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)

Fruit bromelain FA2, the main proteinase component of the juice of pineapple fruit, has been purified and characterized. 1. Efficient extraction of this enzyme from the crude material was possible using "Cellulosin AP," a microbial polysaccharidase preparation containing cellulase, hemicellulase, and pectinase. The enzyme was purified mainly by successive applications of anion-exchange chromatography, yielding an apparently homogeneous protein as judged by several physical, chemical, and immunochemical criteria. Properties of FA2 include: molecular weight, 31,000; isoelectric point, pH 4.6; absorbance at 280 nm of a 1% solution at pH 7.0 per cm, 19.2. 2. FA2 gave only alanine phenylthiohydantoin upon amino-terminal group analysis by the Edman procedure. Stepwise degradation yielded the amino-terminal sequence Ala-Val-Pro-Gln-Ser-Ile-Asp-Trp-Arg-Asp-Tyr-Gly-Ala. The amino acid composition of FA2 was not markedly different from that of stem bromelain, except for a much smaller lysine content and a smaller alanine content relative to glycine in FA2. FA2 contained neither amino sugars nor neutral carbohydrates as determined by several methods, so FA2 is not a glycoprotein. 3. By labeling the reactive cysteine residue (CYS) with [14C]iodoacetate, the following partial amino acid sequence has been determined. Asn-Glx-Asn-Pro-Cys-Gly-Ala-CYS.
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PMID:Purification and characterization of a proteinase from pineapple fruit, fruit bromelain FA2. 95 52

Soybean (Glycine max [L.] Merr. cv. Mandarin) root cells (SB-1 cell line) grown in suspension culture containing Glycyl-Arginyl-Glycyl-Aspartyl-Seryl-Proline (GRGDSP) (0.25 mg/ml), a synthetic peptide containing the RGD sequence found in many extracellular matrix adhesive proteins, demonstrated (a) significantly enhanced growth rate, and (b) aberrant cell wall/plasma membrane interactions and organization. Substitution of the Asp (D) by a Glu (E) amino acid in the hexapeptide, or inversion of the RGD sequence to GDR, abolished the morphological and growth effects observed for GRGDSP in plant cells. Immunoblots, which were prepared from beta-octylglucoside extracts of whole soybean cells and protoplasts, probed with polyclonal antibodies raised against human vitronectin receptor (hVNR) complex, demonstrated a single band with an apparent molecular mass of 70-72 kD. Chromatography of beta-octylglucoside extracts of SB-1 cells on a Gly-Arg-Gly-Asp-Ser-Pro-Lys-Sepharose affinity column demonstrated the retention of a single 70-72 kD polypeptide that reacted specifically with anti-hVNR antiserum. In contradistinction, no cross-reactivity was observed with antifibronectin receptor antiserum. Epifluorescence microscopy of whole soybean cells, after moderate treatment with pectinase, demonstrated punctate fluorescent patches at the cell membrane/wall boundary when probed with anti-hVNR and rhodamine-derivatized secondary antibodies. We propose that coordination and control of plant cell division and proper cell wall biosynthesis may be mediated by an RGD-dependent recognition system in which RGD binding protein(s) promote cell membrane-cell wall attachment.
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PMID:RGD-dependent linkage between plant cell wall and plasma membrane: consequences for growth. 271 84

Phylogenetic relationships among 18 isolates in the genus Verticillium, representing 13 species of diverse econutritional groups (pathogens of insects, plants, mushrooms, nematodes and spiders, and saprobes), were examined by using sequences from the internal transcribed spacer (ITS) and small nuclear (NS) rRNA regions. The isolates were also assessed for their abilities to infect insect larvae (Galleria mellonella) and to cause necrosis in alfalfa (Medicago sativa), and for their proteolytic, chitinolytic and pectinolytic activities. The phylogenetic data suggested that Verticillium is polyphyletic in origin and is therefore a form genus. However, the phylogenetic tree supported the plant pathogens (V. dahliae, V. albo-atrum and V. nigrescens) as a clade. The alfalfa isolate of V. albo-atrum (isolate 595) was an interesting outlier to the main body of plant pathogens as it clustered with the insect pathogen V. indicum. Strains of V. lecanii and V. indicum were able to infect insects and are present in divergent groups in the consensus tree, suggesting that the ability to infect insects may have evolved independently many times. Similarly, the nematophagous Verticillium species appear to have evolved independently along several different routes and one isolate, V. chlamydosporium, was able to infect insects. V. albo-atrum, V. nigrescens and V. dahliae all produced high levels of enzymes capable of degrading pectin, a major component of plant cell walls. The ability to excrete pectinase was a broad indicator of the ability to produce lesions on alfalfa. In the plant pathogens, the functions of a broad-spectrum protease were assumed by trypsins which degrade Bz-AA-AA-Arg-NA substrates (Bz, benzoyl; AA, various amino acids; NA, p-nitroanilide). The insect pathogens and mushroom pathogen (V. fungicola) were characterized by production of high levels of subtilisin-like proteases active against a chymotrypsin substrate (succinyl-Ala2-Pro-Phe-NA) and the inability to clear pectin. The insect and mushroom pathogens, and several nematode pathogens, were distinguishable from the plant pathogens in their ability to produce chitinases.
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PMID:Nuclear rDNA phylogeny in the fungal genus Verticillium and its relationship to insect and plant virulence, extracellular proteases and carbohydrases. 1022 Jan 75

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.
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PMID:1.68-A crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis. 1052 27

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K(m) and V(max)) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.
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PMID:The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis. 1061 68

Most structures of neutral lipases and esterases have been found to adopt the common alpha/beta hydrolase fold and contain a catalytic Ser-His-Asp triad. Some variation occurs in both the overall protein fold and in the location of the catalytic triad, and in some enzymes the role of the aspartate residue is replaced by a main-chain carbonyl oxygen atom. Here, we report the crystal structure of pectin methylesterase that has neither the common alpha/beta hydrolase fold nor the common catalytic triad. The structure of the Erwinia chrysanthemi enzyme was solved by multiple isomorphous replacement and refined at 2.4 A to a conventional crystallographic R-factor of 17.9 % (R(free) 21.1 %). This is the first structure of a pectin methylesterase and reveals the enzyme to comprise a right-handed parallel beta-helix as seen in the pectinolytic enzymes pectate lyase, pectin lyase, polygalacturonase and rhamnogalacturonase, and unlike the alpha/beta hydrolase fold of rhamnogalacturonan acetylesterase with which it shares esterase activity. Pectin methylesterase has no significant sequence similarity with any protein of known structure. Sequence conservation among the pectin methylesterases has been mapped onto the structure and reveals that the active site comprises two aspartate residues and an arginine residue. These proposed catalytic residues, located on the solvent-accessible surface of the parallel beta-helix and in a cleft formed by external loops, are at a location similar to that of the active site and substrate-binding cleft of pectate lyase. The structure of pectin methylesterase is an example of a new family of esterases.
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PMID:Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site. 1116 5

Fusarium moniliforme NCIM 1276 isolated from a tropical mangrove ecosystem produces a single extracellular endo-polygalacturonase with an M(r) of 38 kDa and a carbohydrate content of 4%. It has an alkaline pI of 8.1. The K(m) is 0.12 mg.mL(-1), V(max) is 111.1 micromol.min(-1).mg(-1) and the kcat is 4200 min-1. It has a pH optimum of 4.8. Kinetic and fluorescence data show that tryptophan is involved in binding. An arginine residue at or near the active site may be involved in extended binding of the substrate. A carboxylate and a histidine residue are involved in catalysis. These data are discussed with reference to current literature.
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PMID:Active site characterization of the single endo-polygalacturonase produced by Fusarium moniliforme NCIM 1276. 1116 25

Processivity, also known as multiple attack on a single chain, is a feature commonly encountered only in enzymes in which the substrate binds in a tunnel. However, of the seven Aspergillus niger endopolygalacturonases, which have an open substrate binding cleft, four enzymes show processive behavior, whereas the other endopolygalacturonases are randomly acting enzymes. In a previous study (Benen, J.A.E., Kester, H.C.M., and Visser, J. (1999) Eur. J. Biochem. 259, 577-585) we proposed that the high affinity for the substrate of subsite -5 of processive endopolygalacturonase I constitutes the origin of the multiple attack behavior. Based on primary sequence alignments of A. niger endopolygalacturonases and three-dimensional structure analysis of endopolygalacturonase II, an arginine residue was identified in the processive enzymes at a position commensurate with subsite -5, whereas a serine residue was present at this position in the non-processive enzymes. In endopolygalacturonase I mutation R95S was introduced, and in endopolygalacturonase II mutation S91R was introduced. Product progression analysis on polymer substrate and bond cleavage frequency studies using oligogalacturonides of defined chain length for the mutant enzymes revealed that processive/non-processive behavior is indeed interchangeable by one single amino acid substitution at subsite -5, Arg-->Ser or Ser-->Arg.
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PMID:Changing a single amino acid residue switches processive and non-processive behavior of Aspergillus niger endopolygalacturonase I and II. 1144 90

To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes; among them, endopolygalacturonase (PG) catalyzes the fragmentation and solubilization of homogalacturonan. Polygalacturonase-inhibiting proteins (PGIPs), found in the cell wall of many plants, counteract fungal PGs by forming specific complexes with them. We report the crystal structure at 1.73 A resolution of PG from the phytopathogenic fungus Fusarium moniliforme (FmPG). The structure of FmPG was useful to study the mode of interaction of the enzyme with PGIP-2 from Phaseolus vulgaris. Several amino acids of FmPG were mutated, and their contribution to the formation of the complex with PGIP-2 was investigated by surface plasmon resonance. The residues Lys-269 and Arg-267, located inside the active site cleft, and His-188, at the edge of the active site cleft, are critical for the formation of the complex, which is consistent with the observed competitive inhibition of the enzyme played by PGIP-2. The replacement of His-188 with a proline or the insertion of a tryptophan after position 270, variations that both occur in plant PGs, interferes with the formation of the complex. We suggest that these variations are important structural requirements of plant PGs to prevent PGIP binding.
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PMID:Structural requirements of endopolygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein). 1168 32

1. The apparent ileal nitrogen (N) and amino acid digestibilities in chaya leaf meal (CLM) (Cnidoscolus aconitifolius) with added enzymes, and the same variables in diets containing different amounts of CLM were studied in chickens. 2. In the first experiment pectinase, beta-glucanase, and pectinase + beta-glucanase were added to CLM. In the second experiment, there were three diets based on maize and soybean: 0, 150 and 250 g/kg CLM. 3. Pectinase significantly increased both lysine and overall amino acid digestibilities in CLM. 4. In experiment 2, the amino acid digestibility in birds fed on CLM250 was lower than that from birds fed on either control or CLM150. Only the digestibilities of alanine, arginine and proline were lower in birds fed on CLM150 than in those fed on the control diet. Nitrogen digestibility was lower in birds fed on the CLM250 diet than on either control or CLM150 diets. These findings were attributed to the increasing concentration of fibre with increasing dietary CLM.
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PMID:The effect of chaya (Cnidoscolus aconitifolius) leaf meal and of exogenous enzymes on amino acid digestibility in broilers. 1296 30


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