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PubMed2005

The in situ observation of the temperature and pressure stability of recombinant Aspergillus aculeatus pectin methylesterase with Fourier transform IR spectroscopy reveals an unusual pressure stability of beta-helices.

The stability of recombinant Aspergillus aculeatus PME (pectin methylesterase), an enzyme with high beta-helix content, was studied as a function of pressure and temperature. The conformational stability was monitored using FTIR (Fourier transform IR) spectroscopy whereas the functional enzyme stability was monitored by inactivation studies. Protein unfolding followed by amorphous aggregation, which makes the process irreversible, was observed at temperatures above 50 degrees C. This could be correlated to the irreversible enzyme inactivation observed at that temperature. Hydrostatic pressure greater than 1 GPa was necessary to induce changes in the enzyme's secondary structure. No enzyme inactivation was observed at up to 700 MPa. Pressure increased PME stability towards thermal denaturation. At 200 MPa, temperatures above 60 degrees C were necessary to cause significant PME unfolding and loss of activity. These results may be relevant for an understanding of the extreme stability of amyloid fibrils for which beta-helices have been proposed as a structural element.

Aspergillus↗
PubMed1996

RecA relieves negative autoregulation of rdgA, which specifies a component of the RecA-Rdg regulatory circuit controlling pectin lyase production in Erwinia carotovora ssp. carotovora.

The production of pectin lyase (Pnl) in Erwinia carotovora ssp. carotovora strain 71 is induced by DNA-damaging agents such as mitomycin C (MC). This induction requires functions of recA, rdgA and rdgB genes. Based upon sequence homology, rdgA was predicted to encode a repressor and rdgB was presumed to specify a transcriptional activator. To elucidate the function of rdgA, the gene has been over-expressed in Escherichia coli, and the 30 kDa product purified by ammonium-sulphate precipitation, heparin-agarose chromatography and gel filtration. The results of gel mobility-shift and DNase I protection assays revealed that purified RdgA specifically binds the rdgA operator sequence located between the -10 and -35 boxes. The expression of a rdgA-lacZ gene fusion in E. coli MC4100 is suppressed upon overproduction of RdgA from a Ptac-rdgA construct induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). However, the suppression of rdgA-tacZ expression is relieved by MC in the RecA+ E. coli strain MC4100, but not in its RecA- derivative, MC4160. An immunoblot analysis revealed RecA-dependent in vivo cleavage of the 30 kDa RdgA protein upon MC treatment. These results demonstrate that the transcription of rdgA is autoregulated, and strongly support the idea that proteolytic activity of RecA* is responsible for the derepression of rdgA expression.

Bacterial Proteins↗
PubMed2003

Systemic movement of a tobamovirus requires host cell pectin methylesterase.

Systemic movement of plant viruses through the host vasculature, one of the central events of the infection process, is essential for maximal viral accumulation and development of disease symptoms. The host plant proteins involved in this transport, however, remain unknown. Here, we examined whether or not pectin methylesterase (PME), one of the few cellular proteins known to be involved in local, cell-to-cell movement of tobacco mosaic virus (TMV), is also required for the systemic spread of viral infection through the plant vascular system. In a reverse genetics approach, PME levels were reduced in tobacco plants using antisense suppression. The resulting PME antisense plants displayed a significant degree of PME suppression in their vascular tissues but retained the wild-type pattern of phloem loading and unloading of a fluorescent solute. Systemic transport of TMV in these plants, however, was substantially delayed as compared to the wild-type tobacco, suggesting a role for PME in TMV systemic infection. Our analysis of virus distribution in the PME antisense plants suggested that TMV systemic movement may be a polar process in which the virions enter and exit the vascular system by two different mechanisms, and it is the viral exit out of the vascular system that involves PME.

Carboxylic Ester Hydrolases↗
PubMed1998

Pectinate ligament dysplasia (PLD) and glaucoma in Flat Coated Retrievers. I. Objectives, technique and results of a PLD survey.

Pectinate ligament dysplasia (PLD) in dogs is a congenital ocular abnormality affecting the iridocorneal angle, the presence of which may be associated with adult-onset primary glaucoma. This paper describes the objectives, methods and results of a survey of PLD in Flat Coated Retrievers in the UK, including the technique used for gonioscopic assessment of the degree of PLD. In a random sample of 389 Flat Coated Retrievers, 34.7% were found to have a degree of PLD. In a 'target' sample of 48 Flat Coated Retrievers (close relatives of dogs from the random sample with high degrees of PLD, or dogs presented with primary glaucoma), 83.3% were found to have a degree of PLD. The percentage of animals with a degree of PLD was only 6.0% in a 'control' sample of 100 dogs from other breeds. The relationship between PLD and canine glaucoma is discussed.

Journal Article↗
PubMed1998

Pectinate ligament dysplasia and glaucoma in Flat Coated Retrievers. II. Assessment of prevalence and heritability.

Pectinate ligament dysplasia (PLD) in dogs is a congenital ocular abnormality affecting the iridocorneal angle, the presence of which may be associated with adult-onset primary glaucoma. This study demonstrates a significant association between PLD and glaucoma in Flat Coated Retrievers, and also a higher prevalence of PLD in Flat Coated Retrievers compared with other breeds. It is shown that the association between PLD and glaucoma was robust and not due to a small increase in PLD with age. There was also a significant association between PLD in offspring and parents and, using the offspring-parents regression, the heritability of PLD was estimated as approximately 0.7. As glaucoma is significantly related to PLD, glaucoma may also be heritable. Thus, glaucoma can be controlled by gonioscopic examination and elimination of animals with a high degree of PLD from a breeding program. Breeding from only those animals with a moderate or low PLD value or score should limit the expected prevalence of glaucoma in the resulting population to < 0.2%. The preceding study describes in detail the PLD survey, gonioscopic technique and some initial results obtained prior to this epidemiological investigation.

Journal Article↗
PubMed2002

Pectinate ligament dysplasia and narrowing of the iridocorneal angle associated with glaucoma in the English Springer Spaniel.

OBJECTIVE: The aim of the study was to evaluate if tonometry and gonioscopy could serve as predictors of later glaucoma development in the English Springer Spaniel, a breed prone to developing primary glaucoma. ANIMALS STUDIED: Gonioscopy was performed on 279 English Springer Spaniel dogs, 119 males and 160 females, with clinically normal eyes. In addition, 14 dogs, five males and nine females were examined for glaucoma. RESULTS: A positive relation between pectinate ligament dysplasia (PLD) and narrowing of the relative width of the ciliary cleft (RWOCC) and also between PLD, RWOCC and age was demonstrated. The prevalence of PLD was 25.5%, which is higher than in other breeds reported. A positive association was demonstrated between PLD and glaucoma, as well as between narrowing of the RWOCC and glaucoma. Glaucoma was not observed in dogs with normal appearance of the iridocorneal angle. English Springer Spaniels related to dogs with glaucoma show more narrowing of the RWOCC and more PLD than unrelated dogs. CONCLUSION: This study demonstrates the positive association between PLD and glaucoma, between narrowing of the iridocorneal angle and glaucoma and the effect of age on the iridocorneal angle. Mating of dogs with normal iridocorneal angles appears to reduce the presence and degree of abnormal appearance of the iridocorneal angle in the offspring. However, breeding only dogs with normal iridocorneal angles without consideration of their relationship to dogs with glaucoma is not a guarantee for preventing glaucoma in the offspring.

Animals↗
PubMed2002

Mode of action of pectin lyase A of Aspergillus niger on differently C(6)-substituted oligogalacturonides.

A thorough investigation of the mode of action of Aspergillus niger (4M-147) pectin lyase A (PLA) on differently C(6)-substituted oligogalacturonides is described. PLA appeared to be very specific for fully methyl-esterified oligogalacturonides: removal of the methyl-ester or changing the type of ester (ethyl esterification) or transamidation resulted in (almost) complete loss of conversion. The PLA activity increased with increasing length of the substrate up to a degree of polymerization (DP) of 8 indicating the presence of at least eight subsites on the enzyme. Product analysis demonstrated the formation of several Delta 4,5 unsaturated products and their saturated counterparts. The Delta 4,5 unsaturated trimer was the main product up to DP 8. For DP 9 and 10 Delta 4,5 unsaturated tetramer was the major product. Based upon the bond cleavage frequencies, a provisional subsite map was calculated, which supports the presence of eight subsites. By limited alkaline de-esterification of fully methyl-esterified pentamer and hexamer two sets of partially methyl-esterified pentamers (x and y methyl groups) and hexamers (a and b methyl groups) were prepared. Matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) analysis demonstrated that the methyl-ester distribution was fully random. Using these partially methyl-esterified oligogalacturonides as substrates for PLA a 10-fold decrease in reaction rate was recorded compared with the fully methyl-esterified counterparts. Analysis of the methyl-ester distribution of the products showed that PLA tolerates carboxyl groups in the substrate binding cleft. At either subsite +2, +4, or -1 to -4 a free carboxyl group could be tolerated, whereas methyl-esters were obligatory at subsite +1 and +3. So PLA is capable to cleave the bond between a methyl-esterified and a non-esterified galacturonic acid residue, where the newly formed Delta 4,5 unsaturated non-reducing end residue always contains a methyl-ester.

Aspergillus niger↗
PubMed2004

Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric.

There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.

Bacteria↗
PubMed2003

Activity of a flax pectin methylesterase promoter in transgenic tobacco pollen.

The regulatory region of the flax Lupme3 gene, which codes for a pectin methylesterase, contains two sequences (PB box) that are putative cis-active sequence elements thought to regulate transcription in pollen. The Lupme3 promoter was fused to the beta-glucuronidase (gus) reporter gene. The chimeric gene fusion was introduced into tobacco via Agrobacterium-mediated transformation. Expression of the reporter gene was monitored using a histochemical X-Gluc assay at different stages of pollen maturation and germination. The Lupme3 promoter was found to be active in germination-competent mature pollen and in pollen tube.

Base Sequence↗
PubMed2000

Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement.

Virus-encoded movement protein (MP) mediates cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata. The molecular pathway by which TMV MP interacts with the host cell is largely unknown. To understand this process better, a cell wall-associated protein that specifically binds the viral MP was purified from tobacco leaf cell walls and identified as pectin methylesterase (PME). In addition to TMV MP, PME is recognized by MPs of turnip vein clearing virus (TVCV) and cauliflower mosaic virus (CaMV). The use of amino acid deletion mutants of TMV MP showed that its domain was necessary and sufficient for association with PME. Deletion of the PME-binding region resulted in inactivation of TMV cell-to-cell movement.

Amino Acid Sequence↗
PubMed1986

Effect of guar gum, lignin and pectin on proteolytic enzyme levels in the gastrointestinal tract of the rat: a time-based study.

The effects of dietary pectin (P), guar gum (G) and lignin (L) on stomach emptying and potential levels of pepsin, trypsin and chymotrypsin during a 2-h period after force-feeding were investigated in growing rats. All of the fibers delayed stomach emptying by 21-26 min. Total potential pepsin activity over 2 h decreased for P (57%), G (44%) and L (20%). In the intestine, total potential trypsin activity over 2 h increased for L (16%) but decreased for P (21%). Total potential chymotrypsin activity over 2 h increased for L (54%) and G (39%). Sixteen to 21% of the variability in intestinal activity over time was statistically attributable to variation in the weight of intestinal contents. The results indicate that fiber components altered proteolytic enzyme levels in the gastrointestinal tract, but the decreased protein utilization previously observed with these fibers is probably not due to reduced levels of intestinal proteases.

Administration, Oral↗
PubMed2006

Sensitization to king scallop (Pectin maximus) and queen scallop (Chlamys opercularis) proteins.

OBJECTIVE: To report a case of occupational asthma and urticaria due to the queen scallop (Chlamys opercularis) and king scallop (Pectin maximus). BACKGROUND: A 40-year-old female worked in a shellfish-processing plant, handling king and queen scallops for 5 years. At the time of investigation, she described a 2-year history of work-related respiratory symptoms. METHODS: Serial peak expiratory flow rate readings were recorded and an OASYS study completed. A workplace visit was undertaken and specific immunoglobulin (IgE) radioallergosorbent (RAST) testing of scallop extracts was performed. RESULTS: The OASYS study was consistent with occupational asthma. RAST testing demonstrated evidence of specific sensitization (IgE) to queen and king scallop. There was also some cross-reactivity observed with other shellfish (prawns and crabs). CONCLUSION: Workers exposed to aerosols from scallop species are at risk of occupational asthma and require effective respiratory health surveillance.

Adult↗
PubMed2000

Male flower-specific expression of genes for polygalacturonase, pectin methylesterase and beta-1,3-glucanase in a dioecious willow (Salix gilgiana Seemen).

We isolated cDNA clones (pSgPG1 through pSgPG4, pSgPME1 and pSgGN1) for the polygalacturonases (PGs), pectin methylesterase (PME) and beta-1,3-glucanase (GN) that are expressed specifically in male flowers of the dioecious willow (Salix gilgiana Seemen). The structural characteristics of the deduced proteins, designated SgPGs, SgPME1 and SgGN1, respectively, suggest that these enzymes function in pollens or anthers. The four SgPGs were more than 91.9% homologous to one another at the amino acid level, indicating that their genes are members of a single family. Although the expression of the SgPGs, SgPME1 and SgGN1 was specific to male catkins (inflorescences), these genes were found in the genomes of both male and female plants. The expression of the transcripts of SgPGs, SgPME1 and SgGN1 was regulated developmentally in male reproductive organs. Maximal expression of SgPGs and SgPME1 was detected when male flowers were fully open and mature, while maximal expression of SgGN1 occurred at an earlier time. In situ hybridization revealed that the expression of SgPGs and SgPME1 was restricted to mature pollen grains after microspore mitosis. These results suggest that the pollen-specific or anther-specific expression of genes for PGs, PME and GN occurs in a dioecious plant, willow, just as it does in monoecious plants, and that the expression of these genes is related to the developmental stage of pollen grains during male gametogenesis.

Amino Acid Sequence↗
PubMed2002

Patterns of pectin methylesterase transcripts in developing stem nodules of Sesbania rostrata.

Differential display was applied to the early stages of the interaction between the tropical legume Sesbania rostrata and its microsymbiont Azorhizobium caulinodans ORS571. An upregulated clone that is similar to pectin methylesterase-encoding genes was isolated (Srpmel). The full-length sequence of Srpme1 was used to localize PME transcripts in situ during S. rostrata stem-nodule development. Several expression patterns were distinguished, hinting at general roles in vascular tissue development and cell division or expansion and at symbiosis-specific functions, such as uninfected cell differentiation.

Amino Acid Sequence↗
PubMed2006

Separation of Arabidopsis pollen tetrads is regulated by QUARTET1, a pectin methylesterase gene.

Arabidopsis (Arabidopsis thaliana) QUARTET (QRT) genes are required for pollen separation during normal floral development. In qrt mutants, the four products of microsporogenesis remain fused and pollen grains are released as tetrads. In Arabidopsis, tetrad analysis in qrt mutants has been used to map all five centromeres, easily distinguish sporophytic from gametophytic mutations, and accurately assess crossover interference. Using a combination of forward and reverse genetics, we have identified the gene responsible for the qrt1 phenotype. Annotation predicts that QRT1 encodes a pectin methylesterase (PME), and enzymatic assays of QRT1 expressed in Escherichia coli indicate that QRT1 has PME activity. Promoter and transcription analysis demonstrate QRT1 is expressed in anther tissues shortly after meiosis is complete. Unexpectedly, the QRT1 promoter is also active in a variety of developmentally unrelated tissues, including developing guard cells, the hypocotyl-root transition zone, areas of lateral root emergence, and floral nectaries. PMEs constitute a large gene family in Arabidopsis, are involved in cell wall loosening, and have been implicated in various aspects of floral development and pollen tube elongation. The identification of QRT1 as a PME contributes to our understanding of pollen development and may help to provide valuable genetic tools in other plant species.

Arabidopsis↗
PubMed1994

Reduction in Pectin Methylesterase Activity Modifies Tissue Integrity and Cation Levels in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits.

Pectin methylesterase (PME, EC 3.1.1.11) is an ubiquitous enzyme in the plant kingdom; however, its role in plant growth and development is not yet understood. Using transgenic tomato (Lycopersicon esculentum Mill.) fruits that show more than 10-fold reduction in PME activity because of expression of an antisense PME gene, we have investigated the role of PME in tomato fruit ripening. Our results show that reduced PME activity causes an almost complete loss of tissue integrity during fruit senescence but shows little effect on fruit firmness during ripening. Low PME activity in the transgenic fruit pericarp modified both accumulation and partitioning of cations between soluble and bound forms and selectively impaired accumulation of Mg2+ over other major cations. Decreased PME activity was associated with a 30 to 70% decrease in bound Ca2+ and Mg2+ in transgenic pericarp. Levels of soluble Ca2+ increase 10 to 60%, whereas levels of soluble Mg2+ and Na+ are reduced by 20 to 60% in transgenic pericarp. Changes in cation levels associated with lowered PME activity do not affect the rate of respiration or membrane integrity of fruit during ripening. Overall, these results suggest that PME plays a role in determining tissue integrity during fruit senescence, perhaps by regulating cation binding to the cell wall.

Journal Article↗
PubMed1995

Cell-Free Synthesis of Pectin (Identification and Partial Characterization of Polygalacturonate 4-[alpha]-Galacturonosyltransferase and Its Products from Membrane Preparations of Tobacco Cell-Suspension Cultures).

Polygalacturonate 4-[alpha]-galacturonosyltransferase (EC 2.4.1.43) activity has been identified in microsomal membranes isolated from tobacco (Nicotiana tabacum L. cv Samsun) cell-suspension cultures. Incubation of UDP-[14C]galacturonic acid with tobacco membranes results in a time-dependent incorporation of [14C]galacturonic acid into a chloroform-methanol-precipitable and 65% ethanol-insoluble product. The optimal synthesis of product occurs at a pH of 7.8, 25 to 30[deg]C, an apparent Km for UDP-D-galacturonic acid of approximately 8.9 [mu]M, and a Vmax of approximately 150 pmol min-1 mg-1 protein. The product was characterized by scintillation counting, thin-layer chromatography, high-performance anion-exchange chromatography, and gel-filtration chromatography in combination with enzymatic and chemical treatments. The intact product has a molecular mass of approximately 105,000 D based on dextran molecular standards. The product was treated with base to hydrolyze ester linkages (e.g. methyl esters), digested with a homogeneous endopolygalacturonase (EPGase), or base and EPGase treated. Base and EPGase treatment results in cleavage of 34 to 89% of 14C-labeled product into components that co-chromatograph with mono-, di-, and trigalacturonic acid, indicating that a large portion of product contains contiguous 1,4-linked [alpha]-D-galactosyluronic acid residues. Optimal EPGase fragmentation of the product requires base treatment prior to enzymatic digestion, suggesting that 45 to 67% of the galacturonic acid residues in the synthesized homogalacturonan are esterified. At least 40% of the base-sensitive linkages were shown to be methyl esters by comparing the sensitivity of base-treated and pectin methylesterase-treated products to fragmentation by EPGase.

Journal Article↗
PubMed1997

Polyamines and Pectins (I. Ion Exchange and Selectivity).

The ion-binding and -exchange properties of putrescine, spermidine, and spermine on purified walls of carrot (Daucus carota L.) cell suspensions were investigated by producing ion-exchange isotherms and comparing them with the behavior of Na+, Mg2+, and Ca2+. The cation exchange capacity of the carrot cell walls was 0.8 equivalent kg-1 dry matter, and the ionic selectivity sequence of the walls for polyamines followed the sequence spermine4+ > spermidine3+ [almost equal to] Ca2+ > putrescine2+. The polyamines were subjected to only electroselectivity and probably did not induce any favorable supramolecular conformation of pectin like the one induced by Ca2+. Triangular ion exchanges were also performed with three diamines: ethanediamine, butanediamine, and octanediamine. The shorter the diamine, the higher the total adsorption and selectivity of the exchange. The lower selectivity of the cell wall for putrescine was partly attributed to its inability to access and displace Ca2+ from higher affinity sites within dimerized pectic sequences. The polyamine adsorption and exchange on pectic sequences could result in pectic signal modulation in pathogenesis and in differentiation.

Journal Article↗