases expressed during saprophytic and parasitic modes.

Five major and several minor PG isoenzymes were identified in a Sclerotinia sclerotiorum isolate from Brassica napus by isoelectric focusing and pectin gel overlays. Using a combination of degenerate PCR and expressed sequence tags (ESTs) four endo-polygalacturonase (PG) genes, designated as sspg1d, sspg3, sspg5, and sspg6, and two exo-PG genes, ssxpg1 and ssxpg2, were identified. SSPG1d is a member of the PG gene family previously described by Fraissinet-Tachet et al. [Curr. Genet. 29 (1995) 96]. The mature SSPG1d is a neutral PG, whereas fully processed SSPG3, SSPG5, and SSPG6 are acidic enzymes. Under saprophytic growth conditions, sspg1d, sspg3, sspg5, and ssxpg1 expression was induced by pectin and galacturonic acid and subject to catabolite repression by glucose. Conditions could not be identified under which sspg6 or ssxpg2 were expressed well. Transfer of mycelia from liquid media to solid substrates induced expression of sspg1d suggesting that it may also be regulated by thigmotrophic interactions. Under pathogenic conditions, sspg1d was highly expressed during infection. sspg3 was also expressed during infection, albeit at lower levels than sspg1d, whereas sspg5, sspg6, and ssxpg1 were expressed only weakly.

Amino Acid Sequence↗