Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1988 Sep;170(9):4055–4064. doi: 10.1128/jb.170.9.4055-4064.1988

Metabolic regulation in Streptomyces parvulus during actinomycin D synthesis, studied with 13C- and 15N-labeled precursors by 13C and 15N nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry.

L Inbar 1, A Lapidot 1
PMCID: PMC211409  PMID: 3410824

Abstract

Recent studies have suggested that the onset of synthesis of actinomycin D in Streptomyces parvulus is due to a release from L-glutamate catabolic repression. In the present investigation we showed that S. parvulus has the capacity to maintain high levels of intracellular glutamate during the synthesis of actinomycin D. The results seem contradictory, since actinomycin D synthesis cannot start before a release from L-glutamate catabolic repression, but a relatively high intracellular pool of glutamate is needed for the synthesis of actinomycin D. Utilizing different labeled precursors, D-[U-13C]fructose and 13C- and 15N-labeled L-glutamate, and nuclear magnetic resonance techniques, we showed that carbon atoms of an intracellular glutamate pool of S. parvulus were not derived biosynthetically from the culture medium glutamate source but rather from D-fructose catabolism. A new intracellular pyrimidine derivative whose nitrogen and carbon skeletons were derived from exogenous L-glutamate was obtained as the main glutamate metabolite. Another new pyrimidine derivative that had a significantly reduced intracellular mobility and that was derived from D-fructose catabolism was identified in the cell extracts of S. parvulus during actinomycin D synthesis. These pyrimidine derivatives may serve as a nitrogen store for actinomycin D synthesis. In the present study, the N-trimethyl group of a choline derivative was observed by 13C nuclear magnetic resonance spectroscopy in growing S. parvulus cells. The choline group, as well as the N-methyl groups of sarcosine, N-methyl-valine, and the methyl groups of an actinomycin D chromophore, arose from D-fructose catabolism. The 13C enrichments found in the peptide moieties of actinomycin D were in accordance with a mechanism of actinomycin D synthesis from L-glutamate and D-fructose.

Full text

PDF
4055

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Eakin R. T., Morgan L. O. Carbon-13 nuclear-magnetic-resonance spectroscopy of whole cells and of cytochrome C from Neurospora crass grown with (S-Me-13C)methionine. Biochem J. 1975 Dec;152(3):529–535. doi: 10.1042/bj1520529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Foster J. W., Katz E. Control of actinomycin D biosynthesis in Streptomyces parvullus: regulation of tryptophan oxygenase activity. J Bacteriol. 1981 Nov;148(2):670–677. doi: 10.1128/jb.148.2.670-677.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. GOLDBERG I. H., RABINOWITZ M., REICH E. Basis of actinomycin action. I. DNA binding and inhibition of RNA-polymerase synthetic reactions by actinomycin. Proc Natl Acad Sci U S A. 1962 Dec 15;48:2094–2101. doi: 10.1073/pnas.48.12.2094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Haran N., Kahana Z. E., Lapidot A. In vivo 15N NMR studies of regulation of nitrogen assimilation and amino acid production by Brevibacterium lactofermentum. J Biol Chem. 1983 Nov 10;258(21):12929–12933. [PubMed] [Google Scholar]
  5. Hitchcock M. J., Katz E. Actinomycin biosynthesis by protoplasts derived from Streptomyces parvulus. Antimicrob Agents Chemother. 1978 Jan;13(1):104–114. doi: 10.1128/aac.13.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Inbar L., Kahana Z. E., Lapidot A. Natural-abundance 13C nuclear magnetic resonance studies of regulation and overproduction of L-lysine by Brevibacterium flavum. Eur J Biochem. 1985 Jun 18;149(3):601–607. doi: 10.1111/j.1432-1033.1985.tb08967.x. [DOI] [PubMed] [Google Scholar]
  7. Inbar L., Lapidot A. 13C-NMR, 1H-NMR and gas-chromatography mass-spectrometry studies of the biosynthesis of 13C-enriched L-lysine by Brevibacterium flavum. Eur J Biochem. 1987 Feb 2;162(3):621–633. doi: 10.1111/j.1432-1033.1987.tb10684.x. [DOI] [PubMed] [Google Scholar]
  8. Jones G. H. Actinomycin synthesis in Streptomyces antibioticus: enzymatic conversion of 3-hydroxyanthranilic acid to 4-methyl-3-hydroxyanthranilic acid. J Bacteriol. 1987 Dec;169(12):5575–5578. doi: 10.1128/jb.169.12.5575-5578.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Juretschke H. P., Lapidot A. Actinomycin D, 1H NMR studies on intramolecular interactions and on the planarity of the chromophore. Eur J Biochem. 1984 Sep 17;143(3):651–658. doi: 10.1111/j.1432-1033.1984.tb08418.x. [DOI] [PubMed] [Google Scholar]
  10. Juretschke H. P., Lapidot A. Intramolecular interactions, mesomerism and dynamics in actinomycin D studied by 15N NMR spectroscopy. Eur J Biochem. 1985 Mar 1;147(2):313–324. doi: 10.1111/j.1432-1033.1985.tb08752.x. [DOI] [PubMed] [Google Scholar]
  11. KATZ E., WEISSBACH H. Incorporation of C14-labeled amino acids into actinomycin and protein by Streptomyces antibioticus. J Biol Chem. 1963 Feb;238:666–675. [PubMed] [Google Scholar]
  12. Kahana Z. E., Lapidot A. Microbial production of L-[15N]glutamic acid and its gas chromatography-mass spectrometry analysis. Anal Biochem. 1983 Jul 1;132(1):160–164. doi: 10.1016/0003-2697(83)90441-4. [DOI] [PubMed] [Google Scholar]
  13. Keller U. Actinomycin synthetases. Multifunctional enzymes responsible for the synthesis of the peptide chains of actinomycin. J Biol Chem. 1987 Apr 25;262(12):5852–5856. [PubMed] [Google Scholar]
  14. Lackner H. Three-dimensional structure of the actinomycins. Angew Chem Int Ed Engl. 1975;14(6):375–386. doi: 10.1002/anie.197503751. [DOI] [PubMed] [Google Scholar]
  15. Lapidot A., Irving C. S. Comparative in vivo nitrogen-15 nuclear magnetic resonance study of the cell wall components of five Gram-positive bacteria. Biochemistry. 1979 Feb 20;18(4):704–714. doi: 10.1021/bi00571a024. [DOI] [PubMed] [Google Scholar]
  16. Lapidot A., Irving C. S. Dynamic structure of whole cells probed by nuclear Overhauser enhanced nitrogen-15 nuclear magnetic resonance spectroscopy. Proc Natl Acad Sci U S A. 1977 May;74(5):1988–1992. doi: 10.1073/pnas.74.5.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lapidot A., Nissim I. Regulation of pool sizes and turnover rates of amino acids in humans: 15N-glycine and 15N-alanine single-dose experiments using gas chromatography-mass spectrometry analysis. Metabolism. 1980 Mar;29(3):230–239. doi: 10.1016/0026-0495(80)90064-5. [DOI] [PubMed] [Google Scholar]
  18. Lewis J. L., Jr Chemotherapy of gestational choriocarcinoma. Cancer. 1972 Dec;30(6):1517–1521. doi: 10.1002/1097-0142(197212)30:6<1517::aid-cncr2820300616>3.0.co;2-8. [DOI] [PubMed] [Google Scholar]
  19. Magasanik B. Genetic control of nitrogen assimilation in bacteria. Annu Rev Genet. 1982;16:135–168. doi: 10.1146/annurev.ge.16.120182.001031. [DOI] [PubMed] [Google Scholar]
  20. Martin J. F., Demain A. L. Control of antibiotic biosynthesis. Microbiol Rev. 1980 Jun;44(2):230–251. doi: 10.1128/mr.44.2.230-251.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Troost T., Hitchcock M. J., Katz E. Distinct kynureninase and hydroxykynureninase enzymes in an actinomycin-producing strain of Streptomyces parvulus. Biochim Biophys Acta. 1980 Mar 14;612(1):97–106. doi: 10.1016/0005-2744(80)90282-x. [DOI] [PubMed] [Google Scholar]
  22. Williams W. K., Katz E. Development of a chemically defined medium for the synthesis of actinomycin D by Streptomyces parvulus. Antimicrob Agents Chemother. 1977 Feb;11(2):281–290. doi: 10.1128/aac.11.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES