The most common mode of bacterial resistance to aminoglycoside antibiotics is the enzyme-catalysed chemical modification of the drug. Over the last two decades, significant efforts in medicinal chemistry have been focused on the design of non- inactivable antibiotics. Unfortunately, this strategy has met with limited success on account of the remarkably wide substrate specificity of aminoglycoside-modifying enzymes. To understand the mechanisms behind substrate promiscuity, we have performed a comprehensive experimental and theoretical analysis of the molecular-recognition processes that lead to antibiotic inactivation by Staphylococcus aureus nucleotidyltransferase 4′(ANT(4′)), a clinically relevant protein. According to our results, the ability of this enzyme to inactivate structurally diverse polycationic molecules relies on three specific features of the catalytic region. First, the dominant role of electrostatics in aminoglycoside recognition, in combination with the significant extension of the enzyme anionic regions, confers to the protein/antibiotic complex a highly dynamic character. The motion deduced for the bound antibiotic seem to be essential for the enzyme action and probably provide a mechanism to explore alternative drug inactivation modes. Second, the nucleotide recognition is exclusively mediated by the inorganic fragment. In fact, even inorganic triphosphate can be employed as a substrate. Third, ANT(4′) seems to be equipped with a duplicated basic catalyst that is able to promote drug inactivation through different reactive geometries. This particular combination of features explains the enzyme versatility and renders the design of non-inactivable derivatives a challenging task.