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Structural mechanism of AadA, a dual specificity aminoglycoside adenylyltransferase from Salmonella enterica
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.ORCID iD: 0000-0002-6831-3105
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2018 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, p. 11481-11490Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2018. Vol. 293, p. 11481-11490
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biology with specialization in Structural Biology
Identifiers
URN: urn:nbn:se:uu:diva-353761DOI: 10.1074/jbc.RA118.003989ISI: 000439449700018PubMedID: 29871922OAI: oai:DiVA.org:uu-353761DiVA, id: diva2:1219024
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council, 2017-03827Swedish Research Council, 2013-05930EU, FP7, Seventh Framework Programme, 283570Available from: 2018-06-15 Created: 2018-06-15 Last updated: 2018-10-08Bibliographically approved

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Kanchugal, Sandesh PuttaswamyNäsvall, JoakimGutiérrez-de-Terán, HugoSelmer, Maria

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