Biochemistry (Moscow)

, Volume 77, Issue 11, pp 1258–1265 | Cite as

Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus

  • S. M. Elizarov
  • M. G. Alekseeva
  • F. N. Novikov
  • G. G. Chilov
  • D. A. Maslov
  • A. A. Shtil
  • V. N. Danilenko
Article

Abstract

We demonstrate for the first time the role of phosphorylation in the regulation of activities of enzymes responsible for inactivation of aminoglycoside antibiotics. The aminoglycoside phosphotransferase VIII (APHVIII) from the actinobacterial strain Streptomyces rimosus ATCC 10970 is an enzyme regulated by protein kinases. Two serine residues in APHVIII are shown to be phosphorylated by protein kinases from extracts of the kanamycin-resistant strain S. rimosus 683 (a derivative of strain ATCC 10970). Using site-directed mutagenesis and molecular modeling, we have identified the Ser146 residue in the activation loop of the enzyme as the key site for Ca2+-dependent phosphorylation of APHVIII. Comparison of the kanamycin kinase activities of the unphosphorylated and phosphorylated forms of the initial and mutant APHVIII shows that the Ser146 modification leads to a 6–7-fold increase in the kanamycin kinase activity of APHVIII. Thus, Ser146 in the activation loop of APHVIII is crucial for the enzyme activity. The resistance of bacterial cells to kanamycin increases proportionally. From the practical viewpoint, our results increase prospects for creation of highly effective test systems for selecting inhibitors of human and bacterial serine/threonine protein kinases based on APHVIII constructs and corresponding human and bacterial serine/threonine protein kinases.

Key words

aminoglycoside phosphotransferase serine/threonine protein kinases antibiotic resistance phosphorylation sites site-directed mutagenesis 

Abbreviations

APHVIII

aminoglycoside phosphotransferase VIII

DTT

dithiothreitol

EGTA

sodium ethylene glycol tetraacetate

MD

molecular dynamics

PKA

protein kinase A

PKC

protein kinase C

STPKs

serine/threonine protein kinases

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References

  1. 1.
    Kannan, N., Taylor, S. S., Zhai, Y., Venter, J. C., and Manning, G. (2007) PLoS Biol., 5, 0466–0478.CrossRefGoogle Scholar
  2. 2.
    Davies, J., and Wright, G. D. (1997) Trends Microbiol., 5, 234–240.PubMedCrossRefGoogle Scholar
  3. 3.
    Wright, G. D. (2007) Nat. Rev. Microbiol., 5, 175–186.PubMedCrossRefGoogle Scholar
  4. 4.
    Sizova, I. A., Lapina, T. V., Frolova, O. N., Alexandrova, N. N., Akopiants, K. E., and Danilenko, V. N. (1996) Gene, 181, 13–18.PubMedCrossRefGoogle Scholar
  5. 5.
    Sizova, I. A., Hegemann, P., Furman, M., and Danilenko, V. N. (2002) Mol. Biol. (Moscow), 36, 18–25.Google Scholar
  6. 6.
    Danilenko, V. N., and Akopiants, K. E. (1995) in Proc. 9th Int. Symp. on Biology of Actinomycetes, Moscow, pp. 104–112.Google Scholar
  7. 7.
    Daigle, D. M., McKay, G. A., Thompson, P. R., and Wright, G. D. (1999) Chem. Biol., 6, 11–18.PubMedCrossRefGoogle Scholar
  8. 8.
    Shakya, T., Stogios, P. J., Waglechner, N., Evdokimova, E., Ejim, L., Blanchard, J. E., McArthur, A. G., Savchenko, A., and Wright, G. D. (2011) Chem. Biol., 18, 1591–1601.PubMedCrossRefGoogle Scholar
  9. 9.
    Fong, D. H., Xiong, B., Hwang, J., and Berghuis, A. M. (2011) PLoS One, 6, e19589.PubMedCrossRefGoogle Scholar
  10. 10.
    Scheeff, E. D., and Bourne, P. E. (2005) PLoS Comput. Biol., 1, 0359–0381.CrossRefGoogle Scholar
  11. 11.
    Kennely, P. (2002) FEMS Microbiol. Lett., 206, 1–8.CrossRefGoogle Scholar
  12. 12.
    Narayan, A., Sachdeva, P., Sharma, K., Saini, A., Tyagi, A., and Singh, Y. (2007) Physiol. Genomics, 29, 66–75.PubMedGoogle Scholar
  13. 13.
    Pereira, S. F., Goss, L., and Dworkin, J. (2011) Microbiol. Mol. Biol. Rev., 75, 192–212.PubMedCrossRefGoogle Scholar
  14. 14.
    Bekker, O., Elizarov, S. M., Alekseeva, M. G., Lyubimova, I., and Danilenko, V. (2008) Mikrobiologiya, 77, 630–638.Google Scholar
  15. 15.
    Elizarov, S. M., Sergienko, O. V., Sizova, I. A., and Danilenko, V. N. (2005) Mol. Biol. (Moscow), 39, 255–263.Google Scholar
  16. 16.
    Inoue, H., Nojima, H., and Okayama, H. (1990) Gene, 96, 23–28.PubMedCrossRefGoogle Scholar
  17. 17.
    Mierendorf, R., Yeager, K., and Novy, R. (1994) Newslett. Novagen Inc., 1, 1–3.Google Scholar
  18. 18.
    Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006) Bioinformatics, 22, 195–201.PubMedCrossRefGoogle Scholar
  19. 19.
    Berendsen, H. J. C., van der Spoel, D., and van Drunen, R. (1995) Comp. Phys. Commun., 91, 43–56.CrossRefGoogle Scholar
  20. 20.
    Lindahl, E., Hess, B., and van der Spoel, D. (2001) J. Mol. Model., 7, 306–317.Google Scholar
  21. 21.
    Nelson, R. M., and Long, G. L. (1989) Anal. Biochem., 180, 147–151.PubMedCrossRefGoogle Scholar
  22. 22.
    Elizarov, S. M., Mironov, V. A., and Danilenko, V. N. (2000) Life, 50, 139–143.PubMedGoogle Scholar
  23. 23.
    Hutchcroft, J. E., Anostario, M., Harrison, M. L., and Geahlen, R. L. (1991) Methods Enzymol., 200, 417–422.PubMedCrossRefGoogle Scholar
  24. 24.
    Kameshita, I., and Fujisawa, H. (1989) Anal. Biochem., 183, 139–143.PubMedCrossRefGoogle Scholar
  25. 25.
    Elizarov, S. M., and Danilenko, V. N. (2001) FEMS Microbiol. Lett., 202, 135–138.PubMedCrossRefGoogle Scholar
  26. 26.
    Azucena, E., and Mobashery, S. (2001) Drug. Resist. Updat., 4, 106–117.PubMedCrossRefGoogle Scholar
  27. 27.
    Nurizzo, D., Shewry, S. C., Perlin, M. H., Brown, S. A., Dholakia, J. N., Fuchs, R. L., Deva, T., Baker, E. N., and Smith, C. A. (2003) J. Mol. Biol., 327, 491–506.PubMedCrossRefGoogle Scholar
  28. 28.
    Taylor, S. S., Yang, J., Wu, J., et al. (2004) Biochim. Biophys. Acta, 1697, 259–269.PubMedCrossRefGoogle Scholar
  29. 29.
    Prisic, S., Dankwa, S., Schwartz, D., Chou, M. F., Locasale, J. W., Kang, C. M., Bemis, G., Church, G. M., Steen, H., and Husson, R. N. (2010) Proc. Natl. Acad. Sci. USA, 107, 7521–7526.PubMedCrossRefGoogle Scholar
  30. 30.
    Danilenko, V. N., Osolodkin, D. I., Lakatosh, S. A., Preobrazhenskaya, M. N., and Shtil, A. A. (2011) Curr. Topics Med. Chem., 11, 1352–1369.CrossRefGoogle Scholar
  31. 31.
    Danilenko, V., Simonov, A., Lakatosh, S., Kubbutat, M., Totzke, F., Schaechtele, C., Elizarov, S., Bekker, O., and Printsevskaya, S. (2008) J. Med. Chem., 51, 7731–7736.PubMedCrossRefGoogle Scholar
  32. 32.
    D’Costa, V. M., McGrann, K. M., Hughes, D. W., and Wright, G. D. (2006) Science, 311, 374–377.PubMedCrossRefGoogle Scholar
  33. 33.
    D’Costa, V. M., Griffiths, E., and Wright, G. D. (2007) Curr. Opin. Microbiol., 10, 481–489.PubMedCrossRefGoogle Scholar
  34. 34.
    Anizon, F., Shtil, A. A., Danilenko, V. N., and Moreau, P. (2010) Curr. Med. Chem., 17, 4114–4133.PubMedCrossRefGoogle Scholar
  35. 35.
    Bekker, O. B., Alekseeva, M. G., Osolodkin, D. I., Palyulin, V. A., Elizarov, S. M., Zefirov, N. S., and Danilenko, V. N. (2010) Acta Naturae, 2, 126–139.Google Scholar
  36. 36.
    Manteca, A., Mader, U., Connolly, B. A., and Sanchez, J. (2006) Proteomics, 6, 6008–6022.PubMedCrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2012

Authors and Affiliations

  • S. M. Elizarov
    • 1
    • 2
  • M. G. Alekseeva
    • 1
    • 3
  • F. N. Novikov
    • 4
  • G. G. Chilov
    • 4
  • D. A. Maslov
    • 1
    • 3
  • A. A. Shtil
    • 1
  • V. N. Danilenko
    • 1
  1. 1.Vavilov Institute of General GeneticsRussian Academy of SciencesMoscowRussia
  2. 2.Bach Institute of BiochemistryRussian Academy of SciencesMoscowRussia
  3. 3.Research Center of Biotechnology of Antibiotics and Other Biologically Active Substances “Bioan”MoscowRussia
  4. 4.Zelinsky Institute of Organic ChemistryRussian Academy of SciencesMoscowRussia

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