Solution structure of the ribosome recycling factor from Aquifex aeolicus.

The solution structure of ribosome recycling factor (RRF) from hyperthermophilic bacterium, Aquifex aeolicus, was determined by heteronuclear multidimensional NMR spectroscopy. Fifteen structures were calculated using restraints derived from NOE, J-coupling, and T1/T2 anisotropies. The resulting structure has an overall L-shaped conformation with two domains and is similar to that of a tRNA molecule. The domain I (corresponding to the anticodon stem of tRNA) is a rigid three alpha-helix bundle. Being slightly different from usual coiled-coil arrangements, each helix of domain I is not twisted but straight and parallel to the main axis. The domain II (corresponding to the portion with the CCA end of tRNA) is an alpha/beta domain with an alpha-helix and two beta-sheets, that has some flexible regions. The backbone atomic root-mean-square deviation (rmsd) values of both domains were 0.7 A when calculated separately, which is smaller than that of the molecule as a whole (1.4 A). Measurement of 15N-[1H] NOE values show that the residues in the corner of the L-shaped molecule are undergoing fast internal motion. These results indicate that the joint region between two domains contributes to the fluctuation in the orientation of two domains. Thus, it was shown that RRF remains the tRNA mimicry in solution where it functions.

Interaction of ribosome recycling factor and elongation factor EF-G with E. coli ribosomes studied by the surface plasmon resonance technique.

BACKGROUND: Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the post-termination complex of a ribosome after the release of polypeptides. How RRF dissociates the complex has long been puzzling. Crystal structures of RRF molecules have been solved recently and shown to mimic a transfer RNA (tRNA) shape, which prompted us to examine whether RRF binds to the ribosome as tRNA does. RESULTS: The formation of ribosome complexes on the surface-coupled RRF and elongation factor EF-G of Escherichia coli was monitored in real time with a BIACORE 2000 instrument based on the surface plasmon resonance technique. RRF interacted with 70S ribosomes as well as 50S and 30S subunits, although it interacted preferentially with 50S subunits, which was clearly seen under high but physiological ionic conditions. This 50S interaction was diminished by a single amino acid substitutions for Arg132 of RRF, which did not appreciably affect the protein folding but nullified the activity in vivo and in vitro. Moreover, a set of antibiotics that inhibited the RRF-50S interaction were also inhibitory to the polysome breakdown activity of RRF in vitro. The BIACORE technique also worked very well in demonstrating the action of the antibiotics thiostrepton and fusidic acid, which are inhibitory to the RRF function by freezing the pre- and post-translocation intermediates catalysed by EF-G. CONCLUSIONS: These results suggest that the preferential interplay of RRF with the 50S subunit may be of biological significance, probably reflecting the mode of RRF action. The BIACORE technique proved useful for real-time monitoring of the interaction between the ribosome and translation factors, as well as for screening of potential inhibitors for ribosome recycling factor.

Monamidocin, a novel fibrinogen receptor antagonist. I. Production isolation, characterization and structural elucidation.

Monamidocin, a novel fibrinogen receptor antagonist, has been isolated from the culture broth of Streptomyces sp. NR 0637 BY carbon adsorption, n-BuOH extraction, SP-Toyopearl, silica gel 60 silanised and Sephadex LH-20 column chromatographies and preparative HPLC. The molecular formula of monamidocin has been determined to be C15H22N4O4 from HRFAB-MS and 13C NMR spectral data. The structure of monamidocin has been determined to be N-[(S)-5-guanidino-2-hydroxypentanoyl]-L-phenylalanine by 2D NMR experiments.

Monamidocin, a novel fibrinogen receptor antagonist. II. Biological activity and structure-activity relationships.

Monamidocin, a fibrinogen receptor binding inhibitor produced by Streptomyces sp. NR 0637, inhibits the binding of fibrinogen to its receptor with an IC50 of 0.21 microM. It also inhibits ADP-collagen- and thrombin-induced aggregation of human platelet with IC50S of 46, 30, and 77 microM, respectively. To obtain more potent inhibitors, twenty analogs have been synthesized, among which N-[(R)-5-guanidino-2-hydroxypentanoyl]-L-tyrosine is the most potent. It inhibits the binding of fibrinogen to its receptors with an IC50 of 0.022 microM and is about ten times more potent than monamidocin.

Sulfobacins A and B, novel von Willebrand factor receptor antagonists. I. Production, isolation, characterization and biological activities.

Sulfobacins A and B, novel von Willebrand factor (vWF) receptor antagonists, have been isolated from the culture broth of Chryseobacterium sp. (Flavobacterium sp.) NR 2993 by ethyl acetate extraction, and by Sephadex LH-20 and silica gel column chromatographies. The physico-chemical properties of the sulfobacins indicate that their structures are completely different from that of aurintricarboxylic acid, the one known vWF receptor antagonist. Sulfobacins A and B inhibit the binding of vWF to its receptor with IC50S of 0.47 and 2.2 microM, respectively. Sulfobacin A also inhibits ristocetin-induced agglutination in human platelets fixed with paraformaldehyde with an IC50 of 0.58 microM.

Sulfobacins A and B, novel von Willebrand factor receptor antagonists. II. Structural elucidation.

Sulfobacins A and B are novel von Willebrand factor (vWF) receptor antagonists produced by Chryseobacterium sp. NR 2993. The structures of sulfobacins A and B have been determined to be (2R,3R)-3-hydroxy-2-[(R)-3-hydroxy-15-methylhexadecanamido]-15- methylhexadecanesulfonic acid and (2R,3R)-3-hydroxy-15-methyl-2-[13-methyltetradecanamido]- hexadecanesulfonic acid, respectively, by various 2D NMR experiments and by methanolysis. The absolute configurations of the sulfobacins were determined by a modified MOSHER's method. The structures are related to sulfonolipids, major components of the cell envelope of gliding bacteria of the genus Cytophaga.

Panclicins, novel pancreatic lipase inhibitors. II. Structural elucidation.

Panclicins A-E are novel and potent pancreatic lipase inhibitors produced by Streptomyces sp. NR 0619. Their structures have been elucidated based on NMR and FAB-MS experiments. The relative configurations have also been determined by NMR experiments. The absolute stereochemistry has been determined by the chiral HPLC analysis of the hydrolysates of panclicins A and B and by modified Mosher's method on a derivative of panclicin A. They are structurally related to beta-lactone esterase inhibitors of microbial origin, lipstatin, valilactone, ebelactones and esterastin. Panclicins also contain a beta-lactone structure with two alkyl chains, one of which has an N-formylalanyloxy or N-formylglycyloxy substituent.

Bacithrocins A, B and C, novel thrombin inhibitors.

Novel thrombin inhibitors, bacithrocins A, B and C, have been isolated from the culture broth of Bacillus laterosporus Laubach NR 2988. The structures of these inhibitors have been determined to be N-acyl-L-phenylalanyl-DL-arginal by the 2D-NMR experiments on their oxidation products and by amino acid analysis. Bacithrocin A inhibits thrombin, factor Xa and trypsin with IC50s of 48, 13 and 0.65 microM, respectively, which are similar to those of bacithrocins B and C. Bacithrocins prolong the clotting time induced by thrombin and factor Xa.

Fusarium merismoides Corda NR 6356, the source of the protein kinase C inhibitor, azepinostatin. Taxonomy, yield improvement, fermentation and biological activity.

Fungal strain NR 6356, Fusarium merismoides Corda, was discovered as the source of the protein kinase C (PKC) inhibitor, azepinostatin. The strain was identified based on its growth on potato sucrose agar, slender conidial shape, characteristic polyphialide and production of abundant chlamydospores. Fusarium aquaeductuum Lagh. IMI 103658 and Fusarium sp. NR 7222 were also found to produce the same inhibitor. After single colony isolation and medium optimization trials, a more than 30-fold increase in the production of azepinostatin over the original culture was achieved. Azepinostatin selectively and potently inhibited rat brain PKC with an IC50 value of 70 nM. Other enzymes utilizing ATP, including hexokinase, were not affected. The Ki of azepinostatin for PKC was 0.5 nM. The inhibition of PKC was competitive with ATP and uncompetitive with histone.

Cyclothialidine, a novel DNA gyrase inhibitor. II. Isolation, characterization and structure elucidation.

Cyclothialidine is a novel DNA gyrase inhibitor produced by Streptomyces filipinensis NR 0484. It was isolated from the culture broth by charcoal adsorption, Diaion HP-21, Amberlite CG-50, DEAE Toyopearl, and Toyopearl HW-40 SF column chromatography. The structure of cyclothialidine was determined to be a unique twelve membered lactone by amino acid analysis and various 2D-NMR experiments. Cyclothialidine inhibited Escherichia coli DNA gyrase with an IC50 of 30 ng/ml.

Hispidospermidin, a novel phospholipase C inhibitor produced by Chaetosphaeronema hispidulum (Cda) Moesz NR 7127. II. Isolation, characterization and structural elucidation.

Hispidospermidin (1) is a novel phospholipase C inhibitor produced by Chaetosphaeronema hispidulum (Cda) Moesz NR 7127. Its structure (C25H47N3O) has been elucidated as a cage compound with a trimethylspermidine side chain based on various NMR studies, including 1H-1H COSY, 13C-1H COSY, HOHAHA, HMBC, COLOC and long range J C-H resolved 2D spectroscopy. The absolute configuration of 1 has been elucidated by modified Mosher's method on the (R)- and (S)-MTPA amides of a derivative of 1.

Tetrafibricin, a novel fibrinogen receptor antagonist. I. Taxonomy, fermentation, isolation, characterization and biological activities.

Tetrafibricin is a novel fibrinogen receptor antagonist produced by Streptomyces neyagawaensis NR0577. It was isolated from the culture broth by Diaion HP-21 adsorption, MeOH extraction, MCI GEL CHP-20P column chromatography, preparative HPLC and Toyopearl HW-40 SF column chromatography. The physico-chemical properties of tetrafibricin indicated that the structure of tetrafibricin is different from the known peptide fibrinogen receptor antagonists and closely related to the polyene macrolide antibiotics. Tetrafibricin strongly inhibited the binding of fibrinogen to its receptors with an IC50 of 46 nM. It also inhibited ADP-, collagen-, and thrombin-induced aggregation of human platelets with IC50s of 5.6, 11.0 and 7.6 microM, respectively.

Tetrafibricin, a novel fibrinogen receptor antagonist. II. Structural elucidation.

The structure of tetrafibricin, a novel and potent fibrinogen receptor antagonist isolated from the culture broth of Streptomyces neyagawaensis NR0577, was determined. Tetrafibricin has a unique structure containing primary amine, conjugated tetraenoic acid, and polyhydroxy functionalities that is biosynthetically related to the polyene macrolide antibiotics.

Tetronothiodin, a novel cholecystokinin type-B receptor antagonist produced by Streptomyces sp. NR0489. II. Isolation, characterization and biological activities.

A novel cholecystokinin type-B receptor antagonist named tetronothiodin has been isolated by column chromatography and preparative HPLC from the fermentation broth of Streptomyces sp. NR0489. Tetronothiodin inhibited the binding of CCK8 (C-terminal octapeptide of cholecystokinin) to rat cerebral cortex membranes (CCK type-B receptors) with an IC50 of 3.6 nM, whereas it did not inhibit CCK8 binding to rat pancreatic membranes (CCK type-A receptors). It also inhibited CCK8 induced Ca2+ mobilization in GH3 cells, a rat anterior pituitary cell line, but was without effect on the basal cytosolic Ca2+ concentration. This finding indicated tetronothiodin was an antagonist of CCK type-B receptors.

Tetronothiodin, a novel cholecystokinin type-B receptor antagonist produced by Streptomyces sp. NR0489. III. Structural elucidation.

Tetronothiodin (1) is a potent and selective cholecystokinin type B (CCK-B) receptor antagonist produced by Streptomyces sp. NR0489. Its structure was elucidated to be a macrocyclic compound comprising cyclohexene, alpha-acyltetronic acid and tetrahydrothiophene moieties based on various 2D NMR experiments on 1 and its dihydro derivative. The stereochemistries for the cyclohexene and tetrahydrothiophene rings were elucidated based on the analysis of NOEs obtained by NOESY experiments and NOE difference spectroscopy. The relative configuration of the cyclohexene moiety in 1 was revealed to be the same as that of the corresponding part in kijanimicin and chlorothricin, which can be structurally related to 1 in terms of their containing a cyclohexene ring with a spirotetronic acid in the molecule.

Cyclothiazomycin, a novel polythiazole-containing peptide with renin inhibitory activity. Taxonomy, fermentation, isolation and physico-chemical characterization.

Cyclothiazomycin is a novel renin inhibitor produced by Streptomyces sp. NR0516. It was isolated from fermentation broth by extraction with butyl alcohol, QAE-Toyopearl column chromatography and preparative HPLC. Cyclothiazomycin, which was determined to be a unique polythiazole-containing bicyclic peptide, exhibited inhibitory activity against human plasma renin with IC50 being 1.7 microM.