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. 1985 Mar;27(3):380–387. doi: 10.1128/aac.27.3.380

Isopenicillin N synthetase of Penicillium chrysogenum, an enzyme that converts delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N.

F R Ramos, M J López-Nieto, J F Martín
PMCID: PMC176281  PMID: 3922296

Abstract

The tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, an intermediate in the penicillin biosynthetic pathway, is converted to isopenicillin N by isopenicillin N synthetase (cyclase) of Penicillium chrysogenum. The cyclization required dithiothreitol and was stimulated by ferrous ions and ascorbate. Co2+ and Mn2+ completely inhibited enzyme activity. Optimal temperature and pH were 25 degrees C and 7.8, respectively. The reaction required O2 and was stimulated by increasing the dissolved oxygen concentration of the reaction mixture. Purification of the enzyme to a single major band in polyacrylamide gel electrophoresis was achieved by protamine sulfate precipitation, ammonium sulfate fractionation (50 to 80% of saturation), DEAE-Sephacel chromatography, and gel filtration on Sephacryl S-200. The estimated molecular weight was 39,000 +/- 1,000. The apparent Km of isopenicillin N synthetase for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine was 0.13 mM. The enzyme activity was strongly inhibited by glutathione, which acts as a competitive inhibitor. A good correlation was observed between the isopenicillin N synthetase activity in extracts of four different strains of P. chrysogenum (with widely different penicillin-producing capability) and the amount of penicillin production by these strains.

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Selected References

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  1. Adriaens P., Meesschaert B., Wuyts W., Vanderhaeghe H., Eyssen H. Presence of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine in fermentations of Penicillium chrysogenum. Antimicrob Agents Chemother. 1975 Dec;8(6):638–642. doi: 10.1128/aac.8.6.638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anné J., Peberdy J. F. Induced fusion of fungal protoplasts following treatment with polyethylene glycol. J Gen Microbiol. 1976 Feb;92(2):413–417. doi: 10.1099/00221287-92-2-413. [DOI] [PubMed] [Google Scholar]
  3. Chan J. A., Huang F. C., Sih C. J. The absolute configuration of the amino acids in delta-(alpha-aminoadipyl)cysteinylvaline from Penicillium chrysogenum. Biochemistry. 1976 Jan 13;15(1):177–180. doi: 10.1021/bi00646a027. [DOI] [PubMed] [Google Scholar]
  4. Hollander I. J., Shen Y. Q., Heim J., Demain A. L., Wolfe S. A pure enzyme catalyzing penicillin biosynthesis. Science. 1984 May 11;224(4649):610–612. doi: 10.1126/science.6546810. [DOI] [PubMed] [Google Scholar]
  5. Jensen S. E., Westlake D. W., Wolfe S. Cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to penicillins by cell-free extracts of Streptomyces clavuligerus. J Antibiot (Tokyo) 1982 Apr;35(4):483–490. doi: 10.7164/antibiotics.35.483. [DOI] [PubMed] [Google Scholar]
  6. Jensen S. E., Westlake D. W., Wolfe S. High performance liquid chromatographic assay of cyclization activity in cell-free systems from Streptomyces clavuligerus. J Antibiot (Tokyo) 1982 Aug;35(8):1026–1032. doi: 10.7164/antibiotics.35.1026. [DOI] [PubMed] [Google Scholar]
  7. Konomi T., Herchen S., Baldwin J. E., Yoshida M., Hunt N. A., Demain A. L. Cell-free conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine into an antibiotic with the properties of isopenicillin N in Cephalosporium acremonium. Biochem J. 1979 Nov 15;184(2):427–430. doi: 10.1042/bj1840427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kupka J., Shen Y. Q., Wolfe S., Demain A. L. Studies on the ring-cyclization and ring-expansion enzymes of beta-lactam biosynthesis in Cephalosporium acremonium. Can J Microbiol. 1983 May;29(5):488–496. doi: 10.1139/m83-078. [DOI] [PubMed] [Google Scholar]
  9. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  10. Luengo J. M., Revilla G., López M. J., Villanueva J. R., Martín J. F. Inhibition and repression of homocitrate synthase by lysine in Penicillium chrysogenum. J Bacteriol. 1980 Dec;144(3):869–876. doi: 10.1128/jb.144.3.869-876.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Luengo J. M., Revilla G., Villanueva J. R., Martín J. F. Lysine regulation of penicillin biosynthesis in low-producing and industrial strains of Penicillium chrysogenum. J Gen Microbiol. 1979 Nov;115(1):207–211. doi: 10.1099/00221287-115-1-207. [DOI] [PubMed] [Google Scholar]
  12. Martin-Zanca D. M., Martín J. F. Carbon catabolite regulation of the conversion of penicillin N into cephalosporin C. J Antibiot (Tokyo) 1983 Jun;36(6):700–708. doi: 10.7164/antibiotics.36.700. [DOI] [PubMed] [Google Scholar]
  13. Meesschaert B., Adriaens P., Eyssen H. Studies on the biosynthesis of isopenicillin N with a cell-free preparation of Penicillium chrysogenum. J Antibiot (Tokyo) 1980 Jul;33(7):722–730. doi: 10.7164/antibiotics.33.722. [DOI] [PubMed] [Google Scholar]
  14. Sawada Y., Baldwin J. E., Singh P. D., Solomon N. A., Demain A. L. Cell-free cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Antimicrob Agents Chemother. 1980 Sep;18(3):465–470. doi: 10.1128/aac.18.3.465. [DOI] [PMC free article] [PubMed] [Google Scholar]

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