1887

Abstract

A methyltransferase that acts on carminomycin and 13-dihydrocarminomycin, and that is postulated to be the terminal enzyme in the daunomycin biosynthesis pathway, was purified to near-homogeneity from the daunomycin-and baumycin-producing sp. strain C5. The enzyme was obtained in approximately 5% yield with a purification of 114-fold in specific activity over the sample precipitated with 30–50% ammonium sulphate. Polyacrylamide gel electrophoresis under denaturing conditions indicated a subunit of about 41000. The enzyme was shown by gel filtration chromatography to have an of approximately 166000, suggesting that it is a homotetramer. Kinetic analysis indicated an affinity for -adenosyl--methionine typical of antibiotic methyltransferases; the enzyme had a slightly higher affinity for carminomycin than for 13-dihydrocarminomycin. The reaction product from methylation of carminomycin was confirmed by chromatography and mass spectral analysis to be daunomycin. The purified enzyme did not catalyse methylation of the aglycones carminomycinone or 13-dihydrocarminomycinone. -Adenosyl--homocysteine inhibited the methyltransferase, whereas homocysteine, adenosine, adenine, ε-rhodomycinone, daunomycin, and daunomycinone showed little or no inhibitory activity.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-139-6-1353
1993-06-01
2021-10-25
Loading full text...

Full text loading...

/deliver/fulltext/micro/139/6/mic-139-6-1353.html?itemId=/content/journal/micro/10.1099/00221287-139-6-1353&mimeType=html&fmt=ahah

References

  1. Bartel P. L., Connors N. C., Strohl W. R. 1990; Biosynthesis of anthracyclines: analysis of mutants of Streptomyces sp. strain C5 blocked in daunomycin production.. Journal of General Microbiology 136:1877–1886
    [Google Scholar]
  2. Bauer N. J., Kreuzman A. J., Dotzlaf J. E., Yeh W.-K. 1988; Purification, characterization, and kinetic mechanism of S-adenosyl-l-methionine: macrocin O-methyltransferase from Streptomyces fradiae . Journal of Biological Chemistry 263:15619–15625
    [Google Scholar]
  3. Bradford M. M. 1976; A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.. Analytical Biochemistry 72:248–254
    [Google Scholar]
  4. Brazhnikova M. G., Abarsky V. B., Ponomarenko V. I., Potapova N. P. 1974; Physical and chemical characteristics and structure of carminomycin, a new antitumor antibiotic.. Journal of Antibiotics 27:254–259
    [Google Scholar]
  5. Connors N. C., Bartel P. L., Strohl W. R. 1990a; Biosynthesis of anthracyclines: enzymic conversion of aklanonic acid to aklavinone and ε-rhodomycinone by anthracycline-producing streptomycetes.. Journal of General Microbiology 136:1887–1894
    [Google Scholar]
  6. Connors N. C., Bartel P. L., Strohl W. R. 1990b; Biosynthesis of anthracyclines: carminomycin 4-O-methyltransferase, the terminal enzymic step in the formation of daunomycin.. Journal of General Microbiology 136:1895–1898
    [Google Scholar]
  7. Corcoran J. W. 1975; S-Adenosyl-l-methionine: erythromycin O-methyltransferase.. Methods in Enzymology 43:487–498
    [Google Scholar]
  8. Dekleva M. L., Titus J. A., Strohl W. R. 1985; Nutrient effects on anthracycline production by Streptomyces peucetius in a defined medium.. Canadian Journal of Microbiology 31:287–294
    [Google Scholar]
  9. Eckardt K., Wagner C. 1988; Biosynthesis of anthracyclinones.. Journal of Basic Microbiology 28:137–144
    [Google Scholar]
  10. Fawaz F., Jones G. H. 1988; Actinomycin synthesis in Streptomyces antibioticus. Purification and properties of a 3-hydroxyanthra- nilate 4-methyltransferase.. Journal of Biological Chemistry 263:4602–4606
    [Google Scholar]
  11. Haydock S. F., Dowson J. A., Dhillon N., Roberts G. A., Cortes J., Leadlay P. F. 1991; Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases.. Molecular and General Genetics 230:120–128
    [Google Scholar]
  12. Kreuzman A. J., Turner J. R., Yeh W.-K. 1988; Two distinctive Omethyltransferases catalyzing penultimate and terminal reactions of macrolide antibiotic (tylosin) biosynthesis. Substrate specificity, enzyme inhibition, and kinetic mechanism.. Journal of Biological Chemistry 263:15626–15633
    [Google Scholar]
  13. Lacalle R. A., Ruiz D., Jiménez A. 1991; Molecular analysis of the dmpM gene encoding an O-demethyl puromycin O-methyl-transferase from Streptomyces alboniger.. Gene 109:55–61
    [Google Scholar]
  14. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature; London: 227680–685
    [Google Scholar]
  15. Mcguire J. C., Thomas M. C., Stroshane R. M., Hamilton B. K., White R. J. 1980; Biosynthesis of daunorubicin glycosides: role of ε-rhodomycinone.. Antimicrobial Agents and Chemotherapy 18:454–464
    [Google Scholar]
  16. Morrissey J. H. 1981; Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity.. Analytical Biochemistry 117:307–310
    [Google Scholar]
  17. Otten S. L., Stutzman-Engwall K. J., Hutchinson C. R. 1990; Cloning and expression of daunorubicin biosynthesis genes from Streptomyces peucetius and S. peucetius subsp.caesius.. Journal of Bacteriology 172:3427–3434
    [Google Scholar]
  18. Pogell B. M. 1975; S-Adenosyl-l-methionine: O-demethylpuromycin O-methyltransferase.. Methods in Enzymology 43:508–515
    [Google Scholar]
  19. Pohl T. 1990; Concentration of proteins and removal of solutes.. Methods in Enzymology 182:68–83
    [Google Scholar]
  20. Rao M. M., Rebello P. F., Pogell B. M. 1969; Biosynthesis of puromycin in Streptomyces alboniger: enzymatic methylation of O-demethylpuromycin.. Journal of Biological Chemistry 244:112–118
    [Google Scholar]
  21. Speedie M. K., Hornemann U., Floss H. G. 1975; S-Adenosyl-l-methionine: indolepyruvate 3-methyltransferase.. Methods in Enzymology 75:498–502
    [Google Scholar]
  22. Strohl W. R., Connors N. C. 1992; Significance of anthra- quinone formation resulting from the cloning of actinorhodin genes in heterologous streptomycetes.. Molecular Microbiology 6:147–152
    [Google Scholar]
  23. Stutzman-Engwall K. J., Hutchinson C. R. 1989; Multigene families for anthracycline antibiotic production in Streptomyces peucetius.. Proceedings of the National Academy of Sciences of the United States of America 86:3135–3139
    [Google Scholar]
  24. Thompson M. W., Strohl W. R., Floss H. G. 1992; Purification and characterization of TDP-d-glucose 4,6-dehydratase from anthracycline producing streptomycetes.. Journal of General Microbiology 138:779–786
    [Google Scholar]
  25. Vara J. A., Hutchinson C. R. 1988; Purification of thymidine-diphospho-d-glucose 4,6-dehydratase from an erythromycin-producing strain of Saccharopolyspora erythraea by high resolution liquid chromatography.. Journal of Biological Chemistry 263:14992–14995
    [Google Scholar]
  26. Yabe K., Ando Y., Hashimoto J., Hamasaki T. 1989; Two distinct O-methyltransferases in aflatoxin biosynthesis.. Applied and Environmental Microbiology 55:2172–2177
    [Google Scholar]
  27. Zollner H. 1990 Handbook of Enzyme Inhibitors New York: VCH Publishers.;
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-139-6-1353
Loading
/content/journal/micro/10.1099/00221287-139-6-1353
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error