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. DanilenkoEmail author


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 



aminoglycoside phosphotransferase VIII




sodium ethylene glycol tetraacetate


molecular dynamics


protein kinase A


protein kinase C


serine/threonine protein kinases


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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
    Email author
  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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