L-Amino Acid Oxidases From Mushrooms Show Antibacterial Activity Against the Phytopathogen .

is the quarantine plant pathogenic bacterium that causes bacterial wilt in over 200 host plants, which include economically important crops such as potato, tomato, tobacco, banana, and ginger. Alternative biological methods of disease control that can be used in integrated pest management are extensively studied. In search of new proteins with antibacterial activity against , we identified L-amino acid oxidases (LAOs) from fruiting bodies of (LAO) and (LAO).

Trypsin-specific Inhibitors from the Macrolepiota procera, Armillaria mellea and Amanita phalloides wild mushrooms.

Wild growing mushrooms are a rich source of novel proteins with unique features. We have isolated and characterized trypsin inhibitors from two edible mushrooms, the honey fungus (Armillaria mellea) and the parasol mushroom (Macrolepiota procera), and from the poisonous death cap (Amanita phalloides). The trypsin inhibitors isolated: armespin, macrospin and amphaspin, have similar molecular masses, acidic isoelectric points and are not N-glycosylated.

Antibacterial Activity of Wild Mushroom Extracts on Bacterial Wilt Pathogen Ralstonia solanacearum.

In total, 150 protein extracts from 94 different basidiomycete and ascomycete wild mushroom species were tested for antibacterial activity against the quarantine plant-pathogen bacterium Ralstonia solanacearum. In in vitro microtiter plate assays, 15 extracts with moderate to high antibacterial activities were identified: 11 completely inhibited bacterial growth and 4 showed partial inhibition. Of these 15 extracts, 5 were further tested and 3 extracts slowed disease progression and reduced disease severity in artificially inoculated tomato and potato plants.

Clitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases.

Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a major potato pest that adapts readily to insecticides. Several types of protease inhibitors have previously been investigated as potential control agents, but with limited success. Recently, cysteine protease inhibitors from parasol mushroom, the macrocypins, were reported to inhibit growth of CPB larvae.

Inhibition of the growth of colorado potato beetle larvae by macrocypins, protease inhibitors from the parasol mushroom.

Proteins from higher fungi have attracted interest because of their exceptional characteristics. Macrocypins, cysteine protease inhibitors from the parasol mushroom Macrolepiota procera , were evaluated for their adverse effects and their mode of action on the major potato pest Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). They were shown to reduce larval growth when expressed in potato or when their recombinant analogues were added to the diet.

Basidiomycete Clitocybe nebularis is rich in lectins with insecticidal activities.

Basidiomycete mushrooms are a rich source of unique substances, including lectins, that could potentially be useful in biotechnology or biomedical applications. Lectins are a group of carbohydrate-binding proteins with diverse biological activities and functions. Here, we demonstrate the presence of a number of lectins in the basidiomycete mushroom Clitocybe nebularis.

Versatile loops in mycocypins inhibit three protease families.

Mycocypins, clitocypins and macrocypins, are cysteine protease inhibitors isolated from the mushrooms Clitocybe nebularis and Macrolepiota procera. Lack of sequence homology to other families of protease inhibitors suggested that mycocypins inhibit their target cysteine protease by a unique mechanism and that a novel fold may be found. The crystal structures of the complex of clitocypin with the papain-like cysteine protease cathepsin V and of macrocypin and clitocypin alone have revealed yet another motif of binding to papain like-cysteine proteases, which in a yet unrevealed way occludes the catalytic residue.

Trypsin-specific inhibitors from the basidiomycete Clitocybe nebularis with regulatory and defensive functions.

We have isolated serine protease inhibitors from the basidiomycete Clitocybe nebularis, CnSPIs, using trypsin affinity chromatography. Full-length gene and cDNA sequences were determined for one of them, named cnispin, and the recombinant protein was expressed in Escherichia coli at high yield. The primary structure and biochemical properties of cnispin are very similar to those of the Lentinus edodes serine protease inhibitor, until now the only member of the I66 family of protease inhibitors in the MEROPS classification.

Macrocypins, a family of cysteine protease inhibitors from the basidiomycete Macrolepiota procera.

A new family of cysteine protease inhibitors from the basidiomycete Macrolepiota procera has been identified and the family members have been termed macrocypins. These macrocypins are encoded by a family of genes that is divided into five groups with more than 90% within-group sequence identity and 75-86% between-group sequence identity. Several differences in the promoter and noncoding sequences suggest regulation of macrocypin expression at different levels.

Purification, characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells.

Background: Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.

Methods: Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed.

Comparison of natural and recombinant clitocypins, the fungal cysteine protease inhibitors.

A member of the cysteine protease inhibitor clitocypin gene family from basidiomycete Clitocybe nebularis was expressed in Escherichia coli. Following careful optimization of the expression procedure the active inhibitor was purified from inclusion bodies and its properties examined and compared to those of the natural clitocypin. The CD spectrum of recombinant clitocypin was similar to that of natural clitocypin, indicating that protein was properly refolded during purification.

Heterogeneity in the cysteine protease inhibitor clitocypin gene family.

Clitocypin from the basidiomycete Clitocybe nebularis is the first fungal protein cysteine protease inhibitor to be characterised in detail, yet no information on its molecular genetics is available. Owing to its unique characteristics, it was assigned as the only member of a new family of cysteine protease inhibitors in the MEROPS inhibitor classification. Here we describe the full-length sequence of the clitocypin gene.

Basidiomycetes harbour a hidden treasure of proteolytic diversity.

This survey is the first to investigate the proteolytic potential of a large number of basidiomycetes. Aqueous extracts of 43 basidiomycetes were investigated for their content of proteolytic activities, using gelatin zymography. The activities were characterised qualitatively using class specific inhibitors.

Crystallization and preliminary X-ray crystallographic analysis of the cysteine protease inhibitor clitocypin.

Clitocypin is a cysteine protease inhibitor from the mushroom Clitocybe nebularis. The protein has been purified from natural sources and crystallized in a variety of non-isomorphous forms belonging to monoclinic and triclinic space groups. A diffraction data set to 1.

Human brain cathepsin H as a neuropeptide and bradykinin metabolizing enzyme.

Highly purified human brain cathepsin H (EC 3.4.22.

A novel subtilase from common bean leaves.

We describe the isolation of a protease from common bean leaves grown in the field. On the basis of its biochemical properties it was classified as serine proteinase belonging to the subtilisin clan. Isoelectric focusing resulted in a single band at pH 4.

Folding, stability, and secondary structure of a new dimeric cysteine proteinase inhibitor.

Clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, is a 34-kDa homodimer lacking disulphide bonds, reported to have unusual stability properties. Sequence similarity is limited solely to certain proteins from mushrooms. Infrared spectroscopy shows that clitocypin is a high beta-structure protein which was lost at high temperatures.

Effect of cysteine proteinase inhibitors on murine B16 melanoma cell invasion in vitro.

Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in anti-invasive and anti-metastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, class-specific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors.

Aspartic proteinase inhibitors from tomato and potato are more potent against yeast proteinase A than cathepsin D.

The interaction of a variety of aspartic proteinases with a recombinant tomato protein produced in Pichia pastoris was investigated. Only human cathepsin D and, even more potently, proteinase A from Saccharomyces cerevisiae were inhibited. The tomato polypeptide has >80% sequence identity to a previously reported potato inhibitor of cathepsin D.