• The cloning and identification of a streptonigrin resistance gene from Escherichia coli

      Heller, Phillip Ferdinand; Speedie, Marilyn K. (1996)
      Streptonigrin is a antineoplastic antibiotic whose mode of action is dependent on formation of the hydroxyl radical. A streptonigrin resistance gene was cloned from a partial Sau3A digest of E. coli DNA. One to 10 Kilobase (Kb) fragments were ligated into the vector pUC18 and transformed into E. coli (DH5{dollar}\alpha{dollar}). Once prepared, the library was replica plated and then screened with differing streptonigrin concentrations in a top agar overlay. Clones exhibiting growth into the overlay at the highest STN concentrations (pH191, pH192) were rescued from the overlay and grown for plasmid isolation and for determination of theminimum inhibitory concentration (MIC) for streptonigrin in LB broth, 3 and 1 {dollar}\mu{dollar}g/ml respectively. The isolated plasmids were mapped by restriction endonuclease digestion followed by agarose gel electrophoresis. Restriction sites were assigned based on known location in the multicloning site of the vector, leading to the production of an approximate physical map of the insert in the plasmid. The resistance element (StnR) was localized by deletion studies on pH191. Sequencing and analysis of the deoxynucleotide sequence of pH191 led to the identification of an open reading frame (StnR) with a high homology (97.6%) to the first 185 residues of riboflavin synthase (RibC). The resistance gene was expressed in S. lividans on pIJ702 where it yielded streptonigrin resistance in excess of that found in the wild type producing species, S. flocculus (40 {dollar}\mu{dollar}g/ml). Expression of StnR and RibC in E. coli via the vector pET3C resulted in strains with elevated streptonigrin resistance as compared to the pET3C control in both solid and liquid media. A gene imparting resistance to the hydroxyl radical producing drug streptonigrin has been identified as the riboflavin synthase from E. coli.
    • Partial purification and characterization of a C-methyltransferase from streptonigrin-producing Streptomyces flocculus

      Fox, Bonnie Marie; Speedie, Marilyn K. (1991)
      A C-methyltransferase which catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the C-3 position of the aliphatic side chain of L-tryptophan resulting in formation of {dollar}\beta{dollar}-methyl tryptophan, has been isolated from streptonigrin-producing Streptomyces flocculus. The enzyme catalyzes the first step in streptonigrin biosynthesis and is postulated to have a regulatory role in the pathway. The enzyme has been purified 217-fold by ammonium sulfate fractionation, followed by sequential gel filtration through Sephadex G-150 and Sephadex G-100 SF columns. Attempts at further purification have been hindered by very active proteases which co-purify with the enzyme. Protease inhibitors PMSF, pepstatin A, leupeptin, and trypsin inhibitor have failed to inactivate the protease activity. Based on comparison to reference proteins, the C-methyltransferase was estimated to have a molecular weight of 40,000 by Sephadex G-150 gel filtration. A narrow pH optimum of 7.5-8.0 was determined for the enzyme. The Sephadex G-100 SF fraction was highly unstable, losing 90 {dollar}\pm{dollar} 6% of its activity after 12 hours at 4{dollar}\sp\circ{dollar}C. S-Adenosyl-L-methionine and L-cysteine have been found to stabilize activity in the purified fractions. The enzyme is inhibited by sulfhydryl binding reagents, but no such inhibition is observed in the presence of substrate, suggesting an essential {dollar}-{dollar}SH group at or near the active site. Inhibition by carbonyl reagents was exhibited by the C-methyltransferase. Tritiated sodium cyanoborohydride treatment of the Sephadex G-100 SF fraction resulted in tritium incorporation and a concomitant 36 {dollar}\pm{dollar} 1% inactivation of the enzyme. These combined data led to the hypothesis that pyridoxal-5{dollar}\sp\prime{dollar}-phosphate may be involved as a cofactor in the C-methyltransferase. An enzymatic mechanism is proposed, and several studies related to this mechanism are presented.