Advertisement

Applied Biochemistry and Biotechnology

, Volume 172, Issue 8, pp 3964–3977 | Cite as

Kinetic and Spectroscopic Characterization of 1-Naphthol 2-hydroxylase from Pseudomonas sp. Strain C5

  • Vikas D. Trivedi
  • Prabin Majhi
  • Prashant S. PhaleEmail author
Article

Abstract

1-Naphthol 2-hydroxylase (1-NH) catalyzes the conversion of 1-naphthol to 1,2-dihydroxynaphthalene. 1-NH from carbaryl degrading Pseudomonas strain C5 was purified and characterized for its kinetic and spectroscopic properties. The enzyme was found to be NAD(P)H-dependent external flavin monooxygenase. Though the kinetic parameters of 1-NH from strain C5 appear to be similar to 1-NH enzyme from strains C4 and C6, however, they differ in their N-terminal sequences, mole content of flavin adenine dinucleotide (FAD), reconstitution of apoenzyme, and K i. 1-NH showed narrow substrate specificity with comparable hydroxylation efficiency on 1-naphthol and 5-amino 1-naphthol (~30 %) followed by 4-chloro 1-naphthol (~9 %). Salicylate was found to be the nonsubstrate effector. The flavin fluorescence of 1-NH was found to increase in the presence of 1-naphthol (K d = 11.3 μM) and salicylate (K d = 1027 μM). The circular dichroism (CD) spectra showed significant perturbations in the presence of NAD(P)H, whereas no changes were observed in the presence of 1-naphthol. Naphthalene, 1-chloronaphthalene, 2-napthol, and 2-naphthoic acid were found to be the mixed inhibitors. Chemical modification studies showed the probable involvement of His, Cys, and Tyr in the binding of 1-naphthol, whereas Trp was found to be involved in the binding of NAD(P)H.

Keywords

1-Naphthol 2-hydroxylase Flavoenzyme Hydroxylation efficiency Nonsubstrate effector Spectroscopic characterization Pseudomonas Carbaryl metabolism 

Abbreviations

MSM

Minimal salt medium

1-NH

1-Naphthol 2-hydroxylase

DEPC

Diethyl pyrocarbonate

NEM

N-ethyl maleimide

PGO

Phenylglyoxal

NAI

N-acetyl imidazole

NBS

N-bromosuccinimide

TLC

Thin layer chromatography

Notes

Acknowledgments

Thanks are due to DBT, Govt. of India for research grant to PP and CSIR, Govt. of India for Senior Research fellowship to VDT.

References

  1. 1.
    Dasgupta, R., Chakravorty, P. P., & Kaviraj, A. (2012). Journal of Environmental Science and Health. Part. B, 47, 99–103.CrossRefGoogle Scholar
  2. 2.
    Del, O. M., Laserna, J., Romero, D., Rohand, J., & Vilchez, J. L. (1997). Talanta, 44, 443–449.CrossRefGoogle Scholar
  3. 3.
    Perez-Ruiz, T., Martinez-Lozano, C., & Garcia, M. D. (2007). Journal of Chromatography. A, 1164, 174–180.CrossRefGoogle Scholar
  4. 4.
    Smulders, C. J., Bueters, T. J., Van Kleef, R. G., & Vijverberg, H. P. (2003). Toxicology and Applied Pharmacology, 193, 139–146.CrossRefGoogle Scholar
  5. 5.
    Swetha, V. P., & Phale, P. S. (2005). Applied and Environmental Microbiology, 71, 5951–5956.CrossRefGoogle Scholar
  6. 6.
    Sah, S., & Phale, P. S. (2011). Biodegradation, 22, 517–526.CrossRefGoogle Scholar
  7. 7.
    Swetha, V. P., Basu, A., & Phale, P. S. (2007). Journal of Bacteriology, 189, 2660–2666.CrossRefGoogle Scholar
  8. 8.
    Bradford, M. M. (1976). Analytical Biochemistry, 72, 248–254.CrossRefGoogle Scholar
  9. 9.
    Laemmli, U. K. (1970). Nature, 227, 680–685.CrossRefGoogle Scholar
  10. 10.
    Massey, V., & Curti, B. (1966). Journal of Biological Chemistry, 241, 3417–3423.Google Scholar
  11. 11.
    Louis-Jeune, C., Andrade-Navarro, M. A., & Perez-Iratxeta, C. (2011). Proteins, 80, 374–381.CrossRefGoogle Scholar
  12. 12.
    White-Stevens, R. H., & Kamin, H. (1970). Biochemical and Biophysical Research Communications, 38, 882–889.CrossRefGoogle Scholar
  13. 13.
    Whitby, L. G. (1953). Biochemical Journal, 54, 437–442.CrossRefGoogle Scholar
  14. 14.
    Kumagai, Y., Shinkai, Y., Miura, T., & Cho, A. K. (2012). Annual Review of Pharmacology and Toxicology, 52, 221–247.CrossRefGoogle Scholar
  15. 15.
    Patel, T. R., & Barnsley, E. A. (1980). Journal of Bacteriology, 143, 668–673.Google Scholar
  16. 16.
    Jadan, A. P., van Berkel, W. J., Golovleva, L. A., & Golovlev, E. L. (2001). Biochemistry (Moscow), 66, 898–903.CrossRefGoogle Scholar
  17. 17.
    Enroth, C., Neujahr, H., Schneider, G., & Lindqvist, Y. (1998). Structure, 6, 605–617.CrossRefGoogle Scholar
  18. 18.
    Elmorsi, E. A., & Hopper, D. J. (1977). European Journal of Biochemistry, 76, 197–208.CrossRefGoogle Scholar
  19. 19.
    Howell, L. G., & Massey, V. (1970). Biochemical and Biophysical Research Communications, 40, 887–893.CrossRefGoogle Scholar
  20. 20.
    Meneely, K. M., Barr, E. W., Bollinger, J. M., Jr., & Lamb, A. L. (2009). Biochemistry, 48, 4371–4376.CrossRefGoogle Scholar
  21. 21.
    Ohta, Y., Higgins, I., & Ribbons, D. W. (1975). Journal of Biological Chemistry, 250, 3814–3825.Google Scholar
  22. 22.
    Neujahr, H. Y., & Kjellen, K. G. (1978). Journal of Biological Chemistry, 253, 8835–8841.Google Scholar
  23. 23.
    Spector, T., & Massey, V. (1971). Biochemical and Biophysical Research Communications, 45, 1219–1226.CrossRefGoogle Scholar
  24. 24.
    Van, H. R., & Pirie, A. (1967). Biochemical Journal, 102, 842–852.CrossRefGoogle Scholar
  25. 25.
    Patel, T. R., & Gibson, D. T. (1974). Journal of Bacteriology, 119, 879–888.Google Scholar
  26. 26.
    Entsch, B., Ballou, D. P., & Massey, V. (1976). Journal of Biological Chemistry, 251, 2550–2563.Google Scholar
  27. 27.
    Hesp, B., Calvin, M., & Hosokawa, K. (1969). Journal of Biological Chemistry, 244, 5644–5655.Google Scholar
  28. 28.
    Sumathi, S. S., & Dasgupta, D. (2001). Biotechnology Progress, 17, 1026–1031.CrossRefGoogle Scholar
  29. 29.
    Howell, L. G., Spector, T., & Massey, V. (1972). Journal of Biological Chemistry, 247, 4340–4350.Google Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Vikas D. Trivedi
    • 1
  • Prabin Majhi
    • 1
  • Prashant S. Phale
    • 1
    Email author
  1. 1.Department of Biosciences and BioengineeringIndian Institute of Technology-BombayMumbaiIndia

Personalised recommendations