Archives of Pharmacal Research

, Volume 20, Issue 6, pp 590–596 | Cite as

Purification of the NADH reductase component of the steroid 9α-hydroxylase fromMycobacterium fortuitum

  • Hee Kyoung Kang
  • Sang Sup Lee
Research Articles


The NADH reductase component of the steroid 9α-hydroxylase fromMycobacterium fortuitum was purified to homogeneity. Recovery of the enzyme from the 50≈60% ammonium sulfate saturated fraction was 49%, with a purification factor of 100-fold. The NADH reductase has a relative molecular mass of 60 KDa as determined by SDS-PAGE. The absorption maxima at 410 and 450 nm indicate the presence of iron-sulfur group and flavin. These prosthetic groups seemed to function as redox groups that transfer electrons from NADH to the following protein. The KM value for NADH as substrate was 68 μM. The NH2-terminal amino acid sequence of the reductase was determined as Met-Asp-Ala-Ile-Thr-Asn-Val-Pro-Leu-Pro-Ala-Asn-Glu-Pro-Val-His-Asp-Tyr-Ala-Thr. This sequence does not show a homology with the NH2-terminal sequences reported for the reductase component of other monooxygenases, suggesting that the NADH reductase component of the steroid 9α-hydroxylase system is novel.

Key words

Steroid 9α-hydroxylase NADH reductase component Iron-sulfur group and flavin 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References Cited

  1. Axcell, B. C. and Geary, P. J., Purification and some properties of a soluble benzene oxidizing system from a strain ofPseudomonas.Biochem. J., 146, 173–183 (1975).PubMedGoogle Scholar
  2. Batie, C. J., Lahaie, E. and Ballou, D. P., Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase fromPseudomonas cepacia.J. Biol. Chem., 262, 1510–1518 (1987).PubMedGoogle Scholar
  3. Berg, A., Ingelman-Sundberg, M. and Gustafsson, J. A., Purification and characterization of cytochrome P450meg.J. Biol. Chem., 254, 5264–5271 (1979).PubMedGoogle Scholar
  4. Bernhardt, F. H., Pachowsky, H. and Staudinger, H. A 4-methoxybenzoate o-demethylase fromPseudomonas putida, a new type of monooxygenase system.Eur. J. Biochem., 57, 241–256 (1975).PubMedCrossRefGoogle Scholar
  5. Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities of protein u-tilizing the principle of protein-dye binding.Anal. Biochem., 72, 248–254 (1976).PubMedCrossRefGoogle Scholar
  6. Colby, J. and Dalton, H., Resolution of the methane monooxygenase ofMethylococcus capsulatus (Bath) into three components: purification and properties of component C, a flavoprotein.Biochem. J., 171, 461–468 (1978).PubMedGoogle Scholar
  7. Ensley, B. D., Gibson, D. T. and Liu, T. N., Oxidation of naphthalene by a multicomponent enzyme system fromPseudomonas sp. strain NCIB9816.J. Bacteriol., 149, 948–954 (1982).PubMedGoogle Scholar
  8. Fox, B. G., Froland, W. A., Dege, J. E. and Lipscomb, J. D., Methane monooxygenase fromMethylosinus trichosporium OB 3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph.J. Biol. Chem., 264, 10023–10033 (1989).PubMedGoogle Scholar
  9. Fox, B. G., Liu, Y., Dege, J. E. and Lipscomb, J. D., Complex formation between the protein components of methane monooxygenase fromMethylosinus trichosporium OB 3b.J. Biol. Chem., 266, 540–550 (1991).PubMedGoogle Scholar
  10. Frans, J. W., Willen, J. H., Van Berkel, Hartmans, S. and Jan, A. M., De Bont., Purification and properties of the NADH reductase component alkene monooxygenase fromMycobacterium strain E3.J. Bacteriol., 174, 3275–3281 (1992).Google Scholar
  11. Fulco, A. J., P450BM-3 and other inducible bacterial P 450 cytochromes: biochemistry and regulation.Ann. Rev. Pharmacol. Toxicol., 31, 177–203 (1991).CrossRefGoogle Scholar
  12. Geary, P. J., Saboowalla, F., Patil, D. and Cammack, R., An investigation of the iron-sulfur proteins of benzene dioxygenase fromPseudomonas putida by electron-spin-resonance spectroscopy.Biochem. J., 217, 667–673 (1984).PubMedGoogle Scholar
  13. Haigler, B. E. and Gibson, D. T., Purification and properties of the NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase fromPseudomonas sp. strain.J. Bacteriol., 172, 457–464 (1990).PubMedGoogle Scholar
  14. Hultquist, D. E.; Methemoglobin reduction system of erythrocytes, In Fleischer. S. and Packer, L. (Eds.),Methods in Enzymol., 52; Academic Press, New York, pp. 463–473, 1978.Google Scholar
  15. Kang, H. K., Isolation and partial purification of the steroid 9α-hydroxylase fromMycobacterium fortuitum. Yakhak hoeji, in press.Google Scholar
  16. Kang, H. K. and Lee, S. S., Heterogenous nature of the microbial steroid 9α-hydroxylase in nocardioforms.Arch. Pharm. Res., in press.Google Scholar
  17. Katagiri, M., Ganguli, B. N. and Gunsalus, I. C., A soluble cytochrome P450 functional role in methylene hydroxylation.J. Biol. Chem., 243, 3543–3546 (1968).PubMedGoogle Scholar
  18. Laemmli, U. K., Cleavage of the structural proteins during the assembly of the head of bacteriophage T4.Nature, 227, 68–685 (1970).CrossRefGoogle Scholar
  19. Narhi, L. O. and Fulco, A. J., Identification and characterization of two functional domains in cytochrome 450BM-3 a catalytically self-sufficient monooxygenase induced by barbiturates inBacillus megaterium.J. Biol. Chem., 262, 6683–6690 (1987).PubMedGoogle Scholar
  20. Patel, R. N., Methane monooxygenase: Purification and properties of flavoprotein component.Arch. Biochem. Biophys., 252, 229–236 (1987).PubMedCrossRefGoogle Scholar
  21. Pilkington, S. T. and Dalton, H., Purification and characterization of the soluble methane monooxygenase fromMethylosinus sporium 5 demonstrates the highly conserved nature of this enzyme in methanotrophs.FEMS Microbiol. Lett., 78, 103–108 (1991).CrossRefGoogle Scholar
  22. Shaw, J. P. and Harayama, S., Purification and characterization of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWWO ofPseudomonas putida mt-2.Biochem. 209, 51–61 (1992).Google Scholar
  23. Strijewski, A., The steroid 9α-hydroxylation system fromNocardia species.Eur. J. Biochem., 128, 125–135 (1982).PubMedGoogle Scholar
  24. Subramanian, V., Liu, T. N., Yeh, W. K., Narro, M. and Gibson, D. T., Purification and properties of NADH-ferredoxin-TOL reductase, a component of toluene dioxygenase fromPseudomonas putida.J. Biol. chem., 256, 2723–2730 (1981).PubMedGoogle Scholar
  25. Ueda, T. E., Lode, T. and Coon, M. J., Enzymatic ω-oxidation, VI. Isolation of homogeneous reduced diphosphopyridine nucleotide-rubredoxin reductase.J. Biol. Chem., 247, 2109–2116 (1972).Google Scholar
  26. Weber, F. J., Van Berkel, W. J. H., Hartmans, S. and De Bont, J. A. M., Purification and properties of the NADH reductase component of alkene monooxygenase fromMycobacterium strain E3.J. Bacteriol., 174, 3275–3281 (1992).PubMedGoogle Scholar
  27. Yamaguchi, M. and Fujisawa, H., Characterization of NADH-cytochrome c reductase, a component of benzoate 1,2-dioxygenase system fromPseudomonas arvilla c-1.J. Biol. Chem., 253, 8848–8853 (1978).PubMedGoogle Scholar
  28. Yeh, W. K., Gibson, D. T. and Liu, T. N., Toluene dioxygenase: a multicomponent enzyme system.Biochem. Biophys. Res. Commun., 78, 401–410 (1977).PubMedCrossRefGoogle Scholar

Copyright information

© The Pharmaceutical Society of Korea 1997

Authors and Affiliations

  • Hee Kyoung Kang
    • 1
  • Sang Sup Lee
    • 1
  1. 1.College of PharmacySeoul National UniversitySeoulKorea

Personalised recommendations