Biochemistry (Moscow)

, Volume 77, Issue 11, pp 1258–1265

Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus

Authors

  • S. M. Elizarov
    • Vavilov Institute of General GeneticsRussian Academy of Sciences
    • Bach Institute of BiochemistryRussian Academy of Sciences
  • M. G. Alekseeva
    • Vavilov Institute of General GeneticsRussian Academy of Sciences
    • Research Center of Biotechnology of Antibiotics and Other Biologically Active Substances “Bioan”
  • F. N. Novikov
    • Zelinsky Institute of Organic ChemistryRussian Academy of Sciences
  • G. G. Chilov
    • Zelinsky Institute of Organic ChemistryRussian Academy of Sciences
  • D. A. Maslov
    • Vavilov Institute of General GeneticsRussian Academy of Sciences
    • Research Center of Biotechnology of Antibiotics and Other Biologically Active Substances “Bioan”
  • A. A. Shtil
    • Vavilov Institute of General GeneticsRussian Academy of Sciences
    • Vavilov Institute of General GeneticsRussian Academy of Sciences
Article

DOI: 10.1134/S0006297912110041

Cite this article as:
Elizarov, S.M., Alekseeva, M.G., Novikov, F.N. et al. Biochemistry Moscow (2012) 77: 1258. doi:10.1134/S0006297912110041

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

Copyright information

© Pleiades Publishing, Ltd. 2012