Get access

Deciphering interactions of the aminoglycoside phosphotransferase(3′)-IIIa with its ligands

Authors

  • Lingzhi Wu,

    1. Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville 37996, TN
    2. National Laboratory of Solid State Microstructure and Department of Physics, Nanjing University, No. 22, Hankou Road, Nanjing 210093, People's Republic of China
    Search for more papers by this author
  • Engin H. Serpersu

    Corresponding author
    1. Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville 37996, TN
    2. Graduate School of Genome Science and Technology, University of Tennessee and Oak Ridge National Laboratories, Knoxville 37996, TN
    • Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville 37996, TN
    Search for more papers by this author

Errata

This article is corrected by:

  1. Errata: Erratum: “Deciphering interactions of the aminoglycoside phosphotransferase(3')-IIIa with its ligands,” Biopolymers 91(9), 801–809, (2009) Volume 101, Issue 7, 819, Article first published online: 19 April 2014

Abstract

Aminoglycoside phosphotransferase(3′)-IIIa (APH) is the enzyme with broadest substrate range among the phosphotransferases that cause resistance to aminoglycoside antibiotics. In this study, the thermodynamic characterization of interactions of APH with its ligands are done by determining dissociation constants of enzyme–substrate complexes using electron paramagnetic resonance and fluorescence spectroscopy. Metal binding studies showed that three divalent cations bind to the apo-enzyme with low affinity. In the presence of AMPPCP, binding of the divalent cations occurs with 7-to-37-fold higher affinity to three additional sites dependent on the presence and absence of different aminoglycosides. Surprisingly, when both ligands, AMPPCP and aminoglycoside, are present, the number of high affinity metal binding sites is reduced to two with a 2-fold increase in binding affinity. The presence of divalent cations, with or without aminoglycoside present, shows only a small effect (<3-fold) on binding affinity of the nucleotide to the enzyme. The presence of metal–nucleotide, but not nucleotide alone, increases the binding affinity of aminoglycosides to APH. Replacement of magnesium (II) with manganese (II) lowered the catalytic rates significantly while affecting the substrate selectivity of the enzyme such that the aminoglycosides with 2′-NH2 become better substrates (higher Vmax) than those with 2′-OH. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 801–809, 2009.

This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Ancillary