mucidin and myxothiazol

Topics: Acrylates; Animals; Antifungal Agents; Fatty Acids, Unsaturated; Humans; Methacrylates; Strobilurins; Thiazoles

New antibiotic compounds, melithiazols, were isolated from the culture broth of strains of the myxobacteria Melittangium lichenicola, Archangium gephyra, and Myxococcus stipitatus. The compounds belong to the group of beta-methoxyacrylate (MOA) inhibitors and are related to the myxothiazols. The melithiazols show high antifungal activity, but are less toxic than myxothiazol A and its methyl ester in a growth inhibition assay with mouse cell cultures. The melithiazols inhibit NADH oxidation by submitochondrial particles from beef heart. Melithiazol A blocks the electron transport within the bc1-segment (complex III) and causes a red shift in the reduced spectrum of cytochrome b.

Topics: Acrylates; Animals; Antifungal Agents; Cell Respiration; Cytochrome b Group; Drug Evaluation, Preclinical; Energy Metabolism; Fatty Acids, Unsaturated; Fermentation; Humans; Infant; Inhibitory Concentration 50; Methacrylates; Mice; Microbial Sensitivity Tests; Mitochondria, Heart; Myxococcales; NAD; Strobilurins; Structure-Activity Relationship; Thiazoles

Topics: Animals; Antifungal Agents; Diptera; Electron Transport Complex III; Fatty Acids, Unsaturated; Methacrylates; Mitochondria; Mitosporic Fungi; Molecular Structure; Oxygen Consumption; Rats; Saccharomyces cerevisiae; Strobilurins; Structure-Activity Relationship; Thiazoles; Zea mays

Topics: Amino Acid Sequence; Antifungal Agents; Binding Sites; Cytochrome b Group; Fatty Acids, Unsaturated; Methacrylates; Molecular Sequence Data; Saccharomyces cerevisiae; Strobilurins; Thiazoles

The kinetic and circular dichroic properties of two yeast mutants that are resistant towards specific inhibitors of the mitochondrial cytochrome bc1 complex have been characterized. Both of these mutants have an altered cytochrome b gene in which aromatic residues are exchanged with non-polar residues in a highly conserved region of the protein. The mutant resistant to myxothiazol and mucidin that contains the substitution Phe129----Leu is not greatly affected either in its ubiquinol:cytochrome c reductase or in the spectral properties of cytochrome b. On the other hand, the mutant resistant to stigmatellin that contains the substitution Ile147----Phe shows a large decrease of the catalytic efficiency for ubiquinol and of the maximal turnover of its reductase activity. This stigmatellin mutant also shows an altered circular-dichroic spectrum of the low-potential haem of cytochrome b. This study provides biochemical and biophysical information for identifying a region in mitochondrial cytochrome b that may fulfill a crucial role in the binding of ubiquinol to the bc1 complex. The results are discussed also in terms of the structural model of cytochrome b having a core of four transmembrane helices.

Topics: Amino Acid Sequence; Circular Dichroism; Cytochrome b Group; Electron Transport Complex III; Fatty Acids, Unsaturated; Kinetics; Methacrylates; Molecular Sequence Data; Mutation; Polyenes; Protein Conformation; Saccharomyces cerevisiae; Strobilurins; Thiazoles

We describe the genetic and molecular analysis of the first non-Mendelian mutants of Chlamydomonas reinhardtii resistant to myxothiazol, an inhibitor of the respiratory cytochrome bc1 complex. Using a set of seven oligonucleotide probes, restriction fragments containing the mitochondrial cytochrome b (cyt b) gene from C. reinhardtii were isolated from a mitochondrial DNA library. This gene is located adjacent to the gene for subunit 4 of the mitochondrial NADH-dehydrogenase (ND4), near one end of the 15.8-kb linear mitochondrial genome of C. reinhardtii. The algal cytochrome b apoprotein contains 381 amino-acid residues and exhibits a sequence similarity of about 59% with other plant cytochrome b proteins. The cyt b gene from four myxothiazol resistant mutants of C. reinhardtii was amplified for DNA sequence analysis. In comparison to the wild-type strain, all mutants contain an identical point mutation in the cyt b gene, leading to a change of a phenylalanine codon to a leucine codon at amino acid position 129 of the cytochrome b protein. Segregation analysis in tetrads from reciprocal crosses of mutants with wild type shows a strict uniparental inheritance of this mutation from the mating type minus parent (UP-). However, mitochondrial markers from both parents are recovered in vegetative diploids in variable proportions from one experiment to the next for a given cross. On the average, a strong bias is seen for markers inherited from the mating type minus parent.

Topics: Amino Acid Sequence; Antifungal Agents; Base Sequence; Chlamydomonas; Crosses, Genetic; Cytochrome b Group; Diploidy; DNA, Mitochondrial; Fatty Acids, Unsaturated; Haploidy; Methacrylates; Mitochondria; Molecular Sequence Data; Mutation; NADH Dehydrogenase; Oligonucleotide Probes; Polymerase Chain Reaction; Restriction Mapping; Sequence Homology, Nucleic Acid; Strobilurins; Thiazoles

The ubiquinol:cytochrome c reductase activity of Paracentrotus lividus mitochondria is relatively insensitive to the specific inhibitors myxothiazol and mucidin. The I50 of myxothiazol and mucidin are three and two orders of magnitude higher, respectively, in P. lividus than in bovine heart mitochondria. The natural resistance of the P. lividus reductase to these inhibitors can be correlated with a single amino replacement, an alanine for a glycine at position 143, in the sequence of cytochrome b. This position is located in a conserved region of the molecule, believed to be important in the oxidation of ubiquinol by the reductase.

Topics: Alkenes; Amino Acid Sequence; Animals; Cattle; Electron Transport Complex III; Fatty Acids, Unsaturated; Methacrylates; Mitochondria; Mitochondria, Heart; Molecular Sequence Data; Sea Urchins; Sequence Homology, Nucleic Acid; Species Specificity; Strobilurins; Thiazoles

Seven single-site mutants in six residues of the cyt b polypeptide of Rhodobacter capsulatus selected for resistance to the Qo site inhibitors stigmatellin, myxothiazol, or mucidin [Daldal, F., Tokito, M.K., Davidson, E., & Faham, M. (1989) EMBO J. 8, 3951-3961] have been characterized by using optical and EPR spectroscopy and single-turnover kinetic analysis. The strains were compared with wild-type strain MT1131 and with the Ps- strain R126 (G158D), which is dysfunctional in its Qo site [Robertson, D.E., Davidson, E., Prince, R.C., van den Berg, W.H., Marrs, B.L., & Dutton, P.L. (1986) J. Biol. Chem. 261, 584-591]. Mutants selected for stigmatellin resistance induced a weakening in the binding of the inhibitor without discernible loss of ubiquinone(Q)/ubiquinol(QH2) binding affinity to the Qo site or kinetic impairment to catalysis. Mutants selected for myxothiazol or mucidin resistance, inducing weakening of inhibitor binding, all displayed impaired rates of Qo site catalysis: The most severe cases (F144L, F144S) displayed loss of affinity for Q, and evidence suggests that parallel loss of affinity for the substrate QH2 was incurred in these strains. The results provide a view of the nature of the interaction of Q and QH2 of the Qpool with the Qo site. Consideration of the mutational substitutions and their structural positions along with comparisons with the QA and QB sites of the photosynthetic reaction center suggests a model for the structure of the Qo site.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Cytochrome b Group; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Fatty Acids, Unsaturated; Kinetics; Methacrylates; Models, Structural; Molecular Sequence Data; Mutation; Oxidation-Reduction; Polyenes; Protein Conformation; Rhodobacter capsulatus; Strobilurins; Thiazoles; Ubiquinone

Several spontaneous mutants of the photosynthetic bacterium Rhodobacter capsulatus resistant to myxothiazol, stigmatellin and mucidin--inhibitors of the ubiquinol: cytochrome c oxidoreductase (cyt bc1 complex)--were isolated. They were grouped into eight different classes based on their genetic location, growth properties and inhibitor cross-resistance. The petABC (fbcFBC) cluster that encodes the structural genes for the Rieske FeS protein, cyt b and cyt c1 subunits of the cyt bc1 complex was cloned out of the representative isolates and the molecular basis of inhibitor-resistance was determined by DNA sequencing. These data indicated that while one group of mutations was located outside the petABC(fbcFBC) cluster, the remainder were single base pair changes in codons corresponding to phylogenetically conserved amino acid residues of cyt b. Of these substitutions, F144S conferred resistance to myxothiazol, T163A and V333A to stigmatellin, L106P and G152S to myxothiazol + mucidin and M140I and F144L to myxothiazol + stigmatellin. In addition, a mutation (aer126) which specifically impairs the quinol oxidase (Qz) activity of the cyt bc1 complex of a non-photosynthetic mutant (R126) was identified to be a glycine to an aspartic acid replacement at position 158 of cyt b. Six of these mutations were found between amino acid residues 140 and 163, in a region linking the putative third and fourth transmembrane helices of cyt b. The non-random clustering of several inhibitor-resistance mutations around the non-functional aer126 mutation suggests that this region may be involved in the formation of the Qz inhibitor binding/quinol oxidation domain(s) of the cyt bc1 complex. Of the two remaining mutations, the V333A replacement conferred resistance to stigmatellin exclusively and was located in another region toward the C terminus of cyt b. The L106P substitution, on the other hand, was situated in the transmembrane helix II that carries two conserved histidine residues (positions 97 and 111 in R. capsulatus) considered to be the axial ligands for the heme groups of cyt b. The structural and functional roles of the amino acid residues involved in the acquisition of Qz inhibitor resistance are discussed in terms of the primary structure of cyt b and in relation to the natural inhibitor-resistance of various phylogenetically related cyt bc/bf complexes.

Topics: Alkenes; Amino Acid Sequence; Anti-Bacterial Agents; Antifungal Agents; Drug Resistance, Microbial; Electron Transport Complex III; Fatty Acids, Unsaturated; Genes, Bacterial; Methacrylates; Molecular Sequence Data; Mutation; Phenotype; Polyenes; Protein Conformation; Restriction Mapping; Rhodopseudomonas; Sequence Homology, Nucleic Acid; Strobilurins; Thiazoles

Mucidin and strobilurin A, antifungal antibiotics isolated from the basidiomycetes Oudemansiella mucida and Strobiluris tenacellus, respectively, inhibit electron-transfer reactions in the cytochrome bc1 complex of the mitochondrial respiratory chain. The two compounds have identical effects on oxidation-reduction reactions of the cytochromes b and c1 in isolated succinate-cytochrome c reductase. They inhibit reduction of cytochrome c1 by succinate but do not inhibit reduction of cytochrome b. When added in combination with antimycin, either inhibitor blocks reduction of both cytochromes b and c1. Mucidin and strobilurin A differ from antimycin in that they inhibit, rather than promote, oxidant-induced reduction of cytochrome b. They also differ from antimycin in that they do not block reduction of cytochrome b by succinate when cytochrome c1 is previously reduced by ascorbate and they do not inhibit oxidation of cytochrome b by fumarate. These effects of mucidin and strobilurin A are, however, qualitatively identical with those of myxothiazol, an antibiotic that inhibits respiration by binding to cytochrome b [Von Jagow, G., Ljungdahl, P. O., Graf, P., Ohnishi, T., & Trumpower, B. L. (1984) J. Biol. Chem. 259, 6319-6326]. Mucidin and strobilurin A have identical UV and mass spectra, and they elute together on high-pressure liquid chromatography. We thus conclude that these antibiotics, although isolated from different bacteria, are structurally identical. Our results indicate that strobilurin A and mucidin inhibit electron transport at the same site as myxothiazol and not at the antimycin site, as previously reported [Subik, J., Behren, M., & Musilek, V. (1974) Biochem. Biophys. Res. Commun. 57, 17-22].

Topics: Alkenes; Antifungal Agents; Binding Sites; Chromatography, High Pressure Liquid; Electron Transport; Electron Transport Complex III; Fatty Acids, Unsaturated; Kinetics; Mass Spectrometry; Methacrylates; Mitochondria; Multienzyme Complexes; Oxygen Consumption; Protein Binding; Quinone Reductases; Strobilurins; Structure-Activity Relationship; Thiazoles

Changes are described that are brought about by antimycin, NoHOQnO, funiculosin, myxothiazol and mucidin in the alpha-, beta- and gamma-absorption bands of reduced and oxidized cytochromes b in the isolated complex bc1 form beef heart mitochondria. The inhibitors can be divided into 2 groups. Antimycin, funiculosin and NoHOQnO are likely to shift the spectrum of b-562 and compete for specific binding with complex bc1, with each other but not with myxothiazol and mucidin. The spectral effects of the latter two inhibitors are more difficult to interpret and may involve contributions not only from b-562 but from b-566 as well. The existence of 2 independent inhibitor binding-sites in the complex bc1 corroborates the Q-cycle hypothesis.

Topics: Alkenes; Animals; Anthraquinones; Antimycin A; Cattle; Cytochrome b Group; Electron Transport; Electron Transport Complex III; Fatty Acids, Unsaturated; Hydroxyquinolines; Methacrylates; Mitochondria, Heart; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Quinone Reductases; Spectrophotometry; Strobilurins; Thiazoles

Antimycin, 2-nonyl-4-hydroxyquinoline N-oxide and funiculosin induce O.2(-) generation by submitochondrial particles oxidizing succinate, whereas KCN, mucidin, myxothiazol or 2,3-dimercaptopropanol inhibit O.2(-) generation. Thenoyltrifluoroacetone does not induce superoxide production by itself but slightly stimulates the reaction initiated by antimycin. The results indicate that auto-oxidation of unstable ubisemiquinone formed in centre o of the Q-cycle generates most of the O.2(-) radicals in the cytochrome bc1-site of the mitochondrial respiratory chain.

Topics: Alkenes; Animals; Anthraquinones; Antimycin A; Cattle; Dimercaprol; Electron Transport Complex III; Fatty Acids, Unsaturated; Hydroxyquinolines; Methacrylates; Mitochondria, Heart; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Oxygen; Quinone Reductases; Strobilurins; Superoxides; Thenoyltrifluoroacetone; Thiazoles

Topics: Acrylates; Alkenes; Animals; Antifungal Agents; Antimycin A; Cattle; Cytochrome b Group; Cytochromes; Cytochromes c1; Electron Transport; Fatty Acids, Unsaturated; Methacrylates; Spectrum Analysis; Strobilurins; Structure-Activity Relationship; Submitochondrial Particles; Thiazoles; Valerates