Flexible synthesis and biological activity of uronic acid-type gem-diamine 1-N-iminosugars: a new family of glycosidase inhibitors.

An efficient and flexible synthetic route to four gem-diamine 1-N-iminosugars of uronic acid-type (D-glucuronic, D-mannuronic, L-iduronic, and L-guluronic acid), a new family of glycosidase inhibitor, from l-galactono-1,4-lactone have been developed in an enantiodivergent fashion through a sequence involving as the key steps (a) the formation of gem-diamine 1-N-iminopyranose ring by the Mitsunobu reaction of an aminal and (b) the introduction of a carboxylic acid group by the Wittig reaction of a ketone, hydroboration and oxidation, and the Sharpless oxidation. D-Glucuronic and D-mannuronic acid-type 1-N-iminosugars, (3S,4R,5R, 6R)- and (3S,4R,5R,6S)-4, 5-dihydroxy-6-trifluoroacetamido-3-piperidinecarboxylic acid, were proven to be potent inhibitors for beta-D-glucuronidase (IC(50) 6.5 x 10(-)(8)M) and to affect human heparanase (endo-beta-glucuronidase).

IC202A, a new siderophore with immunosuppressive activity produced by Streptoalloteichus sp. 1454-19. I. Taxonomy, fermentation, isolation and biological activity.

IC202A, a new immunosuppressive compound, was isolated from the culture filtrate of Streptoalloteichus sp. 1454-19. It showed a suppressive effect on mixed lymphocyte culture reaction with an IC50 value of 3.6 microg/ml and mitogen induced lymphocyte blastogenesis in vitro.

Intergenus protoplast fusion between Streptomyces and Micromonospora with reference to the distribution of parental characteristics in the fusants.

The protoplasts of two strains of Micromonospora which were sensitive to kanamycin (KM(s)) and utilized raffinose (Raf+), and one strain of Streptomyces griseus which was resistant to KM (KM(r)) and did not utilize raffinose (Raf-), were prepared, mixed in the presence of polyethylene glycol (PEG) and incubated on regeneration agar plates. Recombinant colonies showing KM(r)*Raf+ were obtained at a frequency of 2 x 10(-6). Their recombinants displayed a significant exchange of taxonomic characteristics between the two genera, although the majority appeared similar to the parent Micromonospora in their morphology as well as growth at 40 degrees C. Their patterns of utilization of carbohydrates, amino acids and diammonium hydrogenphosphate were different from those of the Micromonospora. Intermediate or novel types which different from their parents in their tolerance to NaCl and sensitivity to aminoglycoside antibiotics were also observed. Out of the 31 fusants obtained, two showed antimicrobial activity against Bacillus subtilis PCI 219, without any activity against Escherichia coli K-12 or Candida albicans 3147. The active substance may be a newly formed antibiotic, different from streptomycin in S. griseus.

A novel marine Bacillus with multiple amino acid analog resistance and selenomethionine-dependent antibiotic productivity.

Amino acid analogs (AAA) were used as selective pressures for isolation of marine bacteria with novel physiological properties and as effecters for antibiotic production. Relatively small numbers of isolates were obtained from a minimal medium containing aminoethylcysteine (AC), 3,4-dehydroproline (DP), 5-methyltryptophan (MT), and selenomethionine (SM). These bacteria exhibited a high probability (68%) of antibiotic production in the presence of AAA, which was 10-fold higher than that (7%) in the absence of AAA. Among them, strain 14, obtained as the only SM-resistant and SM-dependent antibiotic (selenohomocystine) producer, was characterized for microbiological properties. It showed taxonomic properties falling into those of the genus Bacillus, required seawater for growth, and exhibited a high level (0.5 mM) of resistance to all the AAAs tested. Neither known Bacillus spp. nor other marine isolates showed such properties. Therefore, the strain 14 appears to be the first marine Bacillus strain with unique AAA resistance and AAA-dependent antibiotic productivity. The AAA-resistance-based strategy was thus demonstrated to be effective for isolation of novel bacteria as well as for screening for antibiotic production.

Fluostatins A and B, new inhibitors of dipeptidyl peptidase III, produced by Streptomyces sp. TA-3391. I. Taxonomy of producing strain, production, isolation, physico-chemical properties and biological properties.

New inhibitors of dipeptidyl peptidaseIII (EC 3.4.14.4) from human placenta, designated as fluostatins A and B, were discovered in the fermentation broth of a strain isolated in our institute. The strain has been identified as Streptomyces sp. TA-3391 on the basis of taxonomic studies. Fluostatins A and B were purified by Diaion HP-20 chromatography, ethyl acetate extraction, silica gel chromatography and reverse phase preparative HPLC. With the synthetic substrate, arginyl-arginine-2-naphthylamide, the IC50 values of fluostatins A and B were 0.44 and 24.0 micrograms/ml, respectively. Fluostatins A and B were slightly inhibitory against other dipeptidyl peptidases. Fluostatin A showed mixed-type (competitive and noncompeptitive) inhibition with human leucine-enkephalin as a substrate, and the inhibition constant (Ki) was 14.2 microM.

Characterization of chitinase C from a marine bacterium, Alteromonas sp. strain O-7, and its corresponding gene and domain structure.

One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.

Cloning and sequence analysis of a protease-encoding gene from the marine bacterium Alteromonas sp. strain O-7.

The gene (aprI) encoding alkaline serine protease (AprI; subtilase) from Alteromonas sp. strain O-7 was cloned and sequenced. The nucleotide sequence of aprI has been identified. The deduced amino acid sequence indicated that aprI codes for a precursor of 715 amino acids and the precursor is composed of four regions including a signal peptide, an N-terminal pro-region, a mature protease region and a C-terminal extension region of 215 amino acids as previously described for aprII [H. Tsujibo et al., Gene, 136, 247-251 (1993)]. The amino acid sequence of the mature AprI (AprI-M) showed high sequence homology with those of other class I subtilases. The C-terminal region was characterized by a repeat of 94 amino acids residues, which showed about 50% similarity with those of the C-terminal pro-region of several known proteases from Gram-negative bacteria.

Mechanism of multiple aminoglycoside resistance of kasugamycin-producing Streptomyces kasugaensis MB273: involvement of two types of acetyltransferases in resistance to astromicin group antibiotics.

The biochemical basis for the multiple resistance to aminoglycoside antibiotics (AGs) of kasugamycin-producing Streptomyces kasugaensis MB273 was studied. The strain was resistant to a wide range of deoxystreptamine (DOS)-containing AGs as well as astromicin (ASTM) group antibiotics. These AGs strongly inhibited in vitro polyU-directed polyphenylalanine-synthesis using ribosomes from the strain, while they were acetylated and inactivated by the MB273 cell free extract supplemented with acetyl-CoA. It seemed thus likely that the acetyltransferase activity played a critical role for the multiple AG resistance. The acetylation was selective to AGs with 2'-NH2, suggesting the involvement of aminoglycoside 2'-N-acetyltransferase, AAC (2'). Interestingly, the acetylation of istamycin B (ISM-B; an ASTM group AG) resulted in the formation of two different products (1-N-acetyl ISM-B and 2"-N-acetyl ISM-B) at a similar ratio. In this context, an AAC (2') gene cloned as an ISM-B resistance gene from the strain MB273 directed the conversion of ISM-B to only 1-N-acetyl ISM-B. It seemed likely that two types of AACs [AAC(2') and a novel one] were involved in the mechanism of resistance to ASTM group AGs.

Identification of the positions of disulfide bonds of chitinase from a marine bacterium, Alteromonas sp. strain O-7.

Extracellular chitinase from marine Alteromonas sp. strain O-7 is unique because of the activation by four major cations contained in sea water, such as Na+, K+, Mg2+, and Ca2+. The positions of S-S bonds of Alteromonas chitinase were identified. Alteromonas chitinase was fragmented by TPCK-trypsin and Staphylococcus aureus V8 protease. The amino acid and sequence analyses of three peptides showed that the positions of disulfide bonds are Cys(94)-Cys(99), Cys(174)-Cys(196), and Cys(386)-Cys(395).

Purification and characterization of beta-N-acetylglucosaminidase from Alteromonas sp. strain O-7.

beta-N-Acetylglucosaminidase (EC 3.2.1.30) was purified from the outer membrane of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (GlcNAcase A) was purified by successive column chromatographies. The purified enzyme was found to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular mass and pI of GlcNAcase A were 92kDa and 4.9, respectively. The optimum pH and temperature were 6.0-7.0 and 45 degrees C, respectively. GlcNAcase A was stable up to 40 degrees C at pH 7.0, and hydrolyzed N-acetylchitooligosaccharides from dimer to hexamer. The amino-terminal 16 amino acid residues of GlcNAcase A were sequenced.

Molecular cloning of the gene which encodes beta-N-acetylglucosaminidase from a marine bacterium, Alteromonas sp. strain O-7.

The gene encoding the periplasmic beta-N-acetylglucosaminidase (GlcNAcase B) from a marine Alteromonas sp. strain, O-7, was cloned and sequenced. The protein sequence of GlcNAcase B revealed a highly significant homology with Vibrio GlcNAcase and alpha- and beta-chains of human beta-hexosaminidase.

Pyrostatins A and B, new inhibitors of N-acetyl-beta-D-glucosaminidase, produced by Streptomyces sp. SA-3501.

Pyrostatins A and B, new inhibitors of N-acetyl-beta-D-glucosaminidase (GlcNAc-ase), have been purified from the culture broth of Streptomyces sp. SA-3501 isolated from a marine environment. They were purified by chromatography on Dowex 50W, silica gel and Capcell Pak C18 (HPLC) followed treatment with active carbon and then isolated as white powders. The structures of pyrostatins A and B were determined by NMR studies to be 4-hydroxy-2-imino-1-methylpyrrolidine-5-carboxylic acid and 2-imino-1-methylpyrrolidine-5-carboxylic acid, respectively. They were competitive with the substrate, and the inhibition constants (Ki) of pyrostatins A and B were 1.7 x 10(-6) M and 2.0 x 10(-6) M respectively.

Gene sequence, purification and characterization of N-acetyl-beta-glucosaminidase from a marine bacterium, Alteromonas sp. strain O-7.

The gene (cht60) encoding N-acetyl-beta-glucosaminidase (Cht; EC 3.2.1.30) from the marine bacterium Alteromonas sp. strain O-7 was cloned into pUC18 in Escherichia coli JM109. The nucleotide (nt) sequence of cht60 was determined. A 1797-bp open reading frame encoded a polypeptide of 598 amino acids (aa) (M(r) 64,535). The aa sequence of the cloned enzyme (Cht60) deduced from the nt sequence showed no significant sequence homologies with available aa sequences from databases. Cht60 was purified from the periplasmic fraction of E. coli cells carrying pCHT982. The enzyme was most active towards p-nitrophenyl-N-acetyl-beta-D-glucosaminide(PNP-beta-GlcNAc) and diacetylchitobiose. The optimum pH and temperature of the enzyme were pH 7.5 and 37 degrees C, respectively. The N-terminal 11 aa residues of Cht60 were sequenced, and the location of the signal peptide cleavage site was clarified.

Cloning and sequence of an alkaline serine protease-encoding gene from the marine bacterium Alteromonas sp. strain O-7.

The gene (aprII) encoding alkaline serine protease (AprII; subtilase) from Alteromonas sp. strain O-7 was cloned in plasmid pUC19 and transformed into Escherichia coli JM109. The nucleotide (nt) sequence of aprII has been determined. A single open reading frame (ORF) encoded a protein consisting of 621 amino acids (aa) with a M(r) of 63,958. The results of aa sequence analysis indicated that AprII is produced as a large precursor consisting of four domains: the signal sequence, the N-terminal pro-region (AprII-N), the mature AprII (AprII-M) and the C-terminal pro-region (AprII-C). The aa sequence of AprII-M shows high sequence homology with those of class-II subtilases. Two conserved sequences were found in AprII-N which might play a critical role in the maintenance of chaperone-like activity. Repeated aa sequences were observed in AprII-C (AprII-C1 and AprII-C2). The aa sequences of AprII-C1 and AprII-C2 show high sequence homology with those of the C-terminal pro-region of the other known proteases.

Site-directed mutagenesis of chitinase from Alteromonas sp. strain O-7.

Chitinase (Chi85) from Alteromonas sp. strain O-7 contains the two conserved regions common to microbial and plant chitinases. We did site-directed mutagenesis of Chi85 to investigate the effects of the conserved amino acid residues on chitinase activity. We suggest that Asp-290 and Glu-292 of Chi85 may be the essential amino acid residues for the cleavage of beta-glycosidic linkage of chitin.

Cloning, sequence, and expression of a chitinase gene from a marine bacterium, Altermonas sp. strain O-7.

The gene encoding an extracellular chitinase from marine Alteromonas sp. strain O-7 was cloned in Escherichia coli JM109 by using pUC18. The chitinase produced was not secreted into the growth medium but accumulated in the periplasmic space. A chitinase-positive clone of E. coli produced two chitinases with different molecular weights from a single chitinase gene. These proteins showed almost the same enzymatic properties as the native chitinase of Alteromonas sp. strain O-7. The N-terminal sequences of the two enzymes were identical. The nucleotide sequence of the 3,394-bp SphI-HindIII fragment that included the chitinase gene was determined. A single open reading frame was found to encode a protein consisting of 820 amino acids with a molecular weight of 87,341. A putative ribosome-binding site, promoter, and signal sequence were identified. The deduced amino acid sequence of the cloned chitinase showed sequence homology with chitinases A (33.4%) and B (15.3%) from Serratia marcescens. Regardless of origin, the enzymes of the two bacteria isolated from marine and terrestrial environments had high homology, suggesting that these organisms evolved from a common ancestor.