Masa Hamada

Bacilysocin, a Novel Phospholipid Antibiotic Produced by Bacillus subtilis 168

ABSTRACT We have found a novel phospholipid antibiotic (named bacilysocin) which accumulates within (or associates with) the cells of Bacillus subtilis 168 and determined the structure by nuclear magnetic resonance and mass spectrometry analyses. The structure of bacilysocin elucidated was 1-(12-methyltetradecanoyl)-3-phosphoglyceroglycerol. Bacilysocin demonstrated antimicrobial activity, especially against certain fungi. Production of bacilysocin commenced immediately after growth ceased and before the formation of heat-resistant spores. The production of bacilysocin was completely blocked when the ytpA gene, which encodes a protein homologous to lysophospholipase, was disrupted, but blockage of the ytpA gene did not significantly affect growth. Sporulation was also impaired, with a 10-fold reduction in heat-resistant spore titers being detected. Since the ytpA disruptant actually lacked phospholipase activity, we propose that the YtpA protein functions as an enzyme for the biosynthesis of bacilysocin.

Labeling of v-Src and BCR-ABL tyrosine kinases with [14C]herbimycin A and its use in the elucidation of the kinase inactivation mechanism

The ansamycin antibiotic, herbimycin A, selectively inactivates cytoplasmic tyrosine kinases, most likely by binding irreversibly to the reactive SH group(s) of kinases. To further investigate the mechanism of herbimycin A action, we attempted to label tyrosine kinases with [14C]herbimycin A. p60 v‐src and p2 10 BCR‐ABL in immune complexes were labeled with [14C]herbimycin A, demonstrating that the antibiotic binds directly to tyrosine kinases. Digestion of [14C]herbimycin A‐labeled p60 v‐src with Staphylococcus taureus V8 protease revealed that the herbimycin A binding site is within the C‐terminal 26‐kDa fragment of p60 v‐src , which contains the tyrosine kinase domain. Herbimycin A treatment inhibited labeling of p60 v‐src by [14]C]fluorosulfonylbenzoyl adenosine, an affinity labeling reagent of nucleotide binding sites, indicating that herbimycin A‐modified p60 v‐src cannot interact with ATP. The results suggest that herbimycin A inactivates tyrosine kinases by binding directly to the kinase domain, thereby inhibiting access to ATP.

Polyoxypeptin isolated from Streptomyces: A bioactive cyclic depsipeptide containing the novel amino acid 3-hydroxy-3-methylproline

Abstract Polyoxypeptin, a potent inducer of apoptosis in human pancreatic carcinoma cells, was isolated from an ethyl acetate extract of a Streptomyces culture broth. Structural determination by 2D-NMR and X-ray crystallographic analysis revealed that it is a novel cyclic hexadepsipeptide containing five hydroxylated amino acids. The unusual and hitherto unreported amino acid 3-hydroxy-3-methylproline was one of them.

Bequinostatins C and D, new inhibitors of glutathione S-transferase, produced by Streptomyces sp. MI384-DF12.

Benastatins have been isolated as part of a program designed to find microorganism-produced inhibitors of glutathione S-transferase from Streptomyces sp. MI384-DF12. They were purified by chromatography of reversed-phase silica gel, silica gel and Capcell Pak C18 (HPLC) followed by solvent extraction and then isolated as yellow powders. Benastatins A and B have the molecular formulae, C30H28O7 and C30H30O7, respectively. They were competitive with 3,4-dichloronitrobenzene as the substrate, and the inhibition constants (Ki) of benastatins A and B were 5.0 x 10(-6) and 3.7 x 10(-6), respectively.

DNA Sequencing and Transcriptional Analysis of the Kasugamycin Biosynthetic Gene Cluster from Streptomyces kasugaensis M338-M1

Streptomyces kasugaensis M338-M1 produces the aminoglycoside antibiotic kasugamycin (KSM). We previously cloned, sequenced and characterized the KSM acetyltransferase, transporter, and some of the biosynthetic genes from this strain. To identify other potential genes in a chromosome walk experiment, a 6.8-kb EcoRI-PstI region immediately downstream from the KSM transporter genes was sequenced. Five open reading frames (designated as kasN, kasO, kasP, kasQ, kasR) and the 5′ region of kasA were found in this region. The genes are apparently co-transcribed as bicistrons, all of which are co-directional except for the kasPQ transcript. Homology analysis of the deduced products of kasN, kasP, kasQ and kasR revealed similarities with known enzymes: KasN, D-amino acid oxidase from Pseudomonas aeruginosa (35% identity); KasP, F420-dependent H4MPT reductase from Streptomyces lavendulae (33% identity); KasQ, UDP-N-acetylglucosamine 2-epimerase from Streptomyces verticillus (45% identity); and KasR, NDP-hexose 3,4-dehydratase from Streptomyces cyanogenus (38% identity); respectively. A gel retardation assay showed that KasT, a putative pathway-specific regulator for this gene cluster, bound to the upstream region of kasN and to the intergenic region of kasQ-kasR, suggesting that the expression of these operons is under the control of the regulator protein.

Amycolamicin: A Novel Broad‐Spectrum Antibiotic Inhibiting Bacterial Topoisomerase

The abuse of antibacterial drugs imposes a selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens. Our efforts to discover novel classes of antibiotics to combat these pathogens resulted in the discovery of amycolamicin (AMM). The absolute structure of AMM was determined by NMR spectroscopy, X-ray analysis, chemical degradation, and modification of its functional groups. AMM consists of trans-decalin, tetramic acid, two unusual sugars (amycolose and amykitanose), and dichloropyrrole carboxylic acid. The pyranose ring named as amykitanose undergoes anomerization in methanol. AMM is a potent and broad-spectrum antibiotic against Gram-positive pathogenic bacteria by inhibiting DNA gyrase and bacterial topoisomerase IV. The target of AMM has been proved to be the DNA gyrase B subunit and its binding mode to DNA gyrase is different from those of novobiocin and coumermycin, the known DNA gyrase inhibitors.

A novel cyclic lipoundecapeptide, pholipeptin, isolated from Pseudomonas sp.

An inhibitor of phosphatidylinositol-specific phospholipase C, pholipeptin, was purified from the culture broth of Pseudomonas sp. Structural determination by 2D NMR spectroscopy with addition of a small amount of trifluoroacetic acid revealed that it was a novel cyclic lipodepsipeptide (1) consisting of 11 amino acids and a 3-hydroxydecanoic acid moiety.

PIPERASTATIN A, A NEW SELECTIVE SERINE CARBOXYPEPTIDASE INHIBITOR PRODUCED BY ACTINOMYCETE. I. TAXONOMY, PRODUCTION, ISOLATION AND BIOLOGICAL ACTIVITIES

Piperastatin A (structure, N-formyl-allo Ile-Thr-Leu-Val-Pip-Leu-Pip, Pip = hexahydropyridadine-3-carboxylic acid; molecular weight, 809), a new inhibitor of serine carboxypeptidase was discovered in the fermentation broth of Streptomyces lavendofoliae MJ908-WF13. It was purified by activated charcoal chromatography, YMC gel ODS-A chromatography and centrifugal partition chromatography (CPC) by monitoring its inhibitory activity against carboxypeptidase Y (CP-Y), and finally obtained as colourless needles. Piperastatin A is a competitive inhibitor of the enzyme with Ki = 52 +/- 6.2 nM. Piperastatin A is a highly specific inhibitor of the serine carboxypeptidases, CP-Y and platelet deamidase with little effect on related enzymes, has no antimicrobial activity and has low toxicity.

Paleic acid, a fatty acid from Paenibacillus sp.: taxonomy, fermentation, isolation, structure determination, and anti-Mannheimia and -Pasteurella activity.

Paleic acid (1), an antibiotic, was obtained from a fermentation broth of Paenibacillus sp. BMK771-AF3. The compound is a fatty acid (9Z,16R)-16-hydroxy-9-octadecenoic acid ((R)-16-hydroxyoleic acid), whose isolation required protection of its polar functional groups. Mosher esters of paleic acid yielded information on the absolute configuration of secondary alcohol, and well-resolved 1H NMR peaks around the double bond suggested that olefin adopted a Z geometry. Paleic acid showed potent antibacterial activity and narrow spectrum against Mannheimia haemolytica with MIC values ranging between 0.78 and 1.56 μg ml−1.

BELACTINS A AND B, NEW SERINE CARBOXYPEPTIDASE INHIBITORS PRODUCED BY ACTINOMYCETE. I: TAXONOMY, PRODUCTION, ISOLATION AND BIOLOGICAL ACTIVITIES

Belactins A and B, new inhibitors of serine carboxypeptidase were discovered in the fermentation broth of Saccharopolyspora sp. MK19-42F6. They were purified by ethyl acetate extraction, silica gel chromatography, Sephadex LH20 chromatography, Capcellpak C18 SG120 reversed phase HPLC and centrifugal partition chromatography (CPC) following their inhibitory activity against carboxypeptidase Y (CP-Y). The inhibition constants (Ki) of belactins A and B against CP-Y are 0.14 and 0.27 microM respectively. Belactins A and B have highly specific inhibitory activities for CP-Y among various peptidases, have no antimicrobial activities at 100 micrograms/ml and have low toxicities.