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• W2805300175 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. 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. Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and interfere with protein synthesis through effects on decoding and translocation (for reviews, see Refs. 1.Vakulenko S.B. Mobashery S. Versatility of aminoglycosides and prospects for their future.Clin. Microbiol. Rev. 2003; 16 (12857776): 430-45010.1128/CMR.16.3.430-450.2003Crossref PubMed Scopus (475) Google Scholar, 2.Wilson D.N. Ribosome-targeting antibiotics and mechanisms of bacterial resistance.Nat. Rev. Microbiol. 2014; 12 (24336183): 35-4810.1038/nrmicro3155Crossref PubMed Scopus (587) Google Scholar, 3.Becker B. Cooper M.A. Aminoglycoside antibiotics in the 21st century.ACS Chem. Biol. 2013; 8 (23110460): 105-11510.1021/cb3005116Crossref PubMed Scopus (235) Google Scholar). They belong to the aminoglycoside and aminocyclitol families of antibiotics, both carrying amino groups that make the drugs positively charged, allowing their specific binding to negatively charged binding sites in 16S rRNA (4.Carter A.P. Clemons W.M. Brodersen D.E. Morgan-Warren R.J. Wimberly B.T. Ramakrishnan V. Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics.Nature. 2000; 407 (11014183): 340-34810.1038/35030019Crossref PubMed Scopus (1288) Google Scholar, 5.Borovinskaya M.A. Shoji S. Holton J.M. Fredrick K. Cate J.H.D. A steric block in translation caused by the antibiotic spectinomycin.ACS Chem. Biol. 2007; 2 (17696316): 545-55210.1021/cb700100nCrossref PubMed Scopus (103) Google Scholar). Streptomycin and spectinomycin are both in clinical use (6.Durante-Mangoni E. Grammatikos A. Utili R. Falagas M.E. Do we still need the aminoglycosides?.Int. J. Antimicrob. Agents. 2009; 33 (18976888): 201-20510.1016/j.ijantimicag.2008.09.001Crossref PubMed Scopus (154) Google Scholar) and included in the World Health Organization model list of essential medicines. The closely related derivative dihydrostreptomycin (dhs) 4The abbreviations used are: dhsdihydrostreptomycinANTaminoglycoside nucleotidyltransferaseITCisothermal titration calorimetryMDmolecular dynamicsr.m.s.d.root mean square deviationAMPCPPadenosine 5′-(α,β-methylene)triphosphateAMPNPPadenosine 5′-[(α,β)-imido]triphosphatesrystreptomycinMICminimum inhibitory concentrationBicineN,N-bis(2-hydroxyethyl)glycineOPLS-AAall-atom optimized potentials for liquid simulationsPDBProtein Data Bank. is only used in veterinary medicine due to its ototoxicity (7.Minkenhof J.E. Dihydrostreptomycine tegenaangewezen bij “lange” kuren. (Dihydrostreptomycin contraindicated in long courses of treatment.).Ned. Tijdschr. Geneeeskd. 1950; 94: 2129Google Scholar). dihydrostreptomycin aminoglycoside nucleotidyltransferase isothermal titration calorimetry molecular dynamics root mean square deviation adenosine 5′-(α,β-methylene)triphosphate adenosine 5′-[(α,β)-imido]triphosphate streptomycin minimum inhibitory concentration N,N-bis(2-hydroxyethyl)glycine all-atom optimized potentials for liquid simulations Protein Data Bank. Clinically, the most important resistance mechanism to these drugs is their inactivation by aminoglycoside-modifying enzymes, but resistance can also occur by mutations in the drug target or through mechanisms of decreased uptake or increased efflux (3.Becker B. Cooper M.A. Aminoglycoside antibiotics in the 21st century.ACS Chem. Biol. 2013; 8 (23110460): 105-11510.1021/cb3005116Crossref PubMed Scopus (235) Google Scholar, 8.Davies J. Wright G.D. Bacterial resistance to aminoglycoside antibiotics.Trends Microbiol. 1997; 5 (9211644): 234-24010.1016/S0966-842X(97)01033-0Abstract Full Text PDF PubMed Scopus (330) Google Scholar). Modification of streptomycin and spectinomycin by O-phosphorylation or O-nucleotidylation prevents the drugs from binding to their respective binding sites on the ribosome (for reviews, see Refs. 9.Ramirez M.S. Tolmasky M.E. Aminoglycoside modifying enzymes.Drug Resist. Updat. 2010; 13 (20833577): 151-17110.1016/j.drup.2010.08.003Crossref PubMed Scopus (812) Google Scholar and 10.Azucena E. Mobashery S. Aminoglycoside-modifying enzymes: mechanisms of catalytic processes and inhibition.Drug Resist. Updat. 2001; 4 (11512519): 106-11710.1054/drup.2001.0197Crossref PubMed Scopus (101) Google Scholar). Aminoglycoside (3″)(9) adenylyltransferase AadA from Salmonella enterica belongs to the ANT(3″)-Ia family (9.Ramirez M.S. Tolmasky M.E. Aminoglycoside modifying enzymes.Drug Resist. Updat. 2010; 13 (20833577): 151-17110.1016/j.drup.2010.08.003Crossref PubMed Scopus (812) Google Scholar) and catalyzes the magnesium-dependent O-adenylation of streptomycin and spectinomycin at positions 3″ and 9, respectively (11.Hollingshead S. Vapnek D. Nucleotide sequence analysis of a gene encoding a streptomycin/spectinomycin adenylyltransferase.Plasmid. 1985; 13 (2986186): 17-3010.1016/0147-619X(85)90052-6Crossref PubMed Scopus (137) Google Scholar) (Fig. 1). The two drugs are chemically dissimilar with streptomycin containing three O-linked rings with significant conformational freedom, whereas spectinomycin is a conformationally more restrained tricyclic molecule. We previously determined the apo crystal structure of S. enterica AadA at 2.5-Å resolution (12.Chen Y. Näsvall J. Wu S. Andersson D.I. Selmer M. Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3″)(9) adenyltransferase.Acta Crystallogr. D Biol. Crystallogr. 2015; 71 (26527143): 2267-227710.1107/S1399004715016429Crossref PubMed Scopus (13) Google Scholar). In the apo state, AadA has a monomeric two-domain structure with the active site located between an N-terminal adenylyltransferase domain and a C-terminal helical domain. In this inactive conformation, residues predicted to be involved in binding of ATP and magnesium were instead engaged in interdomain interactions. Using isothermal titration calorimetry (ITC), we demonstrated that magnesium and ATP had to bind prior to aminoglycoside substrate, suggesting that ATP binding triggered a conformational change, repositioning the two domains for substrate recognition. We now set out to characterize how AadA binds to ATP and its two different antibiotic substrates. We here present crystal structures of wild type (WT) AadA with ATP or ATP and streptomycin and of an active-site mutant in complex with ATP and streptomycin or dihydrostreptomycin. Using manual docking and molecular dynamics (MD) simulations, we present a model for how spectinomycin explores a partially different part of the active-site pocket. We also confirm the identity of the catalytic base using in vitro assays. Based on structure-based sequence analysis, we propose and experimentally verify sequence determinants that allow classification of ANT(3″)(9) enzymes and the related ANT(9) enzymes, which only mediate resistance to spectinomycin. Cocrystallization of WT AadA with ATP and magnesium yielded well-diffracting crystals. The crystals grew in a new space group, P32, with two molecules in the asymmetric unit, and the complete atomic structure could be built and refined to 1.9-Å resolution. The two molecules of AadA display close-to-identical structures (root mean square deviation (r.m.s.d.) of 0.1 Å over 261 Cα atoms). This structure represents a native state of the enzyme prior to binding of aminoglycoside substrate. To accommodate binding of ATP and magnesium, compared with the apo structure, the main part of the C-terminal domain rotates 11° relative to the N-terminal domain around the first helix of the C-terminal domain (helix 7), resulting in a shift of up to 4 Å (Fig. 2A). There are also local conformational changes of the 202–206 loop region directly linked to the interaction with ATP. The two parts of the structure are very similar to the apo structure (12.Chen Y. Näsvall J. Wu S. Andersson D.I. Selmer M. Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3″)(9) adenyltransferase.Acta Crystallogr. D Biol. Crystallogr. 2015; 71 (26527143): 2267-227710.1107/S1399004715016429Crossref PubMed Scopus (13) Google Scholar) (r.m.s.d. of 0.77 Å over 171 Cα atoms for residues 1–173 and 1.03 Å over 88 Cα atoms for residues 175–262). The structure shows clear electron density for ATP and two magnesium ions in the interdomain cleft (Fig. 2B). In the N-terminal domain, Asp-47, Asp-49, and Glu-87 together with the phosphates of ATP coordinate the two magnesium ions. In addition, the phosphates make interactions with Ser-36 and Ser-46 in the N-terminal domain and Arg-192, Lys-205, and Tyr-231 in the C-terminal domain. The ribose is in C2′ endo conformation with both hydroxyl groups involved in hydrogen bonding to Asp-130. The adenine base is in syn conformation, packing between Leu-133, Leu-166, Thr-189, Arg-192, Ile-193, and Phe-202, and makes a single hydrogen bond to Thr-196 of the protein. The interaction of Arg-192 with the bridging oxygen seems critical to obtain a magnesium-coordinating conformation of ATP because binding of nonhydrolyzable ATP analogues AMPCPP (12.Chen Y. Näsvall J. Wu S. Andersson D.I. Selmer M. Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3″)(9) adenyltransferase.Acta Crystallogr. D Biol. Crystallogr. 2015; 71 (26527143): 2267-227710.1107/S1399004715016429Crossref PubMed Scopus (13) Google Scholar) and AMPNPP (data not shown) could not be detected. In the apo crystal structure, ATP- and magnesium-binding residues of both domains were involved in interdomain interactions, explaining why that crystal form could not accommodate any ligand binding (12.Chen Y. Näsvall J. Wu S. Andersson D.I. Selmer M. Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3″)(9) adenyltransferase.Acta Crystallogr. D Biol. Crystallogr. 2015; 71 (26527143): 2267-227710.1107/S1399004715016429Crossref PubMed Scopus (13) Google Scholar). The magnesium- and phosphate-coordinating residues in the N-terminal domain are to a large extent conserved in other adenylyltransferase enzymes as well as polymerases and nucleases with two metal ions in the active site (12.Chen Y. Näsvall J. Wu S. Andersson D.I. Selmer M. Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3″)(9) adenyltransferase.Acta Crystallogr. D Biol. Crystallogr. 2015; 71 (26527143): 2267-227710.1107/S1399004715016429Crossref PubMed Scopus (13) Google Scholar, 13.Yang W. Lee J.Y. Nowotny M. Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity.Mol. Cell. 2006; 22 (16600865): 5-1310.1016/j.molcel.2006.03.013Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar) (see below). We can thus rationalize that some related structures, e.g. the structure of kanamycin nucleotidyltransferase in complex with AMPCPP (14.Pedersen L.C. Benning M.M. Holden H.M. Structural investigation of the antibiotic and ATP-binding sites in kanamycin nucleotidyltransferase.Biochemistry. 1995; 34 (7577914): 13305-1331110.1021/bi00041a005Crossref PubMed Scopus (148) Google Scholar), only display one magnesium ion because the bridging carbon of the ATP analogue affects the conformation of the phosphate tail in a manner preventing binding of a second magnesium ion. Previous experiments demonstrated that the E87Q mutant of AadA did not convey resistance to streptomycin but still bound ATP and streptomycin, although with 4- and 20-fold lower affinity than the WT enzyme (12.Chen Y. Näsvall J. Wu S. Andersson D.I. Selmer M. Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3″)(9) adenyltransferase.Acta Crystallogr. D Biol. Crystallogr. 2015; 71 (26527143): 2267-227710.1107/S1399004715016429Crossref PubMed Scopus (13) Google Scholar). We now cocrystallized this presumably catalytically inactive mutant with ATP, magnesium, and antibiotic substrates and obtained well diffracting crystals with streptomycin and dihydrostreptomycin. The crystals grew in the same space group and with similar cell dimensions as the WT crystals with ATP but only appeared in conditions containing calcium. The structure of AadA(E87Q) with ATP and streptomycin (AadA(E87Q)–ATP–sry) was refined to 1.73-Å resolution, and the structure of AadA(E87Q) with ATP and dihydrostreptomycin (AadA(E87Q)–ATP–dhs) was resolved to 1.4-Å resolution. Both structures are modeled with calcium ions instead of magnesium in the active site. The identity of divalent ion B was confirmed in an anomalous difference map (Fig. S1). The second ion (A) is tentatively modeled as calcium based on coordination distances and level of electron density. However, the identity of this ion does not affect any of our conclusions. Dihydrostreptomycin only differs from streptomycin in the reduction of the carbonyl group at the 3′-position (Fig. 1), and because this position does not show any interaction with the protein and the two structures are virtually identical (r.m.s.d. of 0.2 Å over 261 Cα atoms), further analysis will be based on the dihydrostreptomycin structure. The overall structure of AadA(E87Q)–ATP–dhs is very similar to AadA–ATP (r.m.s.d. of 0.3 Å over 252 Cα atoms). Dihydrostreptomycin is bound between the two domains of AadA with the C ring sandwiched between the two domains in proximity of ATP, whereas the B- and A rings extend further toward the surface and form their major interactions with the C-terminal domain (Fig. 3A). AadA hides 417 Å2 (58%) of the van der Waals area of dihydrostreptomycin and makes interactions with all three rings (Fig. 3B). The C ring is the site of modification and is situated in proximity of the suggested catalytic base Glu-87 (mutated to glutamine in the structure) with the 3″-hydroxyl situated in the closest position. Furthermore, the ring is also oriented through a hydrogen bond of Asp-182 with the 6″-hydroxyl group and a stacking interaction with Trp-112. In turn, the B ring is oriented by His-185 through a hydrogen bond with the 3′-hydroxyl group. The enzyme does not interact with the carbonyl at position 3′ of the B ring (Fig. 1), showing why AadA can bind and modify both streptomycin and dihydrostreptomycin. Finally, the A ring displays multiple interactions with the enzyme. Hydrogen bond interactions occur between Trp-173 and hydroxyl groups at positions 5 and 6, between the backbone carbonyl of Ala-177 and the guanidinium group at position 1, and between the backbone carbonyl of Asp-178 and the 6-hydroxyl group. The conformational change of the C-terminal domain relative to the N-terminal domain upon ATP binding (Fig. 2A) is essential to allow streptomycin to fit into the active site. Brief soaking of WT AadA–ATP crystals with streptomycin resulted in a 2.05-Å–resolution structure with clear density for ATP, magnesium, and the major part of streptomycin. The overall conformation of the enzyme is very similar to AadA–ATP (r.m.s.d. of 0.3 Å over 256 Cα atoms) and AadA(E87Q)–dhs (r.m.s.d. of 0.4 Å over 256 Cα atoms). Absence of electron density for part of the C ring (C2″, C3″, and the methylamine; Fig. 3C) suggests that this part of the substrate may move within the active site in connection with catalysis (further discussed below). Sequence analysis and structure comparison with other adenylyltransferase enzymes suggested that Glu-87 is the catalytic base in AadA. This was supported by minimum inhibitory concentration tests of streptomycin with WT AadA and single-amino acid substitution mutants E87Q and E87A (12.Chen Y. Näsvall J. Wu S. Andersson D.I. Selmer M. Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3″)(9) adenyltransferase.Acta Crystallogr. D Biol. Crystallogr. 2015; 71 (26527143): 2267-227710.1107/S1399004715016429Crossref PubMed Scopus (13) Google Scholar). To confirm the enzymatic in vitro activity of AadA toward streptomycin and to further investigate the role of Glu-87, in vitro activity of AadA was tested for the WT and mutant enzymes E87A and E87Q. In the enzymatic assay, pyrophosphatase converted the pyrophosphate by-product of the reaction to phosphate, which was subsequently detected by the malachite green assay. At the conditions assayed, the turnover for the WT enzyme was 0.020 ± 0.001 s−1. To our surprise, both mutant enzymes showed activity in this assay with turnover of 0.032 ± 0.003 s−1 for E87Q and 0.004 ± 0.001 s−1 for E87A. In the absence of streptomycin, none of the WT or mutant enzymes showed detectable activity. To distinguish whether the observed activity was a result of streptomycin adenylation or unproductive ATP hydrolysis, we developed a chromatographic assay to directly detect adenylated streptomycin. The positive charge of adenylated streptomycin allows its separation from negatively charged ATP and AMP by cation-exchange chromatography, and detection by absorbance at 260 nm, where unmodified streptomycin does not absorb. Our results showed that, in contrast to the WT enzyme, the E87Q mutant could not adenylate streptomycin (Fig. 4), consolidating the role of Glu-87 as essential for adenylation activity. However, in the absence of catalytic base, water can apparently act as a nucleophile, turning AadA into a hydrolytic enzyme. One such candidate water molecule is in the AadA–ATP–dhs structure coordinated between the magnesium ion, the 3″-hydroxyl of dihydrostreptomycin, and the α-phosphate of ATP (Fig. S2). We used the H++ server (15.Anandakrishnan R. Aguilar B. Onufriev A.V. H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations.Nucleic Acids Res. 2012; 40 (22570416): W537-W54110.1093/nar/gks375Crossref PubMed Scopus (991) Google Scholar) to computationally predict a pKa of 7.1 for Glu-87 in the ATP structures with magnesium (with or without streptomycin). This indicates that Glu-87 within the catalytic center has a pKa in the range where it could be reversibly protonated and abstract a proton from the 3″-hydroxyl of streptomycin. The adenyl transfer reaction by AadA is equivalent to the reactions carried out by well characterized two-metal-ion polymerases and nucleases that display very similar arrangements of residues in the active site (13.Yang W. Lee J.Y. Nowotny M. Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity.Mol. Cell. 2006; 22 (16600865): 5-1310.1016/j.molcel.2006.03.013Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). In analogy with these associative-mechanism enzymes, we predict that the catalytic base Glu-87 will abstract a proton from the 3″-hydroxyl group of streptomycin to allow the oxygen to make a nucleophilic attack on the α-phosphate of ATP. The geometry of the substrates in the active site is consistent with a single-in-line displacement mechanism as suggested for kanamycin nucleotidyltransferase (14.Pedersen L.C. Benning M.M. Holden H.M. Structural investigation of the antibiotic and ATP-binding sites in kanamycin nucleotidyltransferase.Biochemistry. 1995; 34 (7577914): 13305-1331110.1021/bi00041a005Crossref PubMed Scopus (148) Google Scholar) and LinB (16.Morar M. Bhullar K. Hughes D.W. Junop M. Wright G.D. Structure and mechanism of the lincosamide antibiotic adenylyltransferase LinB.Structure. 2009; 17 (20004168): 1649-165910.1016/j.str.2009.10.013Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), but the two substrates are too far apart to represent a catalytic state according to this mechanism. Thus, the nucleophilic oxygen would normally coordinate the magnesium ion and approach 2.5-Å distance of the ATP phosphorus atom during the reaction, whereas the position of our 3″-hydroxyl in AadA–ATP–sry is at 5.1 Å from the α-phosphorus. It seems necessary for the substrate to move, possibly linked to a local conformational change of the enzyme active site, to allow the deprotonated 3″-hydroxyl to coordinate the magnesium ion before nucleophilic attack. The weak electron density for the reacting part of the substrate in the AadA–ATP–sry structure supports dynamics in this part of the substrate. In our higher-resolution substrate structures, the active site in the presence of calcium is distorted from the native, magnesium-bound state through a cumulative effect of increased coordination distances. Binding of calcium to the first metal-binding site (B in Figs. 2B and 3B) increases the distances to coordinating atoms, and the octahedral coordination geometry becomes nonperfect (Fig. S3, A and B). This has an impact on the second magnesium site (A in Figs. 2B and 3B), which now has a nonoptimal configuration, leading to partial-occupancy binding of a divalent ion modeled as calcium (Fig. S3, C and D). In the WT structure, Glu-87 is positioned to coordinate both the magnesium and the substrate 3″-hydroxyl position, using the same side-chain oxygen in the manner observed in other adenylyltransferase structures (16.Morar M. Bhullar K. Hughes D.W. Junop M. Wright G.D. Structure and mechanism of the lincosamide antibiotic adenylyltransferase LinB.Structure. 2009; 17 (20004168): 1649-165910.1016/j.str.2009.10.013Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 17.Bassenden A.V. Rodionov D. Shi K. Berghuis A.M. Structural analysis of the tobramycin and gentamicin clinical resistome reveals limitations for next-generation aminoglycoside design.ACS Chem. Biol. 2016; 11 (26900880): 1339-134610.1021/acschembio.5b01070Crossref PubMed Scopus (19) Google Scholar), at 3.7-Å distance to the 3″-hydroxyl. According to the Pfam database (18.Finn R.D. Bateman A. Clements J. Coggill P. Eberhardt R.Y. Eddy S.R. Heger A. Hetherington K. Holm L. Mistry J. Sonnhammer E.L. Tate J. Punta M. Pfam: the protein families database.Nucleic Acids Res. 2014; 42 (24288371): D222-D23010.1093/nar/gkt1223Crossref PubMed Scopus (4116) Google Scholar) the two domains of AadA are classified as an NTP_transf_2 domain (PF01909) and a DUF4111 domain (PF13427). Of the 186 proteins classified in Pfam with this architecture, nine have been functionally characterized and can be divided into three classes. The first class of enzymes shows ANT(3″)(9) activity and confers resistance to streptomycin and spectinomycin (AadAs from S. enterica (11.Hollingshead S. Vapnek D. Nucleotide sequence analysis of a gene encoding a streptomycin/spectinomycin adenylyltransferase.Plasmid. 1985; 13 (2986186): 17-3010.1016/0147-619X(85)90052-6Crossref PubMed Scopus (137) Google Scholar), Enterococcus faecalis (19.Clark N.C. Olsvik O. Swenson J.M. Spiegel C.A. Tenover F.C. Detection of a streptomycin/spectinomycin adenylyltransferase gene (aadA) in Enterococcus faecalis.Antimicrob. Agents Chemother. 1999; 43 (9869582): 157-16010.1093/jac/43.1.157Crossref PubMed Scopus (65) Google Scholar), Escherichia coli (20.Shaw K.J. Rather P.N. Hare R.S. Miller G.H. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes.Microbiol. Rev. 1993; 57 (8385262): 138-163Crossref PubMed Google Scholar), Pseudomonas aeruginosa (21.Papadovasilaki M. Oberthür D. Gessmann R. Sarrou I. Betzel C. Scoulica E. Petratos K. Biophysical and enzymatic properties of aminoglycoside adenylyltransferase AadA6 from Pseudomonas aeruginosa.Biochem. Biophys. Rep. 2015; 4 (29124199): 152-15710.1016/j.bbrep.2015.09.011PubMed Google Scholar), and Serratia marcescens (22.Kim C. Hesek D. Zajícek J. Vakulenko S.B. Mobashery S. Characterization of the bifunctional aminoglycoside-modifying enzyme ANT(3″)-Ii/AAC(6′)-IId from Serratia marcescens.Biochemistry. 2006; 45 (16819836): 8368-837710.1021/bi060723gCrossref PubMed Scopus (48) Google Scholar)). The second class of enzymes shows ANT(9) activity and confers resistance to spectinomycin but not to streptomycin (ANT(9)s from E. faecalis (19.Clark N.C. Olsvik O. Swenson J.M. Spiegel C.A. Tenover F.C. Detection of a streptomycin/spectinomycin adenylyltransferase gene (aadA) in Enterococcus faecalis.Antimicrob. Agents Chemother. 1999; 43 (9869582): 157-16010.1093/jac/43.1.157Crossref PubMed Scopus (65) Google Scholar), Staphylococcus aureus, and Campylobacter jejuni (23.Nirdnoy W. Mason C.J. Guerry P. Mosaic structure of a multiple-drug-resistant, conjugative plasmid from Campylobacter jejuni.Antimicrob. Agents Chemother. 2005; 49 (15917546): 2454-245910.1128/AAC.49.6.2454-2459.2005Crossref PubMed Scopus (57) Google Scholar)). The third class of enzymes does not provide resistance to any of the antibiotics (one example from C. jejuni (23.Nirdnoy W. Mason C.J. Guerry P. Mosaic structure of a multiple-drug-resistant, conjugative plasmid from Campylobacter jejuni.Antimicrob. Agents Chemother. 2005; 49 (15917546): 2454-245910.1128/AAC.49.6.2454-2459.2005Crossref PubMed Scopus (57) Google Scholar)). Including the functionally annotated sequences that were not in the Pfam database, 193 sequences of presumably the same monomeric domain arrangement and sequence identity of 27% or higher were subjected to subsequent analysis. To identify the sequence determinants for the ANT(9) and ANT(3″) activities, the substrate-binding residues from the AadA complex structures with streptomycin and dihydrostreptomycin were mapped to the functionally annotated sequences (Fig. 5) and the entire family. The sequence identities between sequences with predicted ANT(3″)(9) activity were 43% or higher, and those between sequences with predicted ANT(9) activity were 38% or higher. Amino acids that interact with the C ring (the adenylation site) of streptomycin, Glu-87, Trp-112, Asp-182, and His/Asn-185, are conserved in all ANT(3″)(9) and ANT(9) enzymes, defining a subgroup of in total 71 sequences. Enzymes with ANT(3″)(9) activity are characterized by conservation of residues that interact with the A ring of streptomycin. Trp-173 and a two-amino-acid insertion at position 177-178, involved in backbone carbonyl interactions, are conserved in 26 of the 71 sequences. Based on this, we propose that the determinants for adenylation activity on spectinomycin are Glu-87, Trp-112, Asp-182, and His/Asn-185 and that the activity toward streptomycin in addition requires Trp-173 and the insertion at position 177-178. In the characterized enzyme without activity toward spectinomycin or streptomycin (23.Nirdnoy W. Mason C.J. Guerry P. Mosaic structure of a multiple-drug-resistant, conjugative plasmid from Campylobacter jejuni.Antimicrob." @default.
• W2805300175 created "2018-06-13" @default.
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• W2805300175 date "2018-07-01" @default.
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• W2805300175 title "Structural mechanism of AadA, a dual-specificity aminoglycoside adenylyltransferase from Salmonella enterica" @default.
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