Structural and molecular basis for resistance to aminoglycoside antibiotics by the adenylyltransferase ANT(2″)-Ia

Abstract

The aminoglycosides are highly effective broad-spectrum antimicrobial agents. However, their efficacy is diminished due to enzyme-mediated covalent modification, which reduces affinity of the drug for the target ribosome. One of the most prevalent aminoglycoside resistance enzymes in Gram-negative pathogens is the adenylyltransferase ANT(2″)-Ia, which confers resistance to gentamicin, tobramycin, and kanamycin. Despite the importance of this enzyme in drug resistance, its structure and molecular mechanism have been elusive. This study describes the structural and mechanistic basis for adenylylation of aminoglycosides by the ANT(2″)-Ia enzyme. ANT(2″)-Ia confers resistance by magnesium-dependent transfer of a nucleoside monophosphate (AMP) to the 2″-hydroxyl of aminoglycoside substrates containing a 2-deoxystreptamine core. The catalyzed reaction follows a direct AMP transfer mechanism from ATP to the substrate antibiotic. Central to catalysis is the coordination of two Mg(2+) ions, positioning of the modifiable substrate ring, and the presence of a catalytic base (Asp86). Comparative structural analysis revealed that ANT(2″)-Ia has a two-domain structure with an N-terminal active-site architecture that is conserved among other antibiotic nucleotidyltransferases, including Lnu(A), LinB, ANT(4')-Ia, ANT(4″)-Ib, and ANT(6)-Ia. There is also similarity between the nucleotidyltransferase fold of ANT(2″)-Ia and DNA polymerase β. This similarity is consistent with evolution from a common ancestor, with the nucleotidyltransferase fold having adapted for activity against chemically distinct molecules. IMPORTANCE : To successfully manage the threat associated with multidrug-resistant infectious diseases, innovative therapeutic strategies need to be developed. One such approach involves the enhancement or potentiation of existing antibiotics against resistant strains of bacteria. The reduction in clinical usefulness of the aminoglycosides is a particular problem among Gram-negative human pathogens, since there are very few therapeutic options for infections caused by these organisms. In order to successfully circumvent or inhibit the activity of aminoglycoside-modifying enzymes, and to thus rejuvenate the activity of the aminoglycoside antibiotics against Gram-negative pathogens, structural and mechanistic information is crucial. This study reveals the structure of a clinically prevalent aminoglycoside resistance enzyme [ANT(2″)-Ia] and depicts the molecular basis underlying modification of antibiotic substrates. Combined, these findings provide the groundwork for the development of broad-spectrum inhibitors against antibiotic nucleotidyltransferases.

FIG 1

(A) 2-DOS (4,6-disubstituted 2-deoxystreptamine)-based aminoglycoside antibiotics amenable to modification by the nucleotidyltransferase ANT(2″)-Ia (23); the 2″-OH site of modification is in red. (B) Crystal structure of gentamicin C1A (in yellow) in complex with a decoding A site oligonucleotide (in grey) (7). Hydrogen bonds between the 2″-OH of gentamicin and the RNA are shown as dashed lines.

FIG 2

Crystal structure of ANT(2″)-Ia. The nucleotidyltransferase (NT) fold is in black, and the C-terminal domain is in cyan; α-helices and β-sheets are labeled.

FIG 3

Molecular basis underlying adenylylation of kanamycin B by ANT(2″)-Ia. (A) Surface representation of ANT(2″)-Ia with kanamycin B present in the large cleft. Catalytic Mg²⁺ ions are shown as orange spheres. The 2″-OH is also labeled and the region of the protein involved in nucleotide binding is in blue (the ternary structure of LinB [36] was used to assist in the identification of residues that may be important in nucleotide binding). (B) Interactions between the ANT(2″)-Ia active site and kanamycin B. The catalytic base is labeled (Asp₈₆), and bond distance (Å) between the 2″-OH of kanamycin B and Asp₈₆ is also shown. Electron density for kanamycin B is a simulated annealing F_o-F_c map contoured at 2.0 σ, and the Mg²⁺ ions are as described above.

FIG 4

Conservation of the NT fold and catalytic architecture between ANT(2″)-Ia and nucleotidyltransferase enzymes. (A) Structures of ANT(2″)-Ia, lincosamide nucleotidyltransferase Lnu(A) (PDB ID: 4FO1), and DNA polymerase β (PDB ID: 2FMS) (37). The NT fold is in black, and the C-terminal domains are in light cyan. (B) Comparison of the catalytic architecture. Residues coordinating the Mg²⁺ ions are shown as sticks, Mg²⁺ ions and water molecules are shown as orange and red spheres, respectively, and dashed lines indicate hydrogen bonds. Kanamycin B (KanB), lincomycin (Lcm), and primer DNA are shown as sticks. DUP (2′-deoxyuridine-5′-α,β-imido-triphosphate) is shown as thin lines, and the α-phosphates are labeled. The modification site for each substrate is labeled.

FIG 5

Magnesium dependent adenylylation of gentamicin C2 by the aminoglycoside-modifying enzyme ANT(2″)-Ia.