NMR-Based Analysis of Aminoglycoside Recognition by the Resistance Enzyme ANT(4′): The Pattern of OH/NH3+ Substitution Determines the Preferred Antibiotic Binding Mode and is Critical for Drug Inactivation

  1. Revuelta, J. 1
  2. Vacas, T. 1
  3. Torrado, M. 1
  4. Corzana, F. 2
  5. Gonzalez, C. 3
  6. Jiménez-Barbero, J. 4
  7. Menendez, Margarita. 3
  8. Bastida, A. 1
  9. Asensio, J.L. 1
  1. 1 Instituto de Química Orgánica General
    info

    Instituto de Química Orgánica General

    Madrid, España

    ROR https://ror.org/05e0q7s59

  2. 2 Universidad de La Rioja
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    Universidad de La Rioja

    Logroño, España

    ROR https://ror.org/0553yr311

  3. 3 Instituto de Química Física Rocasolano
    info

    Instituto de Química Física Rocasolano

    Madrid, España

    ROR https://ror.org/03xk60j79

  4. 4 Centro de Investigaciones Biológicas
    info

    Centro de Investigaciones Biológicas

    Madrid, España

    ROR https://ror.org/04advdf21

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Revista:
Journal of the American Chemical Society

ISSN: 0002-7863

Any de publicació: 2008

Volum: 130

Número: 15

Pàgines: 5086-5103

Tipus: Article

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DOI: 10.1021/JA076835S PMID: 18366171 SCOPUS: 2-s2.0-42149169026 WoS: WOS:000254933000033 GOOGLE SCHOLAR

Altres publicacions en: Journal of the American Chemical Society

Resum

The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic inactivation of the drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4′) from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4′) displays a wide tolerance to conformational variations in the drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Ψ orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4′) against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4′) as aminoglycoside receptors. © 2008 American Chemical Society.