naphthoquinones and rubradirin

Master of your domain: A domain of the bifunctional enzyme RubC1 catalyzes the adenylation of tyrosine. This domain can be activated not only by MbtH-like proteins but also by another domain of RubC1 that has no similarity to MbtH-like proteins.

Topics: Bacterial Proteins; Naphthoquinones; Protein Processing, Post-Translational; Protein Structure, Tertiary; Tyrosine

The biosynthesis of aminocoumarin antibiotics requires two acyladenylate-forming enzymes: one for the activation of L-tyrosine as a precursor of the aminocoumarin moiety and another for the linkage of an acyl moiety to the aminocoumarin moiety. Unexpectedly, the biosynthetic gene cluster of the aminocoumarin antibiotic rubradirin was found to contain three genes for putative acyladenylate-forming enzymes of aminocoumarin biosynthesis and conjugation. We expressed, purified, and investigated these three proteins. Orf4 (55 kDa) was shown to be an active aminocoumarin acyl ligase. RubF6 (56 kDa) was inactive, but could be converted into an active enzyme by site-directed mutagenesis. RubC1 (138 kDa) was shown to be a unique bifunctional enzyme, comprising an aminocoumarin acyl ligase, and tyrosine-adenylation and peptidyl-carrier domains. This natural hybrid enzyme is unique among known proteins. A hypothesis is proposed as to how such an enzyme could offer a particularly effective machinery for aminocoumarin antibiotic biosynthesis.

Topics: Amino Acid Sequence; Aminocoumarins; Biocatalysis; Enzyme Activation; Ligases; Molecular Sequence Data; Naphthoquinones; Open Reading Frames; Streptomyces; Tyrosine

The four overlapping cosmids from the rubradirin producer, Streptomyces achromogenes var rubradiris NRRL 3061, have 58 ORFs within a 105.6 kb fragment. These ORFs harbored essential genes responsible for the formation and attachment of four distinct moieties, along with the genes associated with regulatory, resistance, and transport functions. The PKS (rubA) and glycosyltransferase (rubG2) genes were disrupted in order to demonstrate a complete elimination of rubradirin production. The rubradirin biosynthetic pathway was proposed based on the putative functions of the gene products, the functional identification of sugar genes, and the mutant strains.

Topics: Aminobenzoates; Anti-Bacterial Agents; Base Sequence; Cosmids; DNA, Bacterial; Fermentation; Gene Silencing; Glycosides; Hydroxybenzoates; Molecular Sequence Data; Multigene Family; Naphthoquinones; Open Reading Frames; Sequence Analysis, DNA; Streptomyces

ORF's for rubN6 and rubN4 have been annotated as thymidine diphosphate glucose 4-ketoreductase and thymidine diphosphate glucose 3-aminotransferase by sequence analysis of the rubradirin biosynthetic gene cluster cloned from Streptomyces achromogenes var. rubradiris NRRL 3061. Both ORFs were heterologously expressed in Escherichia coli as His-tagged fusion proteins. The functionalities of TDP-glucose 4-ketoreductase and TDP-glucose 3-aminotransferase were verified by in vitro enzyme assay, and a biosynthetic pathway for TDP-D: -rubranitrose is proposed.

Topics: Amino Acid Sequence; Bacterial Proteins; Carbohydrate Dehydrogenases; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Molecular Sequence Data; Molecular Structure; Multigene Family; Naphthoquinones; Oxidoreductases; Phylogeny; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Streptomyces; Transaminases

An open reading frame, rub52, has been identified as a gene encoding thymidine diphospho-glucose 2,3-dehydratase by sequence analysis of the rubradirin biosynthetic gene cluster of Streptomyces achromogenes var. nibradiris NRRL3061. The gene codes for a protein consisting of 458 amino acids with calculated molecular mass of 50862 Da. The gene was amplified and heterologously expressed in Escherichia coli as a soluble His-tagged fusion protein. C-2 deoxygenation functionality of thymidine diphospho-4-keto-6-deoxyglucose was assigned to the rub52 gene product from in vitro enzyme assay.

Topics: Amino Acid Sequence; Cloning, Molecular; Escherichia coli; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Glycosides; Hydro-Lyases; Molecular Sequence Data; Naphthoquinones; Recombinant Fusion Proteins; Sequence Alignment; Sequence Analysis, Protein; Streptomyces

Rubradirin, an ansamycin antibiotic has been purified from Streptomyces achromogenes var. rubradiris NRRL3061. It consists of four distinct structural moieties, rubransarol, 3-amino-4-hydroxy-coumarin, dihydroxydipicolinic acid, and 2,6-dideoxynitrosugar (DNS). Polymerase chain reaction (PCR) primers were designed based on consensus sequences of dTDP-D-glucose 4,6-dehydratase, one of enzymes involved in the biosynthesis of 2,6-dideoxysugar. A PCR product was obtained from S. achromogenes var. rubradiris. Hybridization of the PCR product to a cosmid library constructed from S. achromogenes genomic DNA has led to the identification of three unlinked regions of DNA. One of three kinds of cosmid clones contains homologues of dTDP-D-glucose 4,6-dehydratase, 3-amino-5-hydroxybenzoic acid (AHBA) synthase, and eryA genes. The size of the gene homologous to eryA is 30 kb, and the AHBA synthase gene homologue resides between the eryA homologous genes. A gene cluster of rubransarol and 2,6-dideoxynitrosugar is around 50 kb. Sequencing of the PCR product from the AHBA synthase gene homologue isolated from S. achromogenes revealed 85% amino acid sequence homology (73/86) with the AHBA synthase from a rifamycin-producer. dTDP-D-glucose 4,6-dehydratase gene homologue was subcloned from one of the isolated cosmid clones and sequenced. It showed 65% homology (43/66) with dTDP-D-glucose 4,6-dehydratase from a streptomycin-producer.

Topics: Amino Acid Sequence; Aminobenzoates; Blotting, Southern; Cloning, Molecular; Consensus Sequence; DNA Primers; Genes, Bacterial; Hydro-Lyases; Hydroxybenzoates; Molecular Sequence Data; Multienzyme Complexes; Multigene Family; Naphthoquinones; Polymerase Chain Reaction; Streptomyces

In an attempt to improve the isolation of the antibiotic rubradirin from fermentations of Streptomyces achromogenes var. rubradiris, the use of preparative reversed-phase chromatography was investigated. The product isolated was a mixture of rubradirin and a new antibiotic named protorubradirin, of extremely similar structure, which is converted into rubradirin on exposure to light and air. Methanolysis of protorubradirin in the dark yields an anomeric mixture of methyl glycosides of a C-nitroso-sugar, converted photo-oxidatively into the methyl rubranitrosides derived from rubradirin. Thus, protorubradirin is the C-nitroso-analogue of rubradirin. It is suggested that the same relationship between protorubradirin and rubradirin may apply to the anthracycline antibiotics viriplanin A and viriplanin D.

Topics: Anti-Bacterial Agents; Bacteria; Naphthoquinones; Photochemistry; Streptomyces

Rubradirin is an antibiotic of complex chemical structure which is active vs. methicillin resistant staphylococci. Its development has been limited due to inadequate production yields. The incorporation of neutral resins into fermentations of Streptomyces achromogenes v. rubradiris, UC 8051 resulted in the enhanced production of rubradirin. Resins HP-20, HP-21, XAD-2, XAD-7 and XAD-16 were employed in flask and tank fermentations. The incorporation of these resins promoted 2- to 4-fold enhancements of the rubradirin activity produced in flask fermentations, and the incorporation of XAD-16 and HP-21 into tank fermentations promoted production titer increases greater than 5 fold.

Topics: Anti-Bacterial Agents; Biological Assay; Culture Media; Fermentation; Micrococcus; Molecular Structure; Naphthoquinones; Resins, Plant; Streptomyces

Topics: Chemical Phenomena; Chemistry; Coumarins; Glycosides; Naphthoquinones

Topics: Adenine Nucleotides; Amino Acids; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Binding Sites; Carbon Isotopes; Cell-Free System; Coliphages; Cytosine Nucleotides; Drug Synergism; Escherichia coli; Formates; Magnesium; Methionine; Naphthoquinones; Osmolar Concentration; Peptide Biosynthesis; Peptide Chain Initiation, Translational; Poly U; Polynucleotides; Puromycin; Ribosomes; RNA, Bacterial; RNA, Messenger; RNA, Transfer; RNA, Viral

Topics: Anti-Bacterial Agents; Antibiotics, Antitubercular; Dermatologic Agents; Fermentation; Metabolism; Naphthoquinones; Pharmacology; Research

Topics: Anti-Bacterial Agents; Antibiotics, Antitubercular; Chromatography; Countercurrent Distribution; Naphthoquinones; Research; Spectrum Analysis