Characterization of the effects of C-terminal pro-sequence on self-inactivation of Stereum purpureum endopolygalacturonase I.

Endpolygalacturonase I from Stereum purpureum has been identified as a causative substance for the silver-leaf disease in apples. It possesses a unique pro-sequence in the C-terminal region that lacks endpolygalacturonases from any other origin. In this study, we analyzed and compared enzymatic characteristics between pro-form (pro-endoPG I) and mature form processed by V8 protease (endoPG I) and described the suppression activity of the pro-sequence.

Molybdophyllysin, a toxic metalloendopeptidase from the tropical toadstool, Chlorophyllum molybdites.

A toxic protein, dubbed molybdophyllysin, was isolated from the tropical toadstool Chlorophyllum molybdites by following its lethal effect in mice. Analysis of the protein using SDS-PAGE revealed a single 23-kDa band. Sequence analysis of molybdophyllysin tryptic fragments showed that this protein is highly homologous to metalloendopeptidases (MEPs) obtained from edible mushrooms, such as Grifola frondosa, Pleurotus ostreatus, and Armillaria mellea.

Active suppression of EndoPG IV by ligation of the pro-sequence from Stereum purpureum Pro-EndoPG I.

We attempted to inactivate endopolygaolacturonase from Stereum purpureum (EndoPG) IV of identical origin by linking the pro-sequence of S. purpureum Pro-EndoPG I to the C-terminus. The recombinant Pro-EndoPG IV, expressed in Escherichia coli, had no polygalacturonase (PG) activity, but activity was acquired after partial degradation of the pro-sequence with V8 protease, as was the case for Pro-EndoPG I.

Isolation and characterization of a novel two-component hemolysin, erylysin A and B, from an edible mushroom, Pleurotus eryngii.

A novel two-component hemolysin, erylysin A and B (EryA and EryB), was isolated from an edible mushroom, Pleurotus eryngii. Hemolytic activity was exhibited only by the EryA and EryB mixture. EryA showed one band at 15 kDa on SDS-PAGE while EryB showed two bands at 15 kDa (EryB1) and 37 kDa (EryB2).

The pro-form of Stereum purpureum endopolygalacturonase I is inactivated by a pro-sequence in the C-terminal region.

The Pro-form (Pro-EndoPG I) of Stereum purpureum endopolygalacturonase I has a unique C-terminal region (pro-sequence) that is lacking in PGs of other origins. Mature EndoPG I purified from the culture filtrate of this fungus does not have the 44-amino-acid pro-sequence present in Pro-EndoPG I. We expressed Pro-EndoPG I in Escherichia coli and examined its activity.

Cloning of the gene encoding an endo-acting pectate lyase from Streptomyces thermocarboxydus.

An endo-acting family 3 pectate lyase (PL I) gene from Streptomyces thermocarboxydus was cloned and expressed in Escherichia coli. The deduced PL I sequence had highest similarity to a putative pectate lyase of S. coelicolor.

Synthesis of a trigalacturonic acid analogue mimicking the expected transition state in the glycosidases.

A trigalacturonic acid analogue carrying a cyclohexene framework in place of the central pyranose ring was synthesized as a molecular probe for the mechanistic investigation of endo-polygalacturonase 1 (endo-PG 1). Preliminary enzymatic studies revealed that this analogue inhibited endo-PG 1 activity by about 30% at 0.3mM concentration.

Expression, purification, and analyses of glycosylation and disulfide bonds of Stereum purpureum endopolygalacturonase I in Pichia pastoris.

We have succeeded in the expression of Stereum purpureum endopolygalacturonase I (EndoPG I) using the Pichia expression system and in purification of the three kinds of recombinant EndoPG I, which have one to three sugar chains by using CM52 column chromatography. The sugar chains which were added to EndoPG I were the M8, M9, and/or M10 high-mannose type. The results of LC-MS analysis showed that recombinant EndoPG Is were randomly glycosylated at four N-glycosylation sites.

Synthesis of D-trigalacturonic acid methylglycoside and conformational comparison with its sulfur analogue.

D-Trigalacturonic acid methylglycoside (3) was synthesized to evaluate the previously synthesized sulfur analogue 1 by comparison. The NOE experiments revealed that both 3 and 1 took on a similar conformation around their glycosyl linkage.

Bolevenine, a toxic protein from the Japanese toadstool Boletus venenatus.

A toxic protein, called bolevenine, was isolated from the toxic mushroom Boletus venenatus based on its lethal effects on mice. On SDS-PAGE, in either the presence or absence of 2-mercaptoethanol, this protein showed a single band of approximately 12 kDa. In contrast, based on gel filtration and MALDI-TOFMS, its relative molecular mass was estimated to be approximately 30 kDa and approximately 33 kDa, respectively, indicating that the protein consists of three identical subunits.

Design, synthesis, and enzymatic property of a sulfur-substituted analogue of trigalacturonic acid.

A sulfur-substituted analogue of trigalacturonic acid (3) was synthesized. The synthesis features the application of 3-cyano-3-(tert-butyldimethylsilyl)oxypropylthioether (CSP) as a novel protective group for thiols. This analogue was designed with the expectation that it would be a stable analogous substrate for endo-polygalacturonase isolated from Stereum purpureum based on computer modeling experiments.

Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli.

Endopolygalacturonases (EC 3.2.1.

Synthetic studies on oligosaccharides composed of 5-thioglucopyranose units.

Glycosylation reactions of 5-thioglucopyranosyl trichloroacetimidates bearing ethereal protective groups at the 2-O-position 14-15, and 37 proceed smoothly to give alpha-glycosides stereoselectively by using a catalytic amount of silyl triflate. This methodology allowed us to achieve syntheses of sulfur-substituted isomaltotetraoside 2 and maltotetraoside 3. These studies also revealed that benzoyl-protected 5-thioglucopyranosyl trichloroacetimidate 12 underwent beta-selective glycosylation with C6-OH glucopyranosyl acceptors upon activation by BF3OEt2.

Enantioselective synthesis of four isomers of 3-hydroxy-4-methyltetradecanoic acid, the constituent of antifungal cyclodepsipeptides W493 A and B.

Four possible stereoisomers of 3-hydroxy-4-methyltetradecanoic acid were enantioselectively synthesized by using Sharpless epoxidation and a subsequent epoxide-ring opening reaction with trimethylaluminum as the key steps. The absolute configuration of the beta-oxyacid component of antifungal cyclodepsipeptides W493 A and B was consequently determined as 3S,4R.

alpha-Selective glycosylation with 5-thioglucopyranosyl donors; synthesis of an isomaltotetraoside mimic composed of 5-thioglucopyranose units.

A general method for alpha-selective glycosylation with 5-thioglucopyranosyl donors followed by efficient deprotection of the resulting products was developed. This methodology was utilized in the synthesis of an isomaltotetraoside analogue.

S-RNases from self-incompatible and -compatible apple cultivars: purification, cloning, enzymic properties, and pollen tube growth inhibitory activity.

Four S-RNases (RNase associated with self-incompatibility) were purified from the styles of two apple cultivars (Malus domestica), a self-incompatible cv., Starking Delicious (SD), and a self-compatible cv., Megumi (MG).

Active-site architecture of endopolygalacturonase I from Stereum purpureum revealed by crystal structures in native and ligand-bound forms at atomic resolution.

Crystal structures of endopolygalacturonase from Stereum purpureum were solved in native and two galacturonic acid complex states at atomic resolution. Endopolygalacturonase catalyzes the hydrolysis of alpha-1,4-glycosidic linkage of polygalacturonate in pectin. The native structure was determined by the multiple wavelength anomalous dispersion method and was refined anisotropically with SHELXL-97, with an R factor of 11.