Structural Basis of APH(3′)-IIIa-Mediated Resistance to N1-Substituted Aminoglycoside Antibiotics

Butirosin is unique among the naturally occurring aminoglycosides, having a substituted amino group at position 1 (N1) of the 2-deoxystreptamine ring with an (S )-4-amino-2-hydroxybutyrate (AHB) group. While bacterial resistance to aminoglycosides can be ascribed chiefly to drug inactivation by plasmid-encoded aminoglycoside-modifying enzymes, the presence of an AHB group protects the aminoglycoside from binding to many resistance enzymes, and hence, the antibiotic retains its bactericidal properties. Consequently, several semisynthetic N1-substituted aminoglycosides, such as amikacin, isepamicin, and netilmicin, were developed. Unfortunately, butirosin, amikacin, and isepamicin are not resistant to inactivation by 3′-aminoglycosideO -phosphotransferase type IIIa [APH(3′)-IIIa]. We report here the crystal structure of APH(3′)-IIIa in complex with an ATP analog, AMPPNP [adenosine 5′-(β,γ-imido)triphosphate], and butirosin A to 2.4-Å resolution. The structure shows that butirosin A binds to the enzyme in a manner analogous to other 4,5-disubstituted aminoglycosides, and the flexible antibiotic-binding loop is key to the accommodation of structurally diverse substrates. Based on the crystal structure, we have also constructed a model of APH(3′)-IIIa in complex with amikacin, a commonly used semisynthetic N1-substituted 4,6-disubstituted aminoglycoside. Together, these results suggest a strategy to further derivatize the AHB group in order to generate new aminoglycoside derivatives that can elude inactivation by resistance enzymes while maintaining their ability to bind to the ribosomal A site.