Axel Zeeck

Concanamycin A, the specific inhibitor of V-ATPases, binds to the V(o) subunit c.

Vacuolar-type ATPase (V-ATPase) purified from the midgut of the tobacco hornworm Manduca sexta is inhibited 50% by 10 nm of the plecomacrolide concanamycin A, the specific inhibitor of V-ATPases. To determine the binding site(s) of that antibiotic in the enzyme complex, labeling with the semisynthetic 9-O-[p-(trifluoroethyldiazirinyl)-benzoyl]-21,23-dideoxy-23-[125I]iodo-concanolide A (J-concanolide A) was performed, which still inhibits the V-ATPase 50% at a concentration of 15–20 μm. Upon treatment with UV light, a highly reactive carbene is generated from this concanamycin derivative, resulting in the formation of a covalent bond to the enzyme. In addition, the radioactive tracer 125I makes the detection of the labeled subunit(s) feasible. Treatment of the V1/Vo holoenzyme, the Vo complex, and the V-ATPase containing goblet cell apical membranes with concanolide resulted in the labeling of only the proteolipid, subunit c, of the proton translocating Vo complex. Binding of J-concanolide A to subunit c was prevented in a concentration-dependent manner by concanamycin A, indicating that labeling was specific. Binding was also prevented by the plecomacrolides bafilomycin A1 and B1, respectively, but not by the benzolactone enamide salicylihalamide, a member of a novel class of V-ATPase inhibitors.

Archazolid and apicularen: Novel specific V-ATPase inhibitors

BackgroundV-ATPases constitute a ubiquitous family of heteromultimeric, proton translocating proteins. According to their localization in a multitude of eukaryotic membranes, they energize many different transport processes. Since their malfunction is correlated with various diseases in humans, the elucidation of the properties of this enzyme for the development of selective inhibitors and drugs is one of the challenges in V-ATPase research.ResultsArchazolid A and B, two recently discovered cytotoxic macrolactones produced by the myxobacterium Archangium gephyra, and apicularen A and B, two novel benzolactone enamides produced by different species of the myxobacterium Chondromyces, exerted a similar inhibitory efficacy on a wide range of mammalian cell lines as the well established plecomacrolidic type V-ATPase inhibitors concanamycin and bafilomycin. Like the plecomacrolides both new macrolides also prevented the lysosomal acidification in cells and inhibited the V-ATPase purified from the midgut of the tobacco hornworm, Manduca sexta, with IC50 values of 20–60 nM. However, they did not influence the activity of mitochondrial F-ATPase or that of the Na+/K+-ATPase. To define the binding sites of these new inhibitors we used a semi-synthetic radioactively labelled derivative of concanamycin which exclusively binds to the membrane Vo subunit c. Whereas archazolid A prevented, like the plecomacrolides concanamycin A, bafilomycin A1 and B1, labelling of subunit c by the radioactive I-concanolide A, the benzolactone enamide apicularen A did not compete with the plecomacrolide derivative.ConclusionThe myxobacterial antibiotics archazolid and apicularen are highly efficient and specific novel inhibitors of V-ATPases. While archazolid at least partly shares a common binding site with the plecomacrolides bafilomycin and concanamycin, apicularen adheres to an independent binding site.

The structures of antioxidant and cytotoxic agents from natural source: anthraquinones and tannins from roots of Rumex patientia

A new anthraquinone glycoside, emodin-6-O-beta-D-glucopyranoside, and a new simple halogenated flavan-3-ol, 6-chlorocatechin, have been isolated from the roots of Rumex patientia L. together with seven known phenolic compounds. Their structures were elucidated on the basis of spectroscopic methods. Cytotoxic effects and radical scavenging properties of the isolated compounds have been demonstrated.

Insights into the Biosynthesis of Hormaomycin, An Exceptionally Complex Bacterial Signaling Metabolite

Hormaomycin produced by Streptomyces griseoflavus is a structurally highly modified depsipeptide that contains several unique building blocks with cyclopropyl, nitro, and chlorine moieties. Within the genus Streptomyces, it acts as a bacterial hormone that induces morphological differentiation and the production of bioactive secondary metabolites. In addition, hormaomycin is an extremely potent narrow-spectrum antibiotic. In this study, we shed light on hormaomycin biosynthesis by a combination of feeding studies, isolation of the biosynthetic nonribosomal peptide synthetase (NRPS) gene cluster, and in vivo and in vitro functional analysis of enzymes. In addition, several nonnatural hormaomycin congeners were generated by feeding-induced metabolic rerouting. The NRPS contains numerous highly repetitive regions that suggest an evolutionary scenario for this unusual bacterial hormone, providing new opportunities for evolution-inspired metabolic engineering of novel nonribosomal peptides.

Endophenazines A-D, New Phenazine Antibiotics from the Arthropod Associated Endosymbiont Streptomyces anulatus

Four new members of the phenazine family, endophenazines A-D, and the already known phenazine-1-carboxylic acid (tubermycin B) were detected in the culture broth of various endosymbiotic Streptomyces anulatus strains by chemical screening in a combination of TLC-staining reagents and HPLC-diode array analysis. The endosymbiotic strains were isolated from four different arthropod hosts at various sites. The new phenazine compounds showed antimicrobial activities against Gram-positive bacteria and some filamentous fungi, and herbicidal activity against Lemna minor (duckweed).

Physiological and Chemical Investigations into Microbial Degradation of Synthetic Poly(cis-1,4-isoprene)

ABSTRACT Streptomyces coelicolor 1A and Pseudomonas citronellolis were able to degrade synthetic high-molecular-weight poly(cis-1,4-isoprene) and vulcanized natural rubber. Growth on the polymers was poor but significantly greater than that of the nondegrading strain Streptomyces lividans 1326 (control). Measurement of the molecular weight distribution of the polymer before and after degradation showed a time-dependent increase in low-molecular-weight polymer molecules forS. coelicolor 1A and P. citronellolis, whereas the molecular weight distribution for the control (S. lividans 1326) remained almost constant. Three degradation products were isolated from the culture fluid of S. coelicolor 1A grown on vulcanized rubber and were identified as (6Z)-2,6-dimethyl-10-oxo-undec-6-enoic acid, (5Z)-6-methyl-undec-5-ene-2,9-dione, and (5Z,9Z)-6,10-dimethyl-pentadec-5,9-diene-2,13-dione. An oxidative pathway from poly(cis-1,4-isoprene) to methyl-branched diketones is proposed. It includes (i) oxidation of an aldehyde intermediate to a carboxylic acid, (ii) one cycle of β-oxidation, (iii) oxidation of the conjugated double bond resulting in a β-keto acid, and (iv) decarboxylation.

Phenols from Fagus sylvatica and their role in defence against cryptococcus fagisuga

Abstract In extracts of inner and outer bark of Fagus sylvatica, qualitative dependence of the phenolic composition on infection with Cryptococcus fagisuga feeding in the parenchyma tissue was observed. The seven major compounds were isolated and completely assigned, mainly by two-dimensional NMR techniques. Highest concentrations of (2R,3R)-(+)-glucodistylin, (2S,3S)-(−)-glucodistylin and 3-O-(β- d -xylopyranosyl)taxifolin occur in European beeches strongly infested with beech scale The concentration of cis-coniferin was lowered after attack, while the concentrations of catechin, cis-isoconiferin and cis-syringin remained unaffected. The changes are discussed as a defence reaction.

Retymicin, galtamycin B, saquayamycin Z and ribofuranosyllumichrome, novel secondary metabolites from Micromonospora sp. Tü 6368. II. Structure elucidation.

A detailed screening of the secondary metabolite pattern from Micromonospora sp. strain Tü 6368 resulted in the isolation of ten compounds belonging to five different structural families. The structures of the novel compounds 1-(α-ribofuranosyl)-lumichrome (3), retymicin (7), galtamycin B (11) and saquayamycin Z (14) were assigned by spectroscopic methods and chemical transformations. This strain fits our hypothesis that the metabolite analysis of biosynthetically talented strains leads readily to novel compounds.

Structural and Biosynthetic Investigations of the Rubromycins

The structure of the known secondary metabolite β-rubromycin was corrected, based on spectroscopic and chemical investigations, from o-quinone 1 to p-quinone 6. By feeding [U-13C3]malonic acid to the rubromycin-producing strain, Streptomyces sp. A1, the polyketide origin of the skeleton was verified, but the identity of the starter unit and the folding mechanism of the polyketide chain are still unclear. From the culture broth of the strain A1, in addition to 6, the co-metabolites γ-rubromycin (3), δ-rubromycin (4) and 3′-hydroxy-β-rubromycin (7) were isolated. Their structures were determined or confirmed by detailed spectroscopic analysis. The rubromycins inhibit HIV-1 reverse transcriptase (RT) and are cytostatically active against different tumor cell lines.

A comparative analysis of the sugar phosphate cyclase superfamily involved in primary and secondary metabolism.

Sugar phosphate cyclases (SPCs) catalyze the cyclization of sugar phosphates to produce a variety of cyclitol intermediates that serve as the building blocks of many primary metabolites, for example, aromatic amino acids, and clinically relevant secondary metabolites, for example, aminocyclitol/aminoglycoside and ansamycin antibiotics. Feeding experiments with isotopically labeled cyclitols revealed that cetoniacytone A, a unique C7N‐aminocyclitol antibiotic isolated from an insect endophytic Actinomyces sp., is derived from 2‐epi‐5‐epi‐valiolone, a product of SPC. By using heterologous probes from the 2‐epi‐5‐epi‐valiolone synthase class of SPCs, an SPC homologue gene, cetA, was isolated from the cetoniacytone producer. cetA is closely related to BE‐orf9 found in the BE‐40644 biosynthetic gene cluster from Actinoplanes sp. strain A40644. Recombinant expression of cetA and BE‐orf9 and biochemical characterization of the gene products confirmed their function as 2‐epi‐5‐epi‐valiolone synthases. Further phylogenetic analysis of SPC sequences revealed a new clade of SPCs that might regulate the biosynthesis of a novel set of secondary metabolites.