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PubMed2000

Impact of UV-C irradiation on the cell wall-degrading enzymes during ripening of tomato (Lycopersicon esculentum L.) fruit.

The effect of a hormic dose of UV-C (254 nm) on changes in fruit firmness and cell wall-degrading enzyme (CWDE) activity was determined using tomato fruit. Throughout the storage period, a decrease in firmness was jointly observed with an increase of the CWDE (polygalacturonase, pectin methyl esterase, cellulase, xylanase, beta-D-galactosidase, and protease) activity for all treatments, suggesting the involvement of these enzymes in the ripening process. However, the enhancement in the activity of the CWDE was significantly less in fruit subjected to the hormic dose of UV-C. This reduction may explain why irradiated fruit were firmer than control and consequently may explain how UV-C could delay the ripening and senescence process. We suggest that the CWDE are one of the targets of the UV-C, and by this action, irradiation contributed to a delay of the cell wall degradation and consequently retarded softening of the tomato fruit tissues.

Carboxylic Ester Hydrolases↗

PubMed2001

Expression and activity of cell-wall-degrading enzymes in the latex of opium poppy, Papaver somniferum L.

The alkaloid-rich latex of the opium poppy, Papaver somniferum L., is valued as a source of pharmaceuticals including thebaine, codeine, and morphine, but is also harvested for heroin production. The poppy laticifer system develops through the gradual disappearance of the common walls between differentiating laticifer elements throughout the plant. Gene homologues for cell-wall-degrading enzymes were found during random sequencing of an opium poppy latex cDNA library. RNA gel blot analysis of cellulase, polygalacturonase beta-subunit, 1,3-beta-glucanase, and xyloglucan endotransglycosylase homologues showed their expression was not limited to laticifers. In contrast, poppy gene homologues to pectin methylesterase (PME), pectin acetylesterase (PAE) and pectate lyase (PL) where all highly expressed and latex-specific. Enzyme assays confirmed the presence of PME, PAE, and PL activities in latex serum. The abundance of transcripts encoding pectin-degrading enzymes in latex suggests that these enzymes may play an important role in laticifer development.

Blotting, Northern↗

PubMed2002

Viscometric method for assaying of total endodepolymerase activity of pectinases.

An improved method for assaying of the total endodepolymerase activity of pectinases has been developed. The method is based on the determination of the viscosity of a citrus pectin solution in the presence of the enzyme using an Ostwald viscometer. The depolymerizing activity of different pectinases can be detected including polygalacturonase, polymethylgalacturonase, pectin lyase, and pectate lyase. One unit of the endodepolymerase activity corresponds to the activity resulting in 50% decrease in the relative viscosity of 0.5% citrus pectin solution for 5 min at 40 degrees C and the appropriate pH. Depending on the pH-optima of the enzymes, two modifications of the method are described: 1) for acid pectinases at pH 5.0, and 2) for neutral (mildly alkaline) pectinases at pH 8.0. The modifications differed in the control and in the calculation of the activity. Six enzyme preparations were used to demonstrate the applicability of the method. The parameter used for the calculation of the enzymatic activity was directly proportional to the enzyme concentration (the dependence was linear in the range of at least 10-fold change in the enzyme concentration). The relative error of the method did not exceed 10%.

Aspergillus↗

PubMed2004

Characterization of cell wall degrading enzymes of Thanatephorus cucumeris.

Thanatephorus cucumeris is a ubiquitous fungus responsible for many types of plant diseases worldwide. All isolates from infected Hevea brasiliensis trees secreted pectolytic enzymes; polygalacturonase (PG), pectin lyase (PL) and cellulolytic enzymes; beta-glucosidase and cellobiase in culture. The extracts of the rubber tree leaf tissues, inoculated with T. cucumeris did not show any PG activity. However, PL activity was detected in tissue with the establishment of the infection. The levels of beta-glucosidase, an inherent enzyme in Hevea spp. increased rapidly following infection. However, cellobiase was detected only with the initiation of infection. Molecular weights of PG in all isolates were similar and in the range of 53,000 to 58,000. PL also followed the same pattern showing a molecular weight around 39,000.

Cell Wall↗

PubMed2003

Biochemistry of fruit ripening of guava (Psidium guajava L.): compositional and enzymatic changes.

Changes in chemical composition and the activities of hydrolytic enzymes during four different stages of maturity, viz. mature green (MG), color turning (CT), ripe (R), and overripe (OR), have been studied in guava fruits cv. Banarsi Surkha. Chlorophyll content decreased while carotenoid content increased during ripening. Starch content decreased with concomitant increase in alcohol-soluble sugars. Cellulose, hemicellulose, and lignin also decreased up to ripe stage, while pectin continued to decrease up to OR stage. PG (polygalacturonase) and cellulase exhibited progressive increase in activity throughout ripening, whereas pectin methyl esterase (PME) activity increased up to CT stage and decreased at R stage. The activities of alpha-amylase and beta-amylase decreased significantly with ripening. The most notable metabolic changes occurred between MG and CT stage, implying that for improved postharvest handling, guava fruits may be harvested at CT stage.

Amylases↗

PubMed2002

A simple fractionation protocol for, and a comprehensive study of the molecular properties of, two major endopolygalacturonases from Aspergillus niger.

A comprehensive study on purification and characterization of the two endopolygalacturonases from Aspergillus niger, PG II and PG IV, accounting for 70% of the total polygalacturonase activity, is reported. These enzymes were purified to homogeneity using ion-exchange chromatography and gel filtration. The enzymes had specific activities of 982 and 3750 units/mg, and their molecular masses were 61 and 38 kDa, respectively. The pH optimum of PG II was pH 3.8-4.3 and for PG IV it was between pH 3 and 4.6, and the temperature optima also differed for the enzymes. The enzymes preferred pectic acid as a substrate, cleaving it at random, leading to the release of oligogalacturonides as products. The K(m) values of the two enzymes were found to be 0.12 and 0.72% respectively. The enzymes were rich in hydrophilic amino acids and relatively low in the sulphur-containing amino acids. Both enzymes were rich in beta-structure and differed in their tertiary folding. The tryptophan residues were in a hydrophobic environment. The enzymes differed in their thermal stability; the midpoint of thermal inactivation, T(m), of the two enzymes was found to be 43 degrees C for PG II and 46 degrees C for PG IV.

Aspergillus niger↗

PubMed2002

A novel enzyme activity involving the demethylation of specific partially methylated oligogalacturonides.

Studies of the enzymic digestion of pectic substrates using different polygalacturonase (PG) preparations have revealed evidence for a previously unreported enzyme activity carried out by a contaminating enzyme in one of the preparations. This observed activity involves the demethylation of specific oligogalacturonides, namely 2-methyltrigalacturonic acid and 2,3-dimethyltetragalacturonic acid. However, no large-scale demethylation of highly methylated polymeric substrates is found, demonstrating that the enzyme responsible is not a conventional pectin methylesterase (PME). Furthermore, it has been shown that a commercial sample of fungal PME from Aspergillus niger demethylates all of the oligogalacturonides present as primary products of endo-PG digestion, in contrast with the activity observed here. On the basis of the known methyl ester distribution of the endo-PG-generated fragments and knowledge of which of these oligogalacturonides are demethylated, it is concluded that the observed activity can be explained by the existence of an exo-acting methylesterase that attacks the non-reducing end of the oligogalacturonide molecules.

Carboxylic Ester Hydrolases↗

PubMed1997

Analysis of conductance responses during depolymerization of pectate by soft rot Erwinia spp. and other pectolytic bacteria isolated from potato tubers.

Different bacteria isolated from potato tubers were screened for their pectolytic properties by examining pitting in polypectate agar, recording conductance responses in polypectate medium and performing potato tuber soft rot tests. For bacteria found positive in conductimetry, the role of polygalacturonase (PG) and pectate lyase (PL) in the generation of conductance changes in a polygalacturonic acid (PGA) medium was further analysed using enzyme activity staining after gel electrophoresis and high-performance anion exchange chromatography. The extent of the conductance changes during depolymerization of PGA was dependent on the amounts of galacturonate monomers and oligomers accumulated in the medium. In comparison with an unidentified saprophyte and a Klebsiella strain, both mainly having PL activity, soft rot Erwinia spp. rapidly produced larger conductance responses, due to a combined action of multiple forms of PG and PL. The responses of Erwinia spp. were initially associated with the accumulation of large amounts of monomers and saturated dimers to heptamers, due to PG activity. Subsequently, as well as monomers and saturated dimers, large amounts of unsaturated dimers were also detected, due to PL activity. The role of PG as an important conductimetric factor was also demonstrated for a pectinase preparation derived from Aspergillus niger. Besides detection, automated conductimetric assays in pectate media may also be useful for monitoring of pectolytic activity in pectinase preparations and for screening of pectolytic activity of microorganisms under different media and growth conditions.

Chromatography, Ion Exchange↗

PubMed2003

The Erwinia chrysanthemi phoP-phoQ operon plays an important role in growth at low pH, virulence and bacterial survival in plant tissue.

We have studied the role of acidic pH as a barrier for the colonization of the plant apoplast by Erwinia chrysanthemi. A minitransposon containing a promoterless reporter gene, gus, was used for random mutagenesis of the bacterial genome. An acid-sensitive mutant, named BT119, was isolated and had the following differential features with respect to the wild-type strain: (i) inability to grow at pH </= 5.5; (ii) decreased survival at acid pH and in plant tissues; (iii) increased susceptibility to antimicrobial peptides; (iv) decreased virulence in chicory leaves and pear fruits; (v) reduced polygalacturonase production; and (vi) reduced ability to alkalinize chicory tissues after infection. The sequence of the interrupted gene was highly similar to the phoQ gene, which is involved in environmental sensing in several bacteria, such as Yersinia pseudotuberculosis, Erwinia carotovora, Salmonella typhimurium and Escherichia coli and thus, this designation was used for the E. chrysanthemi system. This gene was induced at low Mg(2+) concentrations and in planta. These results suggest that E. chrysanthemi PhoP-PhoQ system plays an important role in bacterial survival in plant tissues during the initial infection stages.

Antimicrobial Cationic Peptides↗

PubMed1992

Cloning and characterization of the gene encoding the endopolygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L.

Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein purified from hypocotyls of true bean (Phaseolus vulgaris L.). PGIP inhibits fungal endopolygalacturonases and is considered to be an important factor for plant resistance to phytopathogenic fungi (Albersheim and Anderson, 1971; Cervone et al., 1987). The amino acid sequences of the N-terminus and one internal tryptic peptide of the PGIP purified from P. vulgaris cv. Pinto were used to design redundant oligonucleotides that were successfully utilized as primers in a polymerase chain reaction (PCR) with total DNA of P. vulgaris as a template. A DNA band of 758 bp (a specific PCR amplification product of part of the gene coding for PGIP) was isolated and cloned. By using the 758-bp DNA as a hybridization probe, a lambda clone containing the PGIP gene was isolated from a genomic library of P. vulgaris cv. Saxa. The coding and immediate flanking regions of the PGIP gene, contained on a subcloned 3.3 kb SalI-SalI DNA fragment, were sequenced. A single, continuous ORF of 1026 nt (342 amino acids) was present in the genomic clone. The nucleotide and deduced amino acid sequences of the PGIP gene showed no significant similarity with any known databank sequence. Northern blotting analysis of poly(A)+ RNAs, isolated from various tissues of bean seedlings or from suspension-cultured bean cells, were also performed using the cloned PCR-generated DNA as a probe. A 1.2 kb transcript was detected in suspension-cultured cells and, to a lesser extent, in leaves, hypocotyls, and flowers.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2002

Relationship between small antisense RNAs and aberrant RNAs associated with sense transgene mediated gene silencing in tomato.

We investigated the occurrence and properties of small sense and antisense RNAs and aberrant transcripts in transgenic tomato plants exhibiting post-transcriptional silencing of the endogenous polygalacturonase (PG) sense gene and a truncated homologous transgene. Small antisense RNAs, about 23 nucleotides in size, were present in the leaves and immature green fruit where the transgene was transcribed in the absence of transcripts from the fruit ripening-specific endogenous PG gene. This suggests that the small antisense RNAs were produced from the transgene, which was supported by their failure to hybridise with the 3' half of the endogenous PG gene, the region that was excluded from the transgene. The features of two aberrant RNAs, corresponding to the 5' and 3' halves of the endogenous gene, strongly suggest that they arise by endonucleolytic cleavage of endogenous PG mRNA. The region where cleavage occurs is associated with the preferential production of small RNAs from the 3' end of the transgene. These results suggest a model where small antisense RNAs generated from the 3' end of the transgene anneal to the endogenous mRNA, resulting in cleavage at or near the annealing regions.

Blotting, Northern↗

PubMed2003

Inverted repeat of a heterologous 3'-untranslated region for high-efficiency, high-throughput gene silencing.

This report describes a method for the easy generation of inverted repeat constructs for the silencing of genes of unknown sequence which is applicable to high-throughput studies. This improved procedure for high-efficiency gene silencing is specific for a target gene, but does not require inverted repeat DNA of the target gene in the construct. The method employs an inverted repeat of the 3'-untranslated region (3'-UTR) of a heterologous gene, and has been demonstrated using the 3'-UTR region of the nopaline synthase (nos) gene from Agrobacterium tumefaciens, which is often used as the 3'-UTR for transgene constructs. In a population of independent tomato primary transformants harboring a stably integrated polygalacturonase (PG) transgene driven by a constitutive promoter and linked to an inverted repeat of the nos 3'-UTR, 51 of 56 primary transformants (91% of the population) showed highly effective post-transcriptional silencing of the PG gene, with PG mRNA abundance in ripe fruit reduced by 98% or more. The method was also effective in Arabidopsis, where two different, relatively uncharacterized plant transcription factors were also targeted effectively. This method has the advantage of ease and rapidity in preparation of the constructs, since a gene of interest can be inserted into a binary vector already containing the promoter and the inverted nos domain in a single-cloning step, and does not require any knowledge of the DNA sequence. The approach is suitable for high-throughput gene silencing studies, where it is necessary to investigate the function of hundreds to thousands of uncharacterized genes.

3' Untranslated Regions↗

PubMed2003

Expression of MsPG3-GFP fusions in Medicago truncatula'hairy roots' reveals preferential tip localization of the protein in root hairs.

Tip growth is a specialized type of polar growth where new cell wall is deposited in a localized region of the cell, the growing tip. These cells show a characteristic zonation, with a high accumulation of secretory vesicles containing cell wall components at the tip, followed by an organelle-enriched zone. MsPG3 is a Medicago sativa polygalacturonase gene isolated in our laboratory, specifically expressed during the interaction of this plant with its symbiotic partner Sinorhizobium meliloti and which might participate in tip growth processes during symbiosis. We have used MsPG3-GFP fusions to study in vivo protein transport processes and localization during root hair growth. Different MsPG3-GFP fusions were expressed in Medicago truncatula'hairy roots' following a protocol developed for this study and also tested by transient expression in onion epidermal cells. Preferential accumulation of an MsPG3-GFP fusion protein in the tip of the growing root hair at different developmental stages was found, confirming the delivery of MsPG3 to the newly synthesized cell wall. This indicates that this protein may participate in tip growth processes during symbiosis and, in addition, that this fusion could be a useful tool to study this process in plants.

Gene Transfer Techniques↗

PubMed2000

hexA of Erwinia carotovora ssp. carotovora strain Ecc71 negatively regulates production of RpoS and rsmB RNA, a global regulator of extracellular proteins, plant virulence and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone.

The soft-rotting bacterium, Erwinia carotovora ssp. carotovora (E. c. carotovora), produces an array of extracellular enzymes (= exoenzymes), including pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel) and protease (Prt), as well as HarpinEcc, the elicitor of hypersensitive reaction (HR). The production of these exoenzymes and HarpinEcc responds to plant products and the quorum-sensing signal [N-(3-oxohexanoyl)-L-homoserine lactone; OHL] and is subject to both transcriptional and post-transcriptional regulation. hexA of E. c. carotovora strain Ecc71 (hereafter hexA71), like that of another E. c. carotovora strain, negatively controls the production of exoenzymes, OHL and virulence in E. c. carotovora strain Ecc71. In addition to exoenzymes, HexA71 negatively regulates the expression of hrpNEcc, the structural gene for HarpinEcc. Exoenzyme overproduction is abolished by OHL deficiency in a HexA- and Ohll- double mutant, indicating that HexA and OHL are components of a common regulatory pathway controlling exoenzyme production. HexA71 negatively affects RpoS, as the levels of this alternative sigma factor are higher in the HexA- mutant than in the HexA+ strain. However, a HexA- and RpoS double mutant produces higher levels of exoenzymes and transcripts of pel-1, peh-1 and celVgenes than the HexA- and RpoS+ parent. Thus, the elevated levels of RpoS protein in the HexA- mutant do not account for exoenzyme overproduction. The following evidence associates for the first time the phenotypic changes in the HexA mutant to overproduction of rsmB RNA, a global regulator of exoenzymes, HarpinEcc, OHL and secondary metabolites. Analyses of rsmB transcripts and expression of an rsmB-lacZoperon fusion in E. c. carotovora strain Ecc71 revealed that HexA71 negatively regulates transcription of rsmB. Multiple copies of hexA71+ DNA suppress various phenotypes, including exoenzyme production in E. c. carotovora strain Ecc71, and concomitantly inhibit the production of rsmB, pel-1, peh-1, celV and hrpNEcc transcripts. Multiple copies of rsmB+ DNA, on the other hand, stimulate exoenzyme production by relieving the negative effects of a chromosomal copy of hexA+. The occurrence of hexA homologues and the negative effect of the dosage of hexA71 DNA on rsmB transcripts were also detected in other E. c. carotovora strains as well as Erwinia carotovora atroseptica and Erwinia carotovora betavasculorum. Extrapolating from the findings with LrhA, the Escherichia coli homologue of HexA, and the presence of sprE homologues in E. carotovora subspecies, we propose that HexA71 controls several regulatory pathways in E. carotovora including rsmB transcription and the production of SprEEcc which, in turn, affects RpoS levels. A model is presented that integrates the findings presented here and our current knowledge of the major regulatory network that controls exoprotein production in soft-rotting Erwinia carotovora subspecies.

4-Butyrolactone↗

PubMed1996

Pectic polysaccharides from roots of Glycyrrhiza uralensis: possible contribution of neutral oligosaccharides in the galacturonase-resistant region to anti-complementary and mitogenic activities.

Digestion with endo-alpha-(1-->4)-polygalacturonase liberated the enzyme-resistant region (PG-1c) as an active site of the anti-complementary and mitogenic pectic polysaccharide (GR-2IIc) from Glycyrrhiza uralensis. Partial acid hydrolysis of PG-1c resulted in acidic oligosaccharides, and methylation analysis and GC-MS analysis of the acidic oligosaccharides suggested that PG-1c comprised a rhamnogalacturonan core such as -->2)-Rha-(1-->4)-GalA-(1-->2)-Rha-(1-->4)-GalA-(1-->-->4)-GalA-(1-->4) as the acidic moiety. Degradation of uronic acids by lithium decreased the anti-complementary and mitogenic activities of PG-1c. Although the products from PG-1c were still active, the methylglycoside of alpha-L-Rha-(1-->4)-alpha-D-GalA-(1-->2)-alpha-L-Rha-(1-->4)-alpha-D-Gal A did not show both activities. The products obtained by the lithium degradation from PG-1c gave fractions containing various neutral oligosaccharide-alditols. Among these fractions the longest and the short oligosaccharide-alditol fractions had relatively potent anti-complementary activity, whereas all oligosaccharide-alditol fractions expressed weak but significant mitogenic activity. GC-MS analysis indicated that the short oligosaccharide-alditol fraction contained various kinds of di- to tetrasaccharide-alditols. However, malto-oligosaccharide-alditols, and malto-, isomalto-, and laminari-oligosaccharides did not show anti-complementary and/or mitogenic activities, and these results suggested that certain neutral carbohydrate chains in PG-1c were responsible for the expression of mitogenic activity as well as anti-complementary activity of PG-1c.

Animals↗

PubMed2001

Group 13 grass allergens: structural variability between different grass species and analysis of proteolytic stability.

BACKGROUND: Determination of the allergen composition of an extract is essential for the improvement of hyposensitization therapy. Surprisingly, although grass pollen extracts have been studied intensively for 20 years, a further major allergen, Phl p 13, was detected recently in timothy grass pollen. OBJECTIVES: We sought to determine the occurrence and importance of group 13 allergens in various grass species and to investigate their proteolytic stability. METHODS: The group 13 allergens were determined by means of 2-dimensional PAGE blotting with patient sera and group 13-specific mAbs. The allergens were isolated chromatographically from several pollen extracts and analyzed by means of microsequencing. Cross-reactivity among various grass species was studied by using Western blots and immunoblot inhibition tests. The stability of the allergens was tested under defined extraction conditions. RESULTS: Group 13 allergens are detectable in all common grasses and show IgE cross-reactivity among them. The allergenic components were identified in the neutral pH range with molecular masses of 50 to 60 kd, and in the case of Phl p 13, maximal binding of the isoforms was observed at 55 kd and at an isoelectric point of 6 to 7.5. Protein sequencing clearly confirms structural identities between different grass species, although individual variations are found. If low-molecular-mass components were depleted by means of gel filtration, a rapid degradation of group 13 allergens was observed. This is in contrast to other pollen allergens described thus far. CONCLUSION: Group 13 allergens are widespread and are major allergens in the grasses. Predicted from their primary structures, these allergens are polygalacturonases. This class of enzymes is already known from microorganisms, and these enzymes are recognized as potential inducers of asthma. Our studies indicate that the group 13 allergens show a considerable microheterogeneity and degradation, especially after depletion of low-molecular-mass components. One has to be aware of this pivotal fact when soluble grass pollen extracts are prepared for diagnostics and hyposensitization therapy.

Allergens↗

PubMed1999

Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway.

Hydrogen peroxide (H2O2) generated in response to wounding can be detected at wound sites and in distal leaf veins within 1 hr after wounding. The response is systemic and maximizes at about 4-6 hr in both wounded and unwounded leaves, and then declines. The timing of the response corresponds with an increase in wound-inducible polygalacturonase (PG) mRNA and enzyme activity previously reported, suggesting that oligogalacturonic acid (OGA) fragments produced by PG are triggering the H2O2 response. Systemin, OGA, chitosan, and methyl jasmonate (MJ) all induce the accumulation of H2O2 in leaves. Tomato plants transformed with an antisense prosystemin gene produce neither PG activity or H2O2 in leaves in response to wounding, implicating systemin as a primary wound signal. The antisense plants do produce both PG activity and H2O2 when supplied with systemin, OGA, chitosan, or MJ. A mutant tomato line compromised in the octadecanoid pathway does not exhibit PG activity or H2O2 in response to wounding, systemin, OGA, or chitosan, but does respond to MJ, indicating that the generation of H2O2 requires a functional octadecanoid signaling pathway. Among 18 plant species from six families that were assayed for wound-inducible PG activity and H2O2 generation, 14 species exhibited both wound-inducible PG activity and the generation of H2O2. Four species, all from the Fabaceae family, exhibited little or no wound-inducible PG activity and did not generate H2O2. The time course of wound-inducible PG activity and H2O2 in Arabidopsis thaliana leaves was similar to that found in tomato. The cumulative data suggest that systemic wound signals that induce PG activity and H2O2 are widespread in the plant kingdom and that the response may be associated with the defense of plants against both herbivores and pathogens.

Hydrogen Peroxide↗

PubMed1999

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.

Amino Acid Sequence↗