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. These proteins include a HEXXH+D zinc-binding motif known as aspzincin. Accordingly, molybdophyllysin is a member of the deuterolysin family of zinc proteases. Molybdophyllysin retained its proteolytic activity at temperatures up to 60°C with an optimum pH of 7.0. The activity was inhibited by both 1,10-phenanthroline and N-bromosuccinimide, but molybdophyllysin exhibited strong resistance to SDS.

Termination of the structural confusion between plipastatin A1 and fengycin IX.

Plipastatin A1 and fengycin IX were experimentally proven to be identical compounds, while these had been considered as diastereomers due to the permutation of the enantiomeric pair of Tyr in most papers. The (1)H NMR spectrum changed to become quite similar to that of plipastatin A1, when the sample which provided resembled spectrum of fengycin IX was treated with KOAc followed by LH-20 gel filtration. Our structural investigations disclosed that the structures of these molecules should be settled into that of plipastatin A1 by Umezawa (L-Tyr4 and D-Tyr10).

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. It was found that Pro-EndoPG I had no PG activity, but that PG activity was acquired after the degradation of part of the pro-sequence with V8 protease. These results suggest that the pro-sequence inactivates auto-PG activity. No similar characteristic has been reported for any glycoside hydrolase. We then constructed EndoPG I mutants and identified two Glu residues, E364 and E366, that were related to auto-inactivation. A test involving injection of the enzyme into apple trees showed that Pro-EndoPG I induced the same silver-leaf symptoms as mature EndoPG I.

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. From the thermal denaturation curves of the recombinant enzymes, it was suggested that EndoPG I differing in thermal stability was included in the sample after purification. Therefore, we investigated the disulfide bonds of recombinant EndoPG I by LC-MS analysis. As a result, peptides without a second or third disulfide bond were detected. This result is the first indicating that there are incomplete enzymes in terms of disulfide bonds in the Pichia expression system.

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.

Syntheses of lambertellols and their stable analogues; investigation of the real active species in the mycoparasitism by Lambertella species.

Lambertellols A (1) and B (2), isolated from mycoparasites Lambertella species, were synthesized. The synthesis features intramolecular aldol-type cyclizations of aldehydes 12 and 14 and site specific oxidations of 1-hydroxylambertellols as key steps. The synthesis also provided all diastereomers of 1-hydrolambertellols 17-19 and 25. Chiral resolution made the optically active forms available, which enabled the investigation of the real active species in the mycoparasitism by Lambertella species against Monilinia fructigena. These experiments suggested that lambertellin (3) is responsible for this phenomenon. Chemically labile 1 and 2 should be converted to 3 during the bioassay. The parasite may excrete 1 and 2 as readily diffusible forms, which are then transformed into 3 to inhibit the host M. fructigena. The parasite may have acquired this "drug delivery system" mechanism as an evolutionary enhancement.

Stimulation of the biosynthesis of the antibiotics lambertellols by the mycoparasitic fungus Lambertella corni-maris under the acidic conditions produced by its host fungus in vitro.

The filamentous fungus, Lambertella corni-maris (L. corni-maris), a mycoparasite on Monilinia fructigena, produces the antibiotics, lambertellols A (1), B (2), and lambertellin (3), in a substantial amounts under acidic conditions, whereas these antibiotics were hardly detected when the fungus was cultured on a potato-sucrose (PS) medium without added acids. Our investigations also revealed that the host, M. fructigena, changed its surroundings into acidic conditions, suggesting that the acidic conditions acted as kairomones that stimulated the production of 1-3.

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. This toxin exhibited its lethal activity following injection at 10mg/kg into mice. The N-terminal amino acid sequence was determined up to 18, and found to be similar to the previously reported bolesatine, a toxic compound isolated from Boletus satanas.

An RNA polymerase inhibitor, cyclothiazomycin B1, and its isomer.

Novel cyclic thiopeptides, cyclothiazomycins B1 (1) and B2 (2), were isolated from Streptomyces sp. A307 as potent hyphal swelling inducing substances. They are stable in the solid state but slowly isomerize with one another in solution. Degradation experiments and spectroscopic analyses disclosed that they comprise unique tricyclic structures each containing a dehydroalanine, and two dehydrohomoalanine residues, along with three thiazolines, three thiazoles, and a trisubstituted pyridine. Cyclothiazomycin B1 (1) is expected to be a powerful tool for DNA-RNA transcription studies, because this cyclopeptide inhibits DNA-dependent RNA synthesis by bacteriophage RNA polymerases.

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. Surface plasmon resonance experiments revealed that 3 forms a stable complex with the target enzyme.

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

Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation.

Isolation, structure elucidation, preparation, and biological properties of neolambertellin.

Neolambertellin (4), a novel biosynthetic congener of lambertellol and lambertellin, was isolated. The structure was confirmed by successful preparation from lambertellol A, which involves a rearrangement of spiro-butenolide moiety.

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. This was applied for preparation of sulfur-substituted gentiobiosides 1 and 46.

Lambertellol C, a labile and novel biosynthetic congener of lambertellols A and B.

Lambertellol C (1), a labile biosynthetic congener of lambertellols A and B, was isolated from a fermentation broth of Lambertella sp. 1346 based on the diagnostic isotope patterns in the mass spectrum of a highly labeled sample. The CD spectrum of 1 indicated that this lambertellol analogue exists as a racemate. The mechanism for this racemization is discussed.

Optimization of isotope-labeling conditions for lambertellin based on isotope patterns observed by mass spectrometry.

A method for the sensitive analysis of the incorporation level of labeled acetate was developed. This method allowed for the optimization of the conditions for lambertellin with up to 48% average incorporation of labeled acetate.

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.

Biosynthesis of lambertellols based on the high specific incorporation of the 13C-labeled acetates and their biological properties.

Biosyntheses of lambertellols A (1) and B (2) as well as lambertellin (3) were investigated by isotope labeling experiments. Nearly 40% of specific incorporation of [1,2-(13)C(2)]acetate was achieved, and all the carbons in 1 and 2 were labeled. This high incorporation of the labeled acetate was realized by providing INADEQUATE spectra by employing only 0.4 and 0.7 mg of 1 and 2, respectively. Our studies revealed that 1-3 are biogenetically synthesized via loss of two carbons from octameric acetate. A biological assay against Monilinia fructicola revealed those remarkably inhibited hyphal germinations. However, neither of them killed the spores immediately, even in high concentration. These conditions induced the formation of microconidia.

Lambertellols A and B, novel 3,4-dihydronaphthalen-1(2H)-ones with spiro-butenolide produced by Lambertella sp.1346.

[structure: see text] Lambertella sp. 1346 was found to produce lambertellols A (1) and B (2) carrying a novel dihydronaphthalen-1(2H)-ones with spiro-1-furan-2(5'H)-one. The spiro-lactone ring moiety of both 1 and 2 were easily migrated to afford lambertellin, a known metabolite of Lambertella corni-maris. The absolute stereochemistry of these compounds was established on the basis of CD spectrum after chemical derivatization.

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.

TMPDB: a database of experimentally-characterized transmembrane topologies.

TMPDB is a database of experimentally-characterized transmembrane (TM) topologies. TMPDB release 6.2 contains a total of 302 TM protein sequences, in which 276 are alpha-helical sequences, 17 beta-stranded, and 9 alpha-helical sequences with short pore-forming helices buried in the membrane. The TM topologies in TMPDB were determined experimentally by means of X-ray crystallography, NMR, gene fusion technique, substituted cysteine accessibility method, N-linked glycosylation experiment and other biochemical methods. TMPDB would be useful as a test and/or training dataset in improving the proposed TM topology prediction methods or developing novel methods with higher performance, and as a guide for both the bioinformaticians and biologists to better understand TM proteins. TMPDB and its subsets are freely available at the following web site: http://bioinfo.si.hirosaki-u.ac.jp/~TMPDB/.