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| | ==AadA in complex with ATP, magnesium and streptomycin== | | ==AadA in complex with ATP, magnesium and streptomycin== |
| - | <StructureSection load='6fzb' size='340' side='right' caption='[[6fzb]], [[Resolution|resolution]] 2.05Å' scene=''> | + | <StructureSection load='6fzb' size='340' side='right'caption='[[6fzb]], [[Resolution|resolution]] 2.05Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[6fzb]] is a 2 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6FZB OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6FZB FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[6fzb]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Salmonella_enterica_subsp._enterica_serovar_Typhimurium_str._LT2 Salmonella enterica subsp. enterica serovar Typhimurium str. LT2]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6FZB OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6FZB FirstGlance]. <br> |
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=ATP:ADENOSINE-5-TRIPHOSPHATE'>ATP</scene>, <scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=GOL:GLYCEROL'>GOL</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene>, <scene name='pdbligand=PEG:DI(HYDROXYETHYL)ETHER'>PEG</scene>, <scene name='pdbligand=SRY:STREPTOMYCIN'>SRY</scene></td></tr> | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.05Å</td></tr> |
| - | <tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[5g4a|5g4a]], [[5luh|5luh]], [[5lpa|5lpa]]</td></tr>
| + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=ATP:ADENOSINE-5-TRIPHOSPHATE'>ATP</scene>, <scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=GOL:GLYCEROL'>GOL</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene>, <scene name='pdbligand=PEG:DI(HYDROXYETHYL)ETHER'>PEG</scene>, <scene name='pdbligand=SRY:STREPTOMYCIN'>SRY</scene></td></tr> |
| - | <tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Streptomycin_3''-adenylyltransferase Streptomycin 3''-adenylyltransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.47 2.7.7.47] </span></td></tr>
| + | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=6fzb FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6fzb OCA], [https://pdbe.org/6fzb PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6fzb RCSB], [https://www.ebi.ac.uk/pdbsum/6fzb PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6fzb ProSAT]</span></td></tr> |
| - | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6fzb FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6fzb OCA], [http://pdbe.org/6fzb PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=6fzb RCSB], [http://www.ebi.ac.uk/pdbsum/6fzb PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=6fzb ProSAT]</span></td></tr> | + | |
| | </table> | | </table> |
| | + | == Function == |
| | + | [https://www.uniprot.org/uniprot/S3AD_SALTY S3AD_SALTY] Mediates bacterial resistance to the antibiotics streptomycin and spectinomycin, does not confer resistance to kanamycin (PubMed:26527143). Binds ATP first, then antibiotic (PubMed:26527143, PubMed:29871922).<ref>PMID:26527143</ref> <ref>PMID:29871922</ref> |
| | <div style="background-color:#fffaf0;"> | | <div style="background-color:#fffaf0;"> |
| | == Publication Abstract from PubMed == | | == Publication Abstract from PubMed == |
| - | Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). Here, the first crystal structure of an ANT(3'')(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5 A resolution. AadA consists of a nucleotidyltransferase domain and an alpha-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.
| + | 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. |
| | | | |
| - | Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3'')(9) adenyltransferase.,Chen Y, Nasvall J, Wu S, Andersson DI, Selmer M Acta Crystallogr D Biol Crystallogr. 2015 Nov 1;71(Pt 11):2267-77. doi:, 10.1107/S1399004715016429. Epub 2015 Oct 31. PMID:26527143<ref>PMID:26527143</ref>
| + | Structural mechanism of AadA, a dual specificity aminoglycoside adenyl transferase from Salmonella enterica.,Stern AL, Van der Verren SE, Kanchugal P S, Nasvall J, Gutierrez de Teran H, Selmer M J Biol Chem. 2018 Jun 5. pii: RA118.003989. doi: 10.1074/jbc.RA118.003989. PMID:29871922<ref>PMID:29871922</ref> |
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| | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> |
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| | __TOC__ | | __TOC__ |
| | </StructureSection> | | </StructureSection> |
| - | [[Category: Streptomycin 3''-adenylyltransferase]] | + | [[Category: Large Structures]] |
| - | [[Category: P, S Kanchugal]] | + | [[Category: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2]] |
| - | [[Category: Selmer, M]] | + | [[Category: Kanchugal P S]] |
| - | [[Category: Aminoglycoside adenyl transferase]] | + | [[Category: Selmer M]] |
| - | [[Category: Antibiotic resistance]]
| + | |
| - | [[Category: Transferase]]
| + | |
| Structural highlights
6fzb is a 2 chain structure with sequence from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
| | Method: | X-ray diffraction, Resolution 2.05Å |
| Ligands: | , , , , , |
| Resources: | FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT |
Function
S3AD_SALTY Mediates bacterial resistance to the antibiotics streptomycin and spectinomycin, does not confer resistance to kanamycin (PubMed:26527143). Binds ATP first, then antibiotic (PubMed:26527143, PubMed:29871922).[1] [2]
Publication Abstract from PubMed
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.
Structural mechanism of AadA, a dual specificity aminoglycoside adenyl transferase from Salmonella enterica.,Stern AL, Van der Verren SE, Kanchugal P S, Nasvall J, Gutierrez de Teran H, Selmer M J Biol Chem. 2018 Jun 5. pii: RA118.003989. doi: 10.1074/jbc.RA118.003989. PMID:29871922[3]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Chen Y, Nasvall J, Wu S, Andersson DI, Selmer M. Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3)(9) adenyltransferase. Acta Crystallogr D Biol Crystallogr. 2015 Nov 1;71(Pt 11):2267-77. doi:, 10.1107/S1399004715016429. Epub 2015 Oct 31. PMID:26527143 doi:http://dx.doi.org/10.1107/S1399004715016429
- ↑ Stern AL, Van der Verren SE, Kanchugal P S, Nasvall J, Gutierrez de Teran H, Selmer M. Structural mechanism of AadA, a dual specificity aminoglycoside adenyl transferase from Salmonella enterica. J Biol Chem. 2018 Jun 5. pii: RA118.003989. doi: 10.1074/jbc.RA118.003989. PMID:29871922 doi:http://dx.doi.org/10.1074/jbc.RA118.003989
- ↑ Stern AL, Van der Verren SE, Kanchugal P S, Nasvall J, Gutierrez de Teran H, Selmer M. Structural mechanism of AadA, a dual specificity aminoglycoside adenyl transferase from Salmonella enterica. J Biol Chem. 2018 Jun 5. pii: RA118.003989. doi: 10.1074/jbc.RA118.003989. PMID:29871922 doi:http://dx.doi.org/10.1074/jbc.RA118.003989
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