Extracellular beta-1,3-Glucanases in Stem Rust-Affected and Abiotically Stressed Wheat Leaves : Immunocytochemical Localization of the Enzyme and Detection of Multiple Forms in Gels by Activity Staining with Dye-Labeled Laminarin

Endo-β-1,3-glucanase activity in intercellular washing fluid (IWF) from leaves of wheat (Triticum aestivum) increased 10-fold 4 days after leaves were infected with the wheat stem rust fungus (Puccinia graminis f.sp. tritici), while exo-β-1,3-glucanase activity remained unchanged at a low level. Heat and ethylene stress had no effect, whereas mercury treatment resulted in a 2-fold increase in endo-β-1,3-glucanase activity. With a new method of activity staining using laminarin-Remazol brilliant blue as substrate in overlay gels, 18 electrophoretic forms of endo-β-1,3-glucanase were detected in IWF from unstressed leaves and up to 24 forms in IWF from stem rust-infected leaves. Most of the increase in β-1,3-glucanase activity and in the number of β-1,3-glucanases after rust infection was due to a nonspecific, stress-related effect on the plant, but two major forms of the enzyme probably originated from the fungus. β-1,3-Glucanase was localized cytochemically with anti-barley-β-1,3-glucanase antibodies. With preembedding labeling, the enzyme was demonstrated on the outside of host and fungal cell walls. Postembedding labeling localized the enzyme in the host plasmalemma and in the domain of host cell walls adjoining the plasmalemma, throughout walls of intercellular hyphal cells and haustoria, in the fungal cytoplasm, and in the extrahaustorial matrix. Cross-reactivity of β-1,3-glucanases from wheat and germinated uredospores of the rust fungus with the anti-barley-β-1,3-glucanase antibodies was confirmed in dot blot assays and on Western blots.     

Molecular modeling and docking characterization of CzR1, a CC-NBS-LRR R-gene from Curcuma zedoaria Loeb. that confers resistance to Pythium aphanidermatum

Plant NBS-LRR R-genes recognizes several pathogen associated molecular patterns (PAMPs) and limit pathogen infection through a multifaceted defense response. CzR1, a coiled-coil-nucleotide-binding-site-leucine-rich repeat R-gene isolated from Curcuma zedoaria L exhibit constitutive resistance to different strains of P. aphanidermatum. Majority of the necrotrophic oomycetes are characterized by the presence of carbohydrate PAMPs β-glucans in their cell walls which intercat with R-genes. In the present study, we predicted the 3D (three dimensional) structure of CzR1 based on homology modeling using the homology module of Prime through the Maestro interface of Schrodinger package ver 2.5. The docking investigation of CzR1 with β-glucan using the Glide software suggests that six amino acid residues, Ser186, Glu187, Ser263, Asp264, Asp355 and Tyr425 act as catalytic residues and are involved in hydrogen bonding with ligand β-(1,3)-D-Glucan. The calculated distance between the carboxylic oxygen atoms of Glu187–Asp355 pair is well within the distance of 5Å suggesting a positive glucanase activity of CzR1. Elucidation of these molecular characteristics will help in in silico screening and understanding the structural basis of ligand binding to CzR1 protein and pave new ways towards a broad spectrum rhizome rot resistance development in the cultivated turmeric.     

Antifungal Hydrolases in Pea Tissue : II. Inhibition of Fungal Growth by Combinations of Chitinase and beta-1,3-Glucanase

Chitinase and β-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and β-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and β-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and β-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by β-1,3-glucanase alone. However, combinations of purified chitinase and β-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and β-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips.     

Analysis of the β-1,3-Glucanolytic System of the Biocontrol Agent Trichoderma harzianum

The biocontrol agent Trichoderma harzianum IMI206040 secretes β-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of β-1,3-glucanase activity secreted was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular β-1,3-glucanases upon induction with laminarin, a soluble β-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60,000 to 80,000, and their pIs are 5.0 to 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted. Glucose appears to inhibit the formation of all of the inducible β-1,3-glucanases detected. A 77-kDa glucanase was partially purified from the laminarin culture filtrate. This enzyme is glycosylated and belongs to the exo-β-1,3-glucanase group. The properties of this complex group of enzymes suggest that the enzymes might play different roles in host cell wall lysis during mycoparasitism.     

Elucidation of the Molecular Basis for Arabinoxylan-Debranching Activity of a Thermostable Family GH62 α-l-Arabinofuranosidase from Streptomyces thermoviolaceus

Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-l-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3 l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-l-arabinofuranoside were also detected. After selective removal of α-1,3 l-Araf substituents from disubstituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2 l-Araf substituents, confirming the ability of SthAbf62A to remove α-l-Araf residues that are (1→2) and (1→3) linked to monosubstituted β-d-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigid l-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking the l-arabinose binding pocket.     

Antifungal Hydrolases in Pea Tissue : I. Purification and Characterization of Two Chitinases and Two beta-1,3-Glucanases Differentially Regulated during Development and in Response to Fungal Infection

Chitinase and β-1,-3-glucanase activities increased coordinately in pea (Pisum sativum L. cv “Dot”) pods during development and maturation and when immature pea pods were inoculated with compatible or incompatible strains of Fusarium solani or wounded or treated with chitosan or ethylene. Up to five major soluble, basic proteins accumulated in stressed immature pods and in maturing untreated pods. After separation of these proteins by chromatofocusing, an enzymic function could be assigned to four of them: two were chitinases and two were β-1,3-glucanases. The different molecular forms of chitinase and β-1,3-glucanase were differentially regulated. Chitinase Ch1 (mol wt 33,100) and β-1,3-glucanase G2 (mol wt 34,300) were strongly induced in immature tissue in response to the various stresses, while chitinase Ch2 (mol wt 36,200) and β-1,3-glucanase G1 (mol wt 33,500) accumulated during the course of maturation. With a simple, three-step procedure, both chitinases and both β-1,3-glucanases were purified to homogeneity from the same extract. The two chitinases were endochitinases. They differed in their pH optimum, in specific activity, in the pattern of products formed from [³H]chitin, as well as in their relative lysozyme activity. Similarly, the two β-1,3-glucanases were endoglucanases that showed differences in their pH optimum, specific activity, and pattern of products released from laminarin.     

Properties of a Clostridium thermocellum Endoglucanase Produced in Escherichia coli

A cellulase gene of Clostridium thermocellum was transferred to Escherichia coli by molecular cloning with bacteriophage lambda and plasmid vectors and shown to be indentical with the celA gene. The celA gene product was purified from extracts of plasmid-bearing E. coli cells by heat treatment and chromatography on DEAE-Trisacryl. It was characterized as a thermophilic endo-β-1,4-glucanase, the properties of which closely resemble those of endoglucanase A previously isolated from C. thermocellum supernatants. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme purified from E. coli exhibited two protein bands with molecular weights of 49,000 and 52,000. It had a temperature optimum at 75°C and was stable for several hours at 60°C. Endoglucanase activity was optimal between pH 5.5 and 6.5. The enzyme was insensitive against end product inhibition by glucose and cellobiose and remarkably resistant to the denaturing effects of detergents and organic solvents. It was capable of degrading, in addition to cellulosic substrates, glucans with alternating β-1,4 and β-1,3 linkages such as barley β-glucan and lichenan.     

Ethylene: Symptom, Not Signal for the Induction of Chitinase and beta-1,3-Glucanase in Pea Pods by Pathogens and Elicitors

Infection of immature pea pods with Fusarium solani f.sp. phaseoli (a non-pathogen of peas) or f.sp. pisi (a pea pathogen) resulted in induction of chitinase and β-1,3-glucanase. Within 30 hours, activities of the two enzymes increased 9-fold and 4-fold, respectively. Chitinase and β-1,3-glucanase were also induced by autoclaved spores of the two F. solani strains and by the known elicitors of phytoalexins in pea pods, cadmium ions, actinomycin D, and chitosan. Furthermore, exogenously applied ethylene caused an increase of chitinase and β-1,3-glucanase in uninfected pods. Fungal infection or treatment with elicitors strongly increased ethylene production by immature pea pods. Infected or elicitor-treated pea pods were incubated with aminoethoxyvinylglycine, a specific inhibitor of ethylene biosynthesis. This lowered stress ethylene production to or below the level of uninfected controls; however, chitinase and β-1,3-glucanase were still strongly induced. It is concluded that ethylene and fungal infection or elicitors are separate, independent signals for the induction of chitinase and β-1,3-glucanase.     

Hormonal regulation of beta1,3-glucanase messenger RNA levels in cultured tobacco tissues

We describe the isolation of a cDNA clone of β1,3-glucanase mRNA from Nicotiana tabacum L. cv. `Havana 425' and its use to measure the kinetics of mRNA accumulation in cultured tobacco tissues treated with the plant hormones auxin and cytokinin. Northern blot analysis showed that the tissues contain a single ˜1.6 kb-sized β1,3-glucanase mRNA. The levels of β1,3-glucanase and β1,3-glucanase mRNA increase by up to seven- and 20-fold, respectively, over a 7-day period in tissues subcultured on hormone-free medium and medium containing auxin or cytokinin added separately. Over the same interval of time, the content of both the enzyme and its mRNA remains at a constant low level in tissues subcultured on medium containing both auxin and cytokinin. The results show that auxin and cytokinin block β1,3-glucanase production at the level of the mRNA.     

Structural and Biochemical Insights into the Peptidoglycan Hydrolase Domain of FlgJ from Salmonella typhimurium

FlgJ is a glycoside hydrolase (GH) enzyme belonging to the Carbohydrate Active enZyme (CAZy) family GH73. It facilitates passage of the bacterial flagellum through the peptidoglycan (PG) layer by cleaving the β-1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid sugars that comprise the glycan strands of PG. Here we describe the crystal structure of the GH domain of FlgJ from bacterial pathogen Salmonella typhimurium (StFlgJ). Interestingly, the active site of StFlgJ was blocked by the C-terminal α-helix of a neighbouring symmetry mate and a β-hairpin containing the putative catalytic glutamic acid residue Glu223 was poorly resolved and could not be completely modeled into the electron density, suggesting it is flexible. Previous reports have shown that the GH73 enzyme Auto from Listeria monocytogenes is inhibited by an N-terminal α-helix that may occlude the active site in similar fashion. To investigate if the C-terminus of StFlgJ inhibits GH activity, the glycolytic activity of StFlgJ was assessed with and without the C-terminal α-helix. The GH activity of StFlgJ was unaffected by the presence or absence of the α-helix, suggesting it is not involved in regulating activity. Removal of the C-terminal α-helix did, however, allow a crystal structure of the domain to be obtained where the flexible β-hairpin containing residue Glu223 was entirely resolved. The β-hairpin was positioned such that the active site groove was fully solvent-exposed, placing Glu223 nearly 21.6 Å away from the putative general acid/base residue Glu184, which is too far apart for these two residues to coordinate glycosidic bond hydrolysis. The mobile nature of the StFlgJ β-hairpin is consistent with structural studies of related GH73 enzymes, suggesting that a dynamic active site may be common to many GH73 enzymes, in which the active site opens to capture substrate and then closes to correctly orient active site residues for catalysis.     

Biochemical Plant Responses to Ozone : III. Activation of the Defense-Related Proteins beta-1,3-Glucanase and Chitinase in Tobacco Leaves

A single pulse of O₃ (0.15 microliter per liter, 5 hours) induced β-1,3-glucanase and chitinase activities in O₃-sensitive and -tolerant tobacco (Nicotiana tabacum L.) cultivars. In the O₃-sensitive cultivar Bel W3, the response was rapid (maximum after 5 to 10 hours) and was far more pronounced for β-1,3-glucanase (40- to 75-fold) than for chitinase (4-fold). In the O₃-tolerant cultivar Bel B, β-1,3-glucanase was induced up to 30-fold and chitinase up to 3-fold under O₃ concentrations that did not lead to visible damage. Northern blot hybridization showed a marked increase in β-1,3-glucanase mRNA in cultivar Bel W3 between 3 and 24 hours following O₃ treatment, a transient induction in cultivar Bel B, and no change in control plants. The induction of β-1,3-glucanase and chitinase activities following O₃ treatment occurred within the leaf cells and was not found in the intercellular wash fluids. In addition, O₃ treatment increased the amount of the β-1,3-glucan callose, which accumulated predominantly around the necrotic spots in cultivar Bel W3. The results demonstrate that near-ambient O₃ levels can induce pathogenesis-related proteins and may thereby alter the disposition of plants toward pathogen attack.     

Purification,characterization and sequencing of the major β-1,3-glucanase from the midgut of Tenebrio molitor larvae

The major β-1,3-glucanase from Tenebrio molitor (TLam) was purified to homogeneity (yield, 6%; enrichment, 113 fold; specific activity, 4.4 U/mg). TLam has a molecular weight of 50 kDa and a pH optimum of 6. It is an endoglucanase that hydrolyzes β-1,3-glucans as laminarin and yeast β-1,3-1,6-glucan, but is inactive toward other polysaccharides (as unbranched β-1,3-glucans or mixed β-1,3-1,4-glucan from cereals) or disaccharides. The enzyme is not inhibited by high substrate concentrations and has low processivity (0.6). TLam has two ionizable groups involved in catalysis, and His, Tyr and Arg residues plus a divalent ion at the active site. A Cys residue important for TLam activity is exposed after laminarin binding. The cDNA coding for this enzyme was cloned and sequenced. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLam activity. TLam efficiently lyses fungal cells, suggesting a role in making available walls and cell contents to digestion and in protecting the midgut from pathogen infections.     

Occurrence of 9.5 cellulase and other hydrolases in flower reproductive organs undergoing major cell wall disruption

The occurrence of enzymes associated with bean leaf abscission was investigated in bean (Phaseolus vulgaris) flower reproductive organs in which catabolic cell wall events are essential during anther and pistil development. Cellulase activity was detected in high levels in both pistil and anthers of bean flowers before anthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified a protein in anthers and pistil with the same size (51 kilodaltons) and serologically closely related to the abscission cellulase. The accumulation of 9.5 cellulase protein in the anther is developmentally regulated and increases from undetectable levels at very young stages of anther development to high levels as the anther matures. In the pistil, the 9.5 cellulase was localized in the upper part of the pistil where the stigma and the stylar neck reside and was detected in the youngest developmental stage analyzed. Antibodies against basic chitinase, which accumulates to high levels in abscission zones after exposure to ethylene, identified a protein with the same size (33 kilodaltons) and serologically closely related, in both anthers and upper portion of the pistil. In contrast, a 45-kilodalton protein and the basic β-1,3-glucanase associated with abscission were undetected in bean reproductive organs. Interestingly, β-1,3-glucanase activity was detected in young bean anthers and decreased at anthesis, but the anther β-1,3-glucanase is serologically unrelated to the basic β-1,3-glucanase. Thus, it appears that the basic cellulase and chitinase occur in combination in many plant processes that require major cell wall disruption, whereas hemicellulases such as β-1,3-glucanase are specific to each process.     

Chitinases and beta-1,3-Glucanases in the Apoplastic Compartment of Oat Leaves (Avena sativa L.)

To isolate chitinases and β-1,3-glucanases from the intercellular space of oats (Avena sativa L.), primary leaves were infiltrated with buffer and subjected to gentle centrifugation to obtain intercellular washing fluid (IWF). Approximately 5% of the chitinase and 10% of the β-1,3-glucanase activity of the whole leaf were released. Only small amounts (0.01-0.03%) of the intracellular marker malate-dehydrogenase were released into the IWF during infiltration. Activities of chitinase and β-1,3-glucanase in the IWF and in the leaf extract were compared by different chromatographic methods. On Sephadex G-75, chitinase appeared as a single peak (M_r 29.8 kD) both in IWF and homogenate. β-1,3-Glucanase, however, showed two peaks in the IWF (M_r 52 and 31.3 kD), whereas the elution pattern of the homogenate showed only one major peak at 22 kD. Chromatofocusing indicated that the IWF contained four chitinases and five β-1,3-glucanases. The elution pattern of the homogenate and IWF were similar with regard to the elution pH, but the peak intensities were distinctly different. Our results demonstrate that extracellular β-1,3-glucanases are different from those located intracellularly. Extracellular and intracellular chitinases do not differ in molecular properties, except for one isozyme which seems to be confined to the extracellular space. We suggest that both enzymes might play a special role in pathogenesis during fungal infection.     

Identification and Characterization of a Novel Intracellular Alkaline α-Amylase from the Hyperthermophilic Bacterium Thermotoga maritima MSB8

The gene for a novel α-amylase, designated AmyC, from the hyperthermophilic bacterium Thermotoga maritima was cloned and heterologously overexpressed in Escherichia coli. The putative intracellular enzyme had no amino acid sequence similarity to glycoside hydrolase family (GHF) 13 α-amylases, yet the range of substrate hydrolysis and the product profile clearly define the protein as an α-amylase. Based on sequence similarity AmyC belongs to a subgroup within GHF 57. On the basis of amino acid sequence similarity, Glu185 and Asp349 could be identified as the catalytic residues of AmyC. Using a 60-min assay, the maximum hydrolytic activity of the purified enzyme, which was dithiothreitol dependent, was found to be at 90°C. AmyC displayed a remarkably high pH optimum of pH 8.5 and an unusual sensitivity towards both ATP and EDTA.     

Biological function of ;pathogenesis-related' proteins: four PR proteins of tobacco have 1,3-beta-glucanase activity

Three of the ten acidic `pathogenesis-related' (PR) proteins known to accumulate in Nicotiana tabacum cv Samsun NN reacting hypersensitively to tobacco mosaic virus, namely −O, −N and −2, have been shown to have 1,3-β-glucanase (EC 3.2.1.39) activity. By using sera raised against each protein purified to homogeneity close serological relationships have been demonstrated between the three proteins. The same specific sera cross-reacted with a basic protein which is also a 1,3-β-glucanase induced by virus infection and which can be considered as a new basic pathogenesis-related protein of tobacco. Protein PR-O and the basic 1,3-β-glucanase display about the same specific enzymatic activity, i.e. 50-fold and 250-fold higher than specific activities of proteins PR-N and -2 respectively.     

台湾家白蚁内切葡聚糖酶活性中心氨基酸的饱和突变

对内切葡聚糖酶的功能改进一直是纤维素酶研究领域的焦点。本研究对台湾家白蚁内切葡聚糖酶(CfEG)的活性位点做了饱和突变。首先,以PDB数据库中高山象白蚁内切葡聚糖酶(NtEG)的三维结构(PDB id=1ks8)为模板,对CfEG进行三维结构同源建模,二者序列一致性高达79%。位于CfEG活性中心的D53、D56、E411,分别与NtEG的催化残基D54、D57、E412重合。用简并引物对CfEG的假定活性位点D53、D56、E411进行定点饱和突变。在位点D53、D56各筛选到羧甲基纤维素酶活有一定提高的突变子D53E、D56C,其中D56C的Km值减小为原始酶的三分之一。双突变子D53L/D56I的比活比原始酶提高了近2倍,同时Km值减小至原始酶的一半。而E411的饱和突变子库均没有活性,进一步将其替换为近似氨基酸的E411D、E411Q定点突变子也丧失了酶活。由突变结果可推断,位点E411为该酶行使功能的必需残基。     

Endo-beta-1,3-galactanase from winter mushroom Flammulina velutipes

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-β-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed β-1,3-galactan, but did not act on β-1,3-glucan, β-1,3:1,4-glucan, xyloglucan, and agarose. It released various β-1,3-galactooligosaccharides together with Gal from β-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on β-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-β-1,3-glucanase and endo-β-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-β-1,3-galactanase.