• Analysis of the role of an acetoacetyl CoA thiolase with a novel acetyl CoA:ACP transacylase activity in the initiation of straight chain fatty acid biosynthesis in Streptomyces collinus

      Lobo, Sandra; Reynolds, Kevin A. (2000)
      Streptomycetes predominantly produce branched-chain fatty acids (BCFA) with a small proportion of straight-chain fatty acids (SCFA). In vivo studies with thiolactomycin, a type-II fatty acid synthase inhibitor suggest that beta-ketoacyl ACP synthase (KAS III) is the major enzyme initiating BCFA biosynthesis, while a KAS III-independent pathway might be partially responsible for initiating SCFA biosynthesis. An acetyl CoA:ACP transacylase (ACT), putatively responsible for initiating the SCFA biosynthetic pathway, has been separated from KASIII and purified to near homogeneity (85 fold) from the crude extract of Streptomyces collinus. This enzyme was found to be less sensitive to inhibition by thiolactomycin (IC50 0.34 +/- 0.04 mM) as compared to KAS III from S. glaucescens (IC50 20 muM). The N-terminal peptide sequence of this enzyme revealed high sequence identity to members of the thiolase superfamily of enzymes. The gene (fadA) encoding the enzyme (FadA) with ACT activity was sequenced, cloned and overexpressed in Escherichia coli. The native purified and the recombinant FadA were found to have both thiolase activity and a novel ACT activity, which has never been observed before. Kinetic studies revealed that in an ACT assay FadA had a substrate specificity for the 2-carbon precursor acetyl CoA, but did not have a specificity for the type of ACP available, using S. glaucescens FAS ACP, E. coli FAS ACP and frenolicin (type-II polyketide) ACP as substrates. Evidence indicates that the formation of the same covalent S-acetyl enzyme intermediate in the active site, and similar enzyme mechanisms may be involved in both ACT and thiolase activities. The overexpression of fadA in S. collinus, however, did not reveal a significant change in phenotype from the wild type strain, and studies with a fadA gene disruption strain of S. collinus revealed only a minor role for the ACT activity of FadA in the initiation of the SCFA biosynthetic pathway. KAS III, thus appears to be the major enzyme initiating both the SCFA and BCFA pathway, and its broad substrate specificity together with intracellular pool sizes of precursors is the major determinant of the type of fatty acids produced by S. collinus.
    • 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.
    • Structure/function analysis of Escherichia coli DNA topoisomerase III

      Zhang, HongLiang; DiGate, Russell J. (1994)
      The generalized nucleic acid-binding domain of Escherichia coli DNA topoisomerase III (Topo III) has been identified using a selection procedure designated to isolate inactive Topo III polypeptides. Deletion of this binding domain, contained within the carboxyl terminal 50 amino acid residues of Topo III, results in a drastic reduction in the ability of the enzyme to bind to single-stranded DNA and RNA substrates. Successive truncation of the enzyme within this region results in the gradual loss of nucleic binding activity and in a gradual change in the mechanism of Topo III-catalyzed relaxation of negatively supercoiled DNA. Truncation of the enzyme to 605 amino acids (Topo III contains 653 amino acids) reduced DNA relaxation activity and binding activity to 1-2% the activity of the full length polypeptide. The reduction of nucleic acid binding activity of the truncated polypeptides does not result in a loss of cleavage site specificity for the enzyme, suggesting other amino acids are involved in the positioning of the nucleic acid within the nicking/closing site of the topoisomerase. Topo III binds asymmetrically to its substrate relative to its cleavage site. Topo III protects 14 nucleotides (12 nucleotides upstream of the cleavage site and 2 nucleotides downstream) from nuclease P1 digestion. The minimum substrate requirement for Topo III-catalyzed cleavage of DNA is only 7 nucleotides; however, this minimum substrate exhibits the same asymmetric sequence preference (6 nucleotides upstream of the cleavage site and 1 nucleotide downstream). In addition, a substrate that contained a Topo III cleavage/binding site formed a more stable complex with the enzyme than a substrate that did not contain a Topo III cleavage/binding site. An oligonucleotide with a Topo III cleavage/binding site was a better competitor for Topo III than an oligonucleotide without the site. Based on these results, a model is proposed in which Topo III binds to DNA and diffuses along the substrate until a cleavage site is located. When a cleavage site is found, Topo III halts and stably binds to the cleavage site. A hybrid of molecule consisting of Topo III cleavage domain and the Topo I binding domain has been used to demonstrate that the domains for cleavage and binding could be separated. The hybrid was created by fusing the first 604 amino acids of Topo III with the carboxyl-terminal 313 amino acids of Topo I. Addition of the C-terminal amino acids of Topo I restored both DNA relaxation activity and DNA binding activity to the enzyme; however, the ability of the enzyme to segregate newly synthesized replication molecules was not drastically enhanced. This suggests that C-terminal domain of Topo III may specifically contribute to its ability to decatenate newly replicated chromosomes.