Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.15 (pectinase)
 2,440 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lei et al. [Gene 117 (1992) 119-124] recently published the nucleotide sequence of the peh gene of Erwinia carotovora subsp. carotovora (Ecc) and a characterization of its product endopolygalacturonase (Peh). The gene appears highly similar to previously described peh sequences of Ecc [Hinton et al., Mol. Microbiol. 4 (1990) 1029-1036; Saarilahti et al., Mol. Microbiol. 4 (1990) 1037-1044] which were not cited in the article. Ecc carries a single peh gene whose product, Peh, is here shown to share similarity with the two Pehs characterized thus far and a Peh-like protein of eukaryotic origin at the amino acid (aa) sequence level. Additionally, a highly conserved region within their C-terminal domains was found to share local similarity with two 13-aa segments of an otherwise distinct exo-poly-alpha-D-galacturonosidase (exo-Peh), suggesting that these segments might be required for enzyme activity in both Peh and exo-Peh.
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PMID:Characterization of polygalacturonases. 844 Apr 78

A set of gene fusions was constructed between the pehA gene encoding the secreted endopolygalacturonase (PehA) and the bla gene coding for a normally periplasmic beta-lactamase (Bla). The resulting hybrid proteins were specifically and actively routed out of the cells via the Out-terminal branch of the general secretory pathway (GSP) in Erwinia carotovora subsp. carotovora (Ecc), provided that no more than the last two amino acids (aa) of the PehA domain were excluded from the fusion. However, both PehA-Bla hybrid proteins and PehA variants lacking at least four aa from the C-terminus of the PehA accumulated in the periplasm. Also, overexpression of the gene fusions prevented extracellular targeting of the hybrid proteins. Site-directed mutagenesis of the codons -4 and -3 (encoding Asn-373 and Val-374, respectively) from the end of the pehA gene and analysis of the protein products suggested that the Val-374 was important both for the structure and secretion of PehA, while the Asn-373 proved to be insignificant. We conclude that: (i) the GSP of Ecc is capable of secreting heterologous proteins; (ii) as the PehA protein can accommodate C-terminal extensions, secretion can occur with no part of the proposed targeting signal lying within the C-terminal extremity of a secreted molecule; and (iii) residues within the C-terminus of PehA play a role in secretion, possibly through stabilization of a structure needed for proper exposition of the proposed targeting motif.
Mol Microbiol 1995 Aug
PMID:The extreme C-terminus is required for secretion of both the native polygalacturonase (PehA) and PehA-Bla hybrid proteins in Erwinia carotovora subsp. carotovora. 855 64

A gene, CEL1, in the maize pathogen Cochliobolus carbonum was identified using the cbh1-3 gene of Phanerochaete chrysosporium as a heterologous probe. The predicted product of CEL1, Cel1, is 62% identical and 71% similar to the product of cbh1-3 and 54 to 62% identical to five cellobiohydrolases from other filamentous fungi. The location of the polyadenylation site 221 bp downstream of the stop codon and the location of a single intron of 55 bp were identified by comparison of the sequences of genomic and cDNA copies of CEL1. The transcriptional start site was determined by rapid amplification of cDNA ends (RACE) to be 39 bp upstream of the putative translational start site. CEL1 mRNA abundance is high when C. carbonum is grown on cellulose or maize cell walls but is undetectable when grown on 2% sucrose or cellulose plus sucrose. Cel1 has a predicted signal peptide of 18 amino acids and therefore a mature size of 46.4 kDa. Like the product of cbh1-1 of P. chrysosporium, but unlike most other endoglucanases and cellobiohydrolases (including the predicted product of cbh1-3), Cel1 does not have a putative cellulose binding domain or associated hinge region. The codon bias of CEL1 is stronger than the bias of cbh1-1 and comparable to that of cbh1-3 and that of the C. carbonum genes PGN1 and XYL1, (encoding endopolygalacturonase and endo-xylanase, respectively). A strain of C. carbonum specifically mutated at CEL1 was produced by transformation with a truncated copy of CEL1. Integration and disruption of CEL1 in the mutant was confirmed by DNA and RNA blotting. Pathogenicity of the CEL1 mutant was indistinguishable from the wild-type, indicating that CEL1 by itself is not a critical disease determinant. Culture filtrates of C. carbonum grown on cellulose or maize cell walls had several cellobiohydrolase, endoglucanase, and beta-glucosidase activities that were separable by chromatofocusing, hydrophobic interaction, or ion-exchange high-performance liquid chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Plant Microbe Interact
PMID:Characterization and disruption of a gene in the maize pathogen Cochliobolus carbonum encoding a cellulase lacking a cellulose binding domain and hinge region. 858 15

The effects of extended heat stress on polygalacturonase (PG; EC 3.2.1.15) and pectin methylesterase (PME; EC 3.1.1.11) gene expression at mRNA, protein and activity levels in ripening tomato fruits were investigated. Steady state levels of PG mRNA declined at temperatures of 27 degrees C and above, and a marked reduction in PG protein and activity was observed at temperatures of 32 degrees C and above. Exogenous ethylene treatment did not reverse heat stress-induced inhibition of PG gene expression. Transfer of heat-stressed fruits to 20 degrees C partly restored PG mRNA accumulation, but the rate of PG mRNA accumulation declined exponentially with duration of heat stress. Heat stress-induced inhibition of PME mRNA accumulation was recoverable even after 14 days of heat stress. In fruits held at 34 degrees C, both PG and PME protein and activity continued to accumulate for about 4 days, but thereafter PG protein and activity declined while little change was observed in PME protein and activity. In spite of increases in mRNA levels of both PG and PME during the recovery of heat-stressed fruit at 20 degrees C, levels of PG protein and activity declined in fruits heat-stressed for four or more days while PME protein and activity levels remained unchanged. Collectively, these data suggest that PG gene expression is being gradually and irreversibly shut off during heat stress, while PME gene expression is much less sensitive to heat stress.
Plant Mol Biol 1995 Dec
PMID:Differential regulation of polygalacturonase and pectin methylesterase gene expression during and after heat stress in ripening tomato (Lycopersicon esculentum Mill.) fruits. 861 11

Site-selected insertion (SSI) is a PCR-based technique which uses primers located within the transposon and a target gene for detection of transposon insertions into cloned genes. We screened tomato plants bearing single or multiple copies of maize Ac or Ds transposable elements for somatic insertions at one close-range target and two long-range targets. Eight close-range Ds insertions near the right border of the T-DNA were recovered. Sequence analysis showed a precise junction between the transposon and the target for all insertions. Two insertions in separate plants occurred at the same site, but others appeared dispersed in the region of the right T-DNA border with no target specificity. However, insertions showed a preference for one orientation of the transposon. Use of plants with multiple Ac (HiAc) or Ds (HiDs) elements allowed detection of somatic insertions at two single-copy genes, PG (polygalacturonase) and DFR (dihydroflavonol 4-reductase). Certain HiDs plants showed much higher rates of insertion into PG than others. Insertions in PG and DFR were found throughout the gene regions monitored and, with the exception of one insertion in PG, the junctions between transposon and target were exact. SSI analysis of progeny from the HiDs parents revealed that in some cases the tendency to incur high levels of somatic insertions in PG was inherited. Inheritance of this character is an indication that SSI could be used to direct a search for germinal PG insertions in tomato.
Mol Gen Genet 1996 Aug 27
PMID:Site-selected insertional mutagenesis of tomato with maize Ac and Ds elements. 880 92

Erwinia carotovora subsp. carotovora wild-type strain Ecc71 does not elicit the hypersensitive reaction (HR) in tobacco leaves. By mini-Tn5-Km and chemical mutagenesis we have isolated RsmA- mutants of Ecc71 that produce high basal levels of pectate lyases, polygalacturonase, cellulase, and protease; they also are hypervirulent. The RsmA- mutants, but not their parent strains, elicit an HR-like response in tobacco leaves. This reaction is characterized by the rapid appearance of water soaking followed by tissue collapse and necrosis. The affected areas remain limited to the region infiltrated with bacterial cells, and the symptoms closely resemble a typical HR, e.g., the reactions caused by Pseudomonas syringae pv. pisi. Moreover, low concentrations of cells of the mini-Tn5-Km insertion RsmA- mutant, AC5070, infiltrated into tobacco leaf tissue prevent elicitation of the rapid necrosis by AC5070 or by P. syringae pv. pisi. Elicitation of the HR-like response by the mutants is not affected by the deficiency of N-(3-oxohexanoyl)-L-homoserine lactone, the cell density (quorum) sensing signal. Cloning and sequence analysis have disclosed that E. carotovora subsp. carotovora strain Ecc71 possesses a homolog of E. chrysanthemi hrpN known to encode an elicitor of the HR; the corresponding Ecc71 gene is designated hrpNEcc. Northern (RNA) blot data show that the level of hrpNEcc mRNA is considerably higher in the RsmA- mutants than in the RsmA+ strains. Moreover, a low copy plasmid carrying the rsmA+ allele severely reduces the level of the hrpNEcc transcripts in the RsmA- mutants. These constructs, like the RsmA+ E. carotovora subsp. carotovora strains, do not elicit the HR-like response. These data taken along with the effects of rsmA on exoenzyme production and pathogenicity (A. Chatterjee et al., 1995, Appl. Environ. Microbiol. 61:1959-1967) demonstrate that this global regulator gene plays a critical role in plant interaction of E. carotovora subsp. carotovora.
Mol Plant Microbe Interact 1996 Sep
PMID:The RsmA- mutants of Erwinia carotovora subsp. carotovora strain Ecc71 overexpress hrpNEcc and elicit a hypersensitive reaction-like response in tobacco leaves. 881 71

The sequence encoding the endopolygalacturonase (PG) of Fusarium moniliforme was cloned into the E. coli/yeast shuttle vector Yepsec1 for secretion in yeast. The recombinant plasmid (pCC6) was used to transform Saccharomyces cerevisiae strain S150-2B; transformed yeast cells were able to secrete PG activity into the culture medium. The enzyme (wtY-PG) was purified, characterized, and shown to possess biochemical properties similar to those of the PG purified from F. moniliforme. The wtY-PG was able to macerate potato medullary tissue disks and was inhibited by the polygalacturonase-inhibiting protein (PGIP) purified from Phaseolus vulgaris. The sequence encoding PG in pCC6 was subjected to site-directed mutagenesis. Three residues in a region highly conserved in all the sequences known to encode PGs were separately mutated: His 234 was mutated into Lys (H 234-->K), and Ser 237 and Ser 240 into Gly (S 237-->G and S 240-->G). Each of the mutated sequences was used to transform S. cerevisiae and the mutated enzymes were purified and characterized. Replacement of His 234 with Lys abolished the enzymatic activity, confirming the biochemical evidence that a His residue is critical for enzyme activity. Replacement of either Ser 237 or Ser 240 with Gly reduced the enzymatic activity to 48% and 6%, respectively, of the wtY-PG. When applied to potato medullary tissue, F. moniliforme PG and wtY-PG caused comparable maceration, while the variant PGs exhibited a limited (S 234-->G and S 240-->G) or null (H 234-->K) macerating activity. The interaction between the variant enzymes and the P. vulgaris PGIP was investigated using a biosensor based on surface plasmon resonance (BIAlite). The three variant enzymes were still able to interact and bind to PGIP with association constants comparable to that of the wild type enzyme.
Mol Plant Microbe Interact 1996 Sep
PMID:Mutagenesis of endopolygalacturonase from Fusarium moniliforme: histidine residue 234 is critical for enzymatic and macerating activities and not for binding to polygalacturonase-inhibiting protein (PGIP). 881 77

Tomato fruit maturation is accompanied by a depolymerization of cell wall pectins which is due to the action of endopolygalacturonase (endoPG) preceded by pectin methylesterase (PE) activity. To investigate the role of endoPG and PE in determining the structure of green bean (Phaseolus vulgaris L.) pectins, these pectinases were studied during pod development. Early developmental stages displayed low endoPG or exoPG activities while PE activities were measurable during all stages of pod and seed development. These results do not favour a possible synergistic action of PE and PG. For seeds, the relatively high PE activities concurred with relatively low levels of pectin methyl esterification. At a molecular level, one partial chromosomal clone of 210 bp (PE1V), two partial PE cDNA clones of 660 bp (PE2V and PE3V) from cv. verona and one full-length PE cDNA clone of 1990 bp (PE3M), from cv. Masai were isolated. The identity of the CDNA clones was confirmed by expression in Escherichia coli and immunodetection with antibodies directed towards a tomato fruit PE. Transcripts corresponding with the genomic clone PE1V were not detected but both PE2 and PE3 cDNAs corresponded with mRNAs 1.8 kb in length. In contrast to PE2, PE3 gene expression levels varied significantly in pods from different cultivars suggesting an involvement in determining pod morphology.
Plant Mol Biol 1996 Sep
PMID:Characterization of pectinases and pectin methylesterase cDNAs in pods of green beans (Phaseolus vulgaris L.). 891 30

A mutant of Erwinia carotovora subsp. carotovora, AH2552, created by a Mud1 insertion was found to be reduced in plant pathogenicity and deficient in extracellular protease and cellulase activity, although it produced normal levels of pectate lyase and polygalacturonase. A cosmid clone, pEC462, was isolated from a wild-type E. carotovora subsp. carotovora DNA library that concomitantly restored pathogenicity and protease and cellulase activities of AH2552 to wild-type levels when present in trans. The genetic locus that was disrupted in AH2552 by insertion of Mud1 has been designated rpfA, for regulator of pathogenicity factors. Sequencing of the rpfA region identified an open reading frame of 2,787 bp, and the predicted 929-amino acid polypeptide shared high identity with several two-component sensor-regulator proteins: BarA from Escherichia coli, ApdA from Pseudomonas fluorescens, PheN from P. tolaasii, RepA from P. viridiflava, LemA from P. syringae pv. syringae, and RpfC from Xanthomonas campestris pv. campestris. The RpfA locus described in this study encodes a putative sensor kinase protein that is involved in both extracellular protease and cellulase production and the pathogenicity of E. carotovora subsp. carotovora on potato tubers.
Mol Plant Microbe Interact 1997 Apr
PMID:Identification of a pathogenicity locus, rpfA, in Erwinia carotovora subsp. carotovora subsp. carotovora that encodes a two-component sensor-regulator protein. 910 Mar 85

Affinity precipitation is being studied as a technique to be introduced at an early stage of downstream processing for the selective isolation of proteins. The technique utilizes a heterobifunctional ligand, which, in addition to having affinity for the target protein(s), possesses another function for controlling precipitation. The latter component is comprised of a polymer which can be made reversibly soluble and insoluble by altering a specific parameter such as pH or temperature. Different polymers of natural and synthetic origin have been used for this purpose. The soluble form of the ligand is used for the affinity binding step and precipitation is induced for obtaining separation of the affinity complex. Some of the polymers used in this laboratory include chitosan, alginate, Eudragit S-100 (copolymer of methacrylic acid and methyl methacrylate) and polyethyleneimine. Chitosan and alginate served as natural ligands for wheat germ agglutinin and pectinase, respectively. The aromatic dye Cibacron Blue 3GA coupled to Eudragit S 100 and polyethyleneimine way used for the affinity precipitation of some model enzymes such as lactate dehydrogenase and alcohol dehydrogenase. As prior removal of cell debris, etc., is essential for affinity precipitation, the possibility of integration of the technique with extraction in aqueous two-phase systems was also demonstrated.
J Mol Recognit
PMID:Affinity precipitation of proteins. 917 9


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