Structural mechanism of AadA, a dual-specificity aminoglycoside adenylyltransferase from Salmonella enterica

Abstract

Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). AadA from Salmonella enterica is an aminoglycoside (3″)(9) adenylyltransferase that O-adenylates position 3″ of streptomycin and position 9 of spectinomycin. We previously reported the apo-AadA structure with a closed active site. To clarify how AadA binds ATP and its two chemically distinct drug substrates, we here report crystal structures of WT AadA complexed with ATP, magnesium, and streptomycin and of an active-site mutant, E87Q, complexed with ATP and streptomycin or the closely related dihydrostreptomycin. These structures revealed that ATP binding induces a conformational change that positions the two domains for drug binding at the interdomain cleft and disclosed the interactions between both domains and the three rings of streptomycin. Spectinomycin docking followed by molecular dynamics simulations suggested that, despite the limited structural similarities with streptomycin, spectinomycin makes similar interactions around the modification site and, in agreement with mutational data, forms critical interactions with fewer residues. Using structure-guided sequence analyses of ANT(3″)(9) enzymes acting on both substrates and ANT(9) enzymes active only on spectinomycin, we identified sequence determinants for activity on each substrate. We experimentally confirmed that Trp-173 and Asp-178 are essential only for streptomycin resistance. Activity assays indicated that Glu-87 is the catalytic base in AadA and that the nonadenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin.

Structural recognition of spectinomycin by resistance enzyme ANT(9) fromEnterococcus faecalis

“…Despite our observation that spectinomycin could bind in absence of ATP in the crystal structure, we could not observe spectinomycin binding in solution in absence of ATP (data not shown). This suggests that in solution, binding of ATP may position the two domains for interaction with the antibiotic substrate, as previously observed for AadA (15).…”

“…Previous site-directed mutagenesis of Asp-182 in AadA, the equivalent of Asp-180 in ANT (9), showed that the Asp182Asn mutation abolishes binding of spectinomycin to AadA while not affecting binding of ATP or streptomycin. In vivo, the Asp182Asn, Asp182Ala and also the Trp112Phe and Trp112Ala mutations are more detrimental for resistance to spectinomycin than streptomycin (15). In the present ANT (9) complex structures, only three amino acids, Glu-86, Asp-180 and Asn-183 ( Figure 2A), form direct hydrogen bonds to spectinomycin, thus explaining why Asp180 is critical for spectinomycin resistance.…”

“…Enzymes with activity on streptomycin contain a characteristic Asp-Val or Asp-Trp insertion in the loop after α5 ( Figure 3B). In this region, Trp-173 and Asp-178 were through site-directed mutagenesis shown to be critical for streptomycin binding and resistance, but not for spectinomycin (15).…”

“…Although spectinomycin is chemically dissimilar to the aminoglycoside streptomycin, both drugs can be adenylated by enzymes of the ANT(3")(9) (also called ANT(3")-Ia) class. Aiming to show how one of these, AadA from Salmonella enterica (13), recognizes the two chemically distinct molecules in the same active site, we previously solved crystal structures of AadA in its apo form and in complex with ATP, magnesium and streptomycin (14,15). We could explain how the enzyme coordinates ATP and streptomycin but failed to obtain a structure of AadA with spectinomycin.…”

“…Based on structure-guided sequence analysis, we could confirm that ANT(9) enzymes, which are only active on spectinomycin, are likely to display a similar overall structure. We could also identify sequence determinants that distinguish them from the ANT(3")(9) enzymes that are active on both antibiotics (15).…”