Dissecting the cosubstrate structure requirements of the Staphylococcus aureus aminoglycoside resistance enzyme ANT(4′)

Vanessa R. Porter, Keith D. Green, Olga E. Zolova, Jacob L. Houghton, Sylvie Garneau-Tsodikova

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

Aminoglycosides are important antibiotics used against a wide range of pathogens. As a mechanism of defense, bacteria have evolved enzymes able to inactivate these drugs by regio-selectively adding a variety of functionalities (acetyl, phospho, and nucelotidyl groups) to their scaffolds. The aminoglycoside nucleotidyltransferase ANT(4′) is one of the most prevalent and unique modifying-enzymes. Here, by TLC, HRMS, and colorimetric assays, we demonstrate that the resistance enzyme ANT(4′) from Staphylococcus aureus is highly substrate and cosubstrate promiscuous. We show that deoxy-ribonucleotide triphosphates (dNTPs) are better cosubstrates than NTPs. We demonstrate that the position of the triphosphate group (5′ and not 3′) on the ribose/deoxyribose ring is important for recognition by ANT(4′), and that NTPs with larger substituents at the 3′-position of the ribose ring are not cosubstrates for ANT(4′). We confirm that for all aminoglycosides tested, the respective nucleotidylated products are completely inactive. These results provide valuable insights into the development of strategies to combat the ever-growing bacterial resistance problem.

Original languageEnglish
Pages (from-to)85-90
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume403
Issue number1
DOIs
StatePublished - Dec 3 2010

Bibliographical note

Funding Information:
This work was supported by start-up funds from the LSI and the College of Pharmacy at the University of Michigan and by a BSF Grant 2008017 (S.G.T.). V.R.P. is the recipient of a Rackham Merit Fellowship. J.L.H. is supported by ACS Division of Medicinal Chemistry Predoctoral Fellowship sponsored by Eli Lilly and a 2010 BSF travel grant for young scientists. We thank Dr. Micha Fridman (Tel Aviv University, Israel) for his help with MS and Wenjing Chen for helping with the bioTLCs. We thank Dr. Juan L. Asensio for the pANT(4′)-pET28b plasmid.

Keywords

  • Aminoglycoside
  • Antibacterial activity
  • Antibiotic
  • Bacterial resistance
  • Nucleotidyltransferase
  • Substrate and cosubstrate promiscuity

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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