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Structural Basis for Dual Nucleotide Selectivity of Aminoglycoside 2″-Phosphotransferase IVa Provides Insight on Determinants of Nucleotide Specificity of Aminoglycoside Kinases*

  • Kun Shi
    Footnotes
    Affiliations
    Department of Biochemistry, McGill University, Montreal, Quebec H3G, 0B1, Canada
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  • Albert M. Berghuis
    Correspondence
    Holds the Canada Research Chair in Structural Biology. To whom correspondence should be addressed: Dept. of Biochemistry and Department of Microbiology and Immunology. McGill University, 3649 Promenade Sir William Osler, Rm. 466, Montreal, Quebec H3G 0B1, Canada. Tel.: 514-398-8795; Fax: 514-398-2036;
    Affiliations
    Department of Biochemistry, McGill University, Montreal, Quebec H3G, 0B1, Canada

    Microbiology and Immunology, Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, Quebec H3G 0B1, Canada
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  • Author Footnotes
    * This work was supported by a grant from the Canadian Institute of Health Research (CIHR) (Grant MOP-13107) (to A. M. B.).
    This article contains supplemental Table S1 and Fig. S1.
    ♦ This article was selected as a Paper of the Week.
    1 Supported by the CIHR Strategic Training Initiative in Chemical Biology. Recipient of a CIHR Banting and Best Graduate Scholarship.
Open AccessPublished:February 24, 2012DOI:https://doi.org/10.1074/jbc.M112.349670
      Enzymatic phosphorylation through a family of enzymes called aminoglycoside O-phosphotransferases (APHs) is a major mechanism by which bacteria confer resistance to aminoglycoside antibiotics. Members of the APH(2″) subfamily are of particular clinical interest because of their prevalence in pathogenic strains and their broad substrate spectra. APH(2″) enzymes display differential preferences between ATP or GTP as the phosphate donor, with aminoglycoside 2″-phosphotransferase IVa (APH(2″)-IVa) being a member that utilizes both nucleotides at comparable efficiencies. We report here four crystal structures of APH(2″)-IVa, two of the wild type enzyme and two of single amino acid mutants, each in complex with either adenosine or guanosine. Together, these structures afford a detailed look at the nucleoside-binding site architecture for this enzyme and reveal key elements that confer dual nucleotide specificity, including a solvent network in the interior of the nucleoside-binding pocket and the conformation of an interdomain linker loop. Steady state kinetic studies, as well as sequence and structural comparisons with members of the APH(2″) subfamily and other aminoglycoside kinases, rationalize the different substrate preferences for these enzymes. Finally, despite poor overall sequence similarity and structural homology, analysis of the nucleoside-binding pocket of APH(2″)-IVa shows a striking resemblance to that of eukaryotic casein kinase 2 (CK2), which also exhibits dual nucleotide specificity. These results, in complement with the multitude of existing inhibitors against CK2, can serve as a structural basis for the design of nucleotide-competitive inhibitors against clinically relevant APH enzymes.

      Introduction

      Aminoglycosides form an important class of bactericidal antibiotics in therapeutic use today. However, bacterial resistance against nearly all known aminoglycosides persistently emerges, which poses a serious clinical threat in cases of pathogenic species (
      • Smith C.A.
      • Baker E.N.
      Aminoglycoside antibiotic resistance by enzymatic deactivation.
      ). A major mechanism of resistance to aminoglycoside antibiotics is the covalent addition of functional groups by a large repertoire of proteins collectively referred to as the aminoglycoside-modifying enzymes. Among them, aminoglycoside O-phosphotransferases specialize in the phosphorylation of specific hydroxyl groups and thereby prevent this class of antibiotics from effectively binding to their intended ribosomal target (
      • Chow J.W.
      Aminoglycoside resistance in enterococci.
      ).
      Several subfamilies of APH
      The abbreviations used are: APH
      aminoglycoside O-phosphotransferase
      APH(2″)-IVa
      aminoglycoside 2″-phosphotransferase IVa
      APH(3′)-IIIa
      3′,5″-aminoglycoside phosphotransferase type IIIa
      CK2
      casein kinase 2
      AMPPNP
      adenosine 5′-(β,γ-imino)triphosphate
      GMPPNP
      guanosine 5′-(β,γ-imino)triphosphate
      r.m.s.
      root mean square.
      enzymes have been discovered, and together they are capable of detoxifying aminoglycosides of diverse chemical structures. Members within the same subfamily, although generally sharing significant sequence similarity and structural homology, often exhibit distinctly different aminoglycoside preferences (
      • Toth M.
      • Chow J.W.
      • Mobashery S.
      • Vakulenko S.B.
      Source of phosphate in the enzymic reaction as a point of distinction among aminoglycoside 2"-phosphotransferases.
      ). Despite diverging antibiotic substrate profiles, all known APHs share a limited set of phosphate donors, namely ATP or GTP. Structurally, this implies that although the aminoglycoside-binding site shows wide variations among the APHs, the nucleotide-binding site is comparatively more conserved. Therefore, the latter has been considered an attractive target for the development of small molecule inhibitors that would ideally be active against a broad range of APHs and could thus serve as adjuvants in combination therapy with existing aminoglycosides (
      • Burk D.L.
      • Berghuis A.M.
      Protein kinase inhibitors and antibiotic resistance.
      ).
      Although some nucleotide-competitive kinase inhibitors have also been shown to inhibit a number of APHs, the prognostic for developing a pan-APH inhibitor is poor due to significant structural divergences among different APH subfamilies (
      • Fong D.H.
      • Xiong B.
      • Hwang J.
      • Berghuis A.M.
      Crystal structures of two aminoglycoside kinases bound with a eukaryotic protein kinase inhibitor.
      ). This does not imply, however, that a common inhibitor against a smaller subset of APHs, such as those belonging to either the APH(3′) or the APH(2″) subfamily, is impossible to find. Such an inhibitor would harbor significant clinical potential because these two subfamilies, beside comprising over half of all known APH enzymes, are both characterized by a broad antibiotic substrate spectrum and contain some of the most prevalent resistance enzymes found in clinical isolates worldwide (
      • Yadegar A.
      • Sattari M.
      • Mozafari N.A.
      • Goudarzi G.R.
      Prevalence of the genes encoding aminoglycoside-modifying enzymes and methicillin resistance among clinical isolates of Staphylococcus aureus in Tehran, Iran.
      ,
      • Chandrakanth R.K.
      • Raju S.
      • Patil S.A.
      Aminoglycoside resistance mechanisms in multidrug-resistant Staphylococcus aureus clinical isolates.
      ,
      • Zarrilli R.
      • Tripodi M.F.
      • Di Popolo A.
      • Fortunato R.
      • Bagattini M.
      • Crispino M.
      • Florio A.
      • Triassi M.
      • Utili R.
      Molecular epidemiology of high-level aminoglycoside-resistant enterococci isolated from patients in a university hospital in southern Italy.
      ).
      A divide between APH(3′) and APH(2″) enzymes lies in their nucleotide specificity. Members of the APH(3′) subfamily are ATP-specific, whereas members of the APH(2″) subfamily are also able to use GTP. Currently, the crystal structures of several APHs in complex with ATP-analogues have been determined (
      • Young P.G.
      • Walanj R.
      • Lakshmi V.
      • Byrnes L.J.
      • Metcalf P.
      • Baker E.N.
      • Vakulenko S.B.
      • Smith C.A.
      The crystal structures of substrate and nucleotide complexes of Enterococcus faecium aminoglycoside-2"-phosphotransferase-IIa (APH(2")-IIa) provide insights into substrate selectivity in the APH(2") subfamily.
      ,
      • Nurizzo D.
      • Shewry S.C.
      • Perlin M.H.
      • Brown S.A.
      • Dholakia J.N.
      • Fuchs R.L.
      • Deva T.
      • Baker E.N.
      • Smith C.A.
      The crystal structure of aminoglycoside-3′-phosphotransferase-IIa, an enzyme responsible for antibiotic resistance.
      ,
      • Fong D.H.
      • Lemke C.T.
      • Hwang J.
      • Xiong B.
      • Berghuis A.M.
      Structure of the antibiotic resistance factor spectinomycin phosphotransferase from Legionella pneumophila.
      ), but APH structures with a bound GTP analog remain elusive. Among aminoglycoside phosphotransferases, APH(2″)-IVa stands out with its nearly identical catalytic efficiencies with either ATP or GTP, which presents the opportunity of contrasting ATP versus GTP binding at the same active site and elucidating key structural features that influence nucleotide specificity.
      We have previously reported the apo and aminoglycoside-bound structures of APH(2″)-IVa (
      • Shi K.
      • Houston D.R.
      • Berghuis A.M.
      Crystal structures of antibiotic-bound complexes of aminoglycoside 2"-phosphotransferase IVa highlight the diversity in substrate binding modes among aminoglycoside kinases.
      ), and kinetic parameters had also been established for this enzyme (
      • Toth M.
      • Frase H.
      • Antunes N.T.
      • Smith C.A.
      • Vakulenko S.B.
      Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase-2"-IVa.
      ). Here, we present four nucleoside-bound crystal structures of wild type and mutant APH(2″)-IVa. These structures shed light on the detailed binding patterns of the different nucleoside substrates and explain the nucleotide specificity of this enzyme. These results, especially in complement with existing data on the nucleotide-bound structures of other APH enzymes, inform avenues for the rational design of small molecule inhibitors that can potentially target multiple subfamilies of aminoglycoside phosphotransferases.

      Acknowledgments

      We thank Dr. Joseph Chow for providing us with the aph(2″)-IVa gene. We also thank past and present members of the Berghuis laboratory, in particular John Ozcelik, for assistance and helpful discussions.

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