Characterization of the exopolygalacturonate lyase PelX of Erwinia chrysanthemi 3937. (1/703)

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.  (+info)

Dietary pectin lowers sphingomyelin concentration in VLDL and raises hepatic sphingomyelinase activity in rats. (2/703)

There is evidence that cholesterol and sphingomyelin metabolism are interrelated, and thus the hypothesis tested was that dietary pectin, because it can alter hepatic cholesterol metabolism, would also alter hepatic sphingomyelin metabolism. For that purpose, 4-wk-old female Wistar rats were fed a diet without or with pectin (20 g/100 g) up to 21 d. In accordance with previous work, pectin consumption caused a significant (P < 0.001) reduction in hepatic (65%), whole plasma (37%), and VLDL (80%) cholesterol levels. Pectin also significantly reduced VLDL sphingomyelin concentrations (57%), but raised the amount of sphingomyelin in the high density lipoproteins (HDL)-2 fractions (58%), so that the level of sphingomyelin in whole plasma remained unaffected. Pectin did not affect the sphingomyelin concentration in the liver. Pectin consumption did not affect the hepatic sphingomyelin synthesizing enzymes, serine palmitoyltransferase, phosphatidylcholine:ceramide phosphocholine transferase, or phosphatidylethanolamine:ceramide phosphoethanolamine transferase. In contrast, dietary pectin activated both lysosomal (28%) and plasma membrane (26%) sphingomyelinase and thus may have enhanced sphingomyelin degradation. An attempt was made to describe the effects of dietary pectin on sphingomyelin metabolism in terms of altered fluxes through liver and plasma, with whole liver and whole plasma concentrations of sphingomyelin remaining unaffected.  (+info)

Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C. (3/703)

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.  (+info)

Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea. (4/703)

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.  (+info)

Azospirillum irakense produces a novel type of pectate lyase. (5/703)

The pelA gene from the N2-fixing plant-associated bacterium Azospirillum irakense, encoding a pectate lyase, was isolated by heterologous expression in Escherichia coli. Nucleotide sequence analysis of the region containing pelA indicated an open reading frame of 1,296 bp, coding for a preprotein of 432 amino acids with a typical amino-terminal signal peptide of 24 amino acids. N-terminal amino acid sequencing confirmed the processing of the protein in E. coli at the signal peptidase cleavage site predicted by nucleotide sequence analysis. Analysis of the amino acid sequence of PelA revealed no homology to other known pectinases, indicating that PelA belongs to a new pectate lyase family. PelA macerates potato tuber tissue, has an alkaline pH optimum, and requires Ca2+ for its activity. Of several divalent cations tested, none could substitute for Ca2+. Methyl-esterified pectin (with a degree of esterification up to 93%) and polygalacturonate can be used as substrates. Characterization of the degradation products formed upon incubation with polygalacturonate indicated that PelA is an endo-pectate lyase generating unsaturated digalacturonide as the major end product. Regulation of pelA expression was studied by means of a translational pelA-gusA fusion. Transcription of this fusion is low under all growth conditions tested and is dependent on the growth phase. In addition, pelA expression was found to be induced by pectin. An A. irakense pelA::Tn5 mutant still displayed pectate lyase activity, suggesting the presence of multiple pectate lyase genes in A. irakense.  (+info)

Potential of short chain fatty acids to modulate the induction of DNA damage and changes in the intracellular calcium concentration by oxidative stress in isolated rat distal colon cells. (6/703)

Short chain fatty acids (SCFA) are considered to be beneficial fermentation products in the gut by exerting trophic effects in non-transformed colon cells and by slowing proliferation and enhancing differentiation in colonic tumour cells. We have studied the further effects of SCFA on cellular events of early carcinogenesis, genotoxicity and cytotoxicity in rat distal colon cells. Cytotoxicity was assessed by measuring trypan blue exclusion and by determining the H2O2-induced changes in intracellular calcium concentration ([Ca2+]i) using a fluorospectrophotometer and the calcium-sensitive fluorescent dye Fura-2. The microgel electrophoresis technique (COMET assay) was used to assess oxidative DNA damage. Individual SCFA and physiological SCFA mixtures were investigated for their potential to prevent DNA and cell damage induced by H2O2. For this, freshly isolated colon cells were treated with H2O2 (100-500 microM) and 6.25 mM SCFA. We have found 100-500 microM H2O2 to cause a fast initial increase in [Ca2+]i, whereafter the levels gradually further increased. Addition of SCFA did not affect [Ca2+]i nor did it reduce the H2O2-induced increase in [Ca2+]i. Butyrate and acetate were able to reduce the induction of DNA damage by 100, 200 and 500 microM H2O2, respectively. In contrast, i-butyrate and propionate were ineffective. The degree of reduction of DNA damage for the two protective SCFA was similar. Physiological mixtures containing acetate, propionate and butyrate in ratios of 41:21:38 or 75:15:10 that are expected to arise in the colon after fermentation of resistant starches and pectin, respectively, did not show significant antigenotoxic effects. The major difference between butyrate and acetate, on one hand, and i-butyrate and propionate, on the other hand, is that the former compounds are utilized best as energy sources by the colon cells. Therefore, our results on antigenotoxicity coupled with the findings on [Ca2+]i homeostasis indicate that molecular effects on the energy system render these non-transformed, freshly isolated colon cells to be less susceptible to H2O2.  (+info)

The plant cell wall polysaccharide rhamnogalacturonan II self-assembles into a covalently cross-linked dimer. (7/703)

The location of the 1:2 borate-diol ester cross-link in the dimer of the plant cell wall polysaccharide rhamnogalacturonan II (RG-II) has been determined. The ester cross-links the apiofuranosyl residue of the 2-O-methyl-D-xylose-containing side chains in each of the subunits of the dimer. The apiofuranosyl residue in each of the two aceric acid-containing side chains is not esterified. The site of borate esterification is identical in naturally occurring and in in vitro synthesized dimer. Pb2+, La3+, and Ca2+ increase dimer formation in vitro in a concentration- and pH-dependent manner. Pb2+ is the most effective cation. The dimer accounts for 55% of the RG-II when the monomer (0.5 mM) is treated for 5 min at pH 3.5 with boric acid (1 mM) and Pb2+ (0.5 mM); at pH 5 the rate of conversion is somewhat slower. Hg2+ does not increase the rate of dimer formation. A cation's charge density and its ability to form a coordination complex with RG-II, in addition to steric factors, may regulate the rate and stability of dimer formation in vitro. Our data provide evidence that the structure of RG-II itself determines which apiofuranosyl residues are esterified with borate and that in the presence of boric acid and certain cations, two RG-II monomers self-assemble to form a dimer.  (+info)

Multiple epoxide hydrolases in Alternaria alternata f. sp. lycopersici and their relationship to medium composition and host-specific toxin production. (8/703)

The production of Alternaria alternata f. sp. lycopersici host-specific toxins (AAL toxins) and epoxide hydrolase (EH) activity were studied during the growth of this plant-pathogenic fungus in stationary liquid cultures. Media containing pectin as the primary carbon source displayed peaks of EH activity at day 4 and at day 12. When pectin was replaced by glucose, there was a single peak of EH activity at day 6. Partial characterization of the EH activities suggests the presence of three biochemically distinguishable EH activities. Two of them have a molecular mass of 25 kDa and a pI of 4.9, while the other has a molecular mass of 20 kDa and a pI of 4.7. Each of the EH activities can be distinguished by substrate preference and sensitivity to inhibitors. The EH activities present at day 6 (glucose) or day 12 (pectin) are concomitant with AAL toxin production.  (+info)

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