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Structural mechanism of AadA, a dual specificity aminoglycoside adenyl transferase from Salmonella enterica

DOI: 10.1074/jbc.RA118.003989 DOI Help

Authors: Ana Laura Stern (Uppsala University) , Sander Egbert Van Der Verren (VIB-VUB Centre for Structural Biology) , Sandesh Kanchugal Puttaswamy (Uppsala University) , Joakim Näsvall (Uppsala University) , Hugo Gutierrez De Teran (Uppsala University) , Maria Selmer (Uppsala University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: June 2018
Diamond Proposal Number(s): 11171

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) adenylyl transferase 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 wildtype 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, critically interacts 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 non-adenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin.

Journal Keywords: streptomycin; spectinomycin; ATP; enzyme structure; enzyme mechanism; antibiotics; antibiotic resistance; X-ray crystallography

Subject Areas: Biology and Bio-materials

Instruments: I03-Macromolecular Crystallography

Other Facilities: ESRF

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