Structural and phylogenetic analyses of resistance to next-generation aminoglycosides conferred by AAC(2′) enzymes

Plazomicin is currently the only next-generation aminoglycoside approved for clinical use that has the potential of evading the effects of widespread enzymatic resistance factors. However, plazomicin is still susceptible to the action of the resistance enzyme AAC(2′)-Ia from Providencia stuartii. As the clinical use of plazomicin begins to increase, the spread of resistance factors will undoubtedly accelerate, rendering this aminoglycoside increasingly obsolete. Understanding resistance to plazomicin is an important step to ensure this aminoglycoside remains a viable treatment option for the foreseeable future. Here, we present three crystal structures of AAC(2′)-Ia from P. stuartii, two in complex with acetylated aminoglycosides tobramycin and netilmicin, and one in complex with a non-substrate aminoglycoside, amikacin. Together, with our previously reported AAC(2′)-Ia-acetylated plazomicin complex, these structures outline AAC(2′)-Ia’s specificity for a wide range of aminoglycosides. Additionally, our survey of AAC(2′)-I homologues highlights the conservation of residues predicted to be involved in aminoglycoside binding, and identifies the presence of plasmid-encoded enzymes in environmental strains that confer resistance to the latest next-generation aminoglycoside. These results forecast the likely spread of plazomicin resistance and highlight the urgency for advancements in next-generation aminoglycoside design.


Results
Structure characteristics. Three high-resolution crystal structures of AAC(2′)-Ia from P. stuartii were solved, two structures containing coenzyme A (CoA) and N2′-acetylated aminoglycosides tobramycin and netilmicin; and one containing acetyl-CoA and the non-substrate aminoglycoside amikacin, which lacks an N2′ moiety. Data collection and final refinement statistics for each of these crystal structures are listed in Table 1. We also recently reported the crystal structure of AAC(2′)-Ia in complex with N2′-acetylated plazomicin and CoA, and have included it here in our analyses 18 . The overall fold of AAC(2′)-Ia, as with all other reported structures of aminoglycoside N-acetyltransferases [19][20][21][22][23][24][25][26][27][28][29] , belongs to the GCN5 related N-acetyltransferase (GNAT) superfamily. Its dimeric structure has an identical fold to another enzyme in this subclass, AAC(2′)-Ic from Mycobacterium tuberculosis, which shares a 55% sequence similarity and 32% sequence identity to AAC(2′)-Ia (Fig. 1a) 19 . The overall structural features of AAC(2′)-Ia are highlighted in Fig. 1b. Acetyl coenzyme A and coenzyme A binding. The CoA binding pocket of AAC(2′)-Ia has not previously been described. The discovery maps for each of the AAC(2′)-Ia crystal structures allow us to unambiguously place acetyl-CoA and CoA within the enzyme's active site (Fig. 2a,b). The crystal structure of AAC(2′)-Ia in complex with acetyl-CoA and amikacin, a non-substrate aminoglycoside, is allowing us to capture the enzyme in a pre-catalysis state. Meanwhile, the crystal structures of AAC(2′)-Ia in complex with CoA and acetylated aminoglycosides describes the enzyme in three product-bound states. While there are a large number of flexible basic residues in the CoA and acetyl-CoA binding regions, most of the interactions within the binding sites observed in each monomer are maintained. In the pre-catalyzed state, the adenine rings of acetyl-CoA are sandwiched in between two flexible loops, with residue Arg89 on one side and Lys121 on the other; however, this portion does not form any inter-or intramolecular hydrogen bonds. The phosphoryl-group of the 3′-phosphorylated adenine diphosphate (ADP) moiety forms hydrogen bond interactions with Arg89, while the ribose ring is void of interactions with the enzyme (Fig. 2d). The α-and β-phosphates of this same moiety form backbone hydrogen bond interactions with Gln90, Gly91 and Arg94, as well as Arg89 and Gly93, respectively (Fig. 2d). In the pantothenic acid moiety, the hydroxyl group of the carboxylic acid hydrogen bonds with Asp118, while the oxygen can interact with Arg88 or Val83. The second oxygen of this moiety interacts with Ser116 (Fig. 2d). The amide groups of acetyl-CoA form hydrogen bonds with Asp118 and the backbone oxygen of Met81, respectively (Fig. 2d). Finally, the oxygen group of the acetyl portion forms a hydrogen bond with the backbone amide of Met81 (Fig. 2d).
In the three product-bound states of AAC(2′)-Ia, the pantothenic acid moiety and the α-and β-phosphates of the 3′-phosphorylated ADP moiety of CoA remain in a similar conformation to that of acetyl-CoA (Fig. 2c). The hydrogen bond interactions remain similar, except towards the tail end of the CoA molecule, where the pantothenic acid moiety forms fewer interactions with the enzyme. (Fig. 2e). The most apparent difference   www.nature.com/scientificreports/ between acetyl-CoA and CoA binding to the pre-catalyzed-state and the product-bound state is the positioning of the 3′-adenosine moiety. In the product-bound state, this portion of CoA flips roughly 270° towards a solventexposed portion of the enzyme (Fig. 2c). The flipping of the 3′-adenosine moiety is likely due to the solventexposed nature of the active site, as well as space group differences between the pre-catalyzed and product-bound states. Clashes from molecules in the asymmetric unit likely inhibit this moiety of acetyl-CoA from adopting the same conformation seen in the pre-catalyzed state. However, the flexibility of the 3′-adenosine moiety does not introduce notable downstream changes in the binding of the acetyl-CoA molecule. As previously noted, the ribose and adenine rings do not interact with the enzyme. Instead, the adenine rings are now adjacent to a loop containing residues Arg89-Gly91.
Structural basis for aminoglycoside binding to AAC(2′)-Ia. The discovery maps for each of the AAC(2′)-Ia crystal structures also allowed us to unambiguously place the aminoglycosides within the enzyme's active site (Fig. 3a-d). The kinetic parameters for each aminoglycoside substrate are highlighted in Table 2. The structural basis for acetylated-tobramycin is similar to that of a previously reported acetylated-gentamicinbound structure of AAC(2′)-Ia 30 . While the aminoglycoside portion of acetylated-gentamicin only hydrogen  www.nature.com/scientificreports/ bonds with AAC(2′)-Ia via three residues, the tobramycin moiety can interact with up to 7 residues. In identical fashion to gentamicin, the central ring of tobramycin is anchored by two hydrogen bond interactions, N-1 with Glu149 and N-3 with the C-terminal carboxylate of Trp178 (Fig. 4a) 30 . Additionally, both aminoglycosides interact with the backbone carbonyl of Ser114 at their site of modification, N-2′ (Fig. 4a) 30 . Aside from these interactions, the prime-ring of tobramycin also interacts with the backbone carbonyl of Asp32 and the side chain of Asp37 at the N-6′ position ( Fig. 4a). At the double-prime ring, tobramycin interacts with the enzyme at its N-4′′ and O-5′′ via hydrogen bond with Asp117 (Fig. 4a). The tobramycin molecule in chain B also forms an additional hydrogen bond interaction between its O-2′′ and Glu148 of AAC(2′)-Ia. The acetyl group modification of the molecule interacts with the enzyme by way of the backbone amine of Met81 (Fig. 4a). The acetylated tobramycin in chain A also interacts with the backbone amine of the adjacent residue, Ala80 (Fig. 4a). AAC(2′)-Ia is capable of modifying three semi-synthetic aminoglycosides i.e., dibekacin, netilmicin, and plazomicin. Netilmicin is a semi-synthetic derivative of sisomicin, an aminoglycoside that is structurally similar and has comparable bacterial targets to gentamicin. Just as for gentamicin, AAC(2′)-Ia utilizes the same three residues, Ser114, Glu149, and Trp178, for hydrogen bonding (Fig. 4b). However, unlike gentamicin, netilmicin's N-6′ can form hydrogen bonds with both Asp32 and 37 (Fig. 4b). This is due to a slight shift in the N-6′ group's positioning. Additionally, Glu148 in chain A flips in towards the active site to interact with the 2′′-OH and 3′′-NH segments of netilmicin (Fig. 4b). Finally, the presence of a bulky ethyl substituent at N-1 does not cause any structural rearrangements of the enzyme, since this portion of the active site is readily solvent accessible.
Amikacin, a structural derivative of kanamycin, incorporates an (S)-HABA group at its N-1 position, but lacks an amine group at its N-2′ position, making it impervious to the effects of AAC(2′)-Ia (K i = 87 ± 10 µM). Although plazomicin is the only aminoglycoside substrate of AAC(2′)-Ia with an N-1 (S)-HABA group, understanding how another N-1 substituted aminoglycoside binds to this enzyme can provide additional reasoning behind this enzyme's specificity. Amikacin's interactions are similar to those between the enzyme and its natural substrates (Fig. 4c). Although interactions are maintained, especially at the prime ring, the slight movement of residues around the active site in the amikacin-bound structure causes deviations in the positioning of the central and double-prime rings of the aminoglycoside. Additionally, the N-1 substitution does not form any hydrogen bond interactions with the enzyme, and instead, residues Glu148 and Glu149 shift to make it so the enzyme can accommodate the (S)-HABA group in a conformation away from the active site (Fig. 4c). This also shifts the binding of the double-prime ring to interact similarly to that of the acetylated-tobramycin-bound structure, as they both have similar chemical groups for this ring.
Comparing netilmicin and amikacin binding to AAC(2′)-Ia with how plazomicin interacts with the enzyme reveals similarities, as well as intriguing differences. Plazomicin, like netilmicin, is a semi-synthetic derivative Figure 3. Binding of naturally occurring, semi-synthetic and next-generation aminoglycosides to AAC(2′)-Ia. Depicted in panels (a), (b), (c), and (d) are the Fo − Fc discovery maps for ligands tobramycin, netilmicin, amikacin, and plazomicin, respectively, at a contour level of 3σ. The plazomicin structure is presented here for comparison 18 . The enzyme is colored purple and the aminoglycosides tobramycin, netilmicin, amikacin, and plazomicin are colored in orange, red, teal, and violet, respectively. www.nature.com/scientificreports/ of sisomicin. As such, its binding is essentially identical, except at its N-1 and N-6′ substituents, where due to chemical differences in the extensions, these two next-generation aminoglycosides form differing interactions (Fig. 4d). Surprisingly, when comparing amikacin with plazomicin, different interactions are noted at the N-1 site. As mentioned above, both amikacin and plazomicin incorporate the (S)-HABA group at the N-1 position; however, while this moiety in amikacin does not form any specific interactions with the enzyme, the (S)-HABA tail of plazomicin forms hydrogen bonds with Glu149 and Asp176 (Fig. 4d).

Substrate specificity of the AAC(2′) enzyme class. Applying a phylogenetic analysis can provide an
understanding of other potential AAC(2′) enzymes that can bind and modify plazomicin, and ultimately spread resistance to this aminoglycoside. A search of sequence databases using the five members of the AAC(2′)-I enzyme class identified 56 additional homologues from unique bacterial species filtered from 5000 psi-BLAST result sequences (1000 sequences from each psi-BLAST search). The identified sequences had at least a 23% sequence identity to the original query sequences (AAC(2′)-Ia-AAC(2′)-Ie). Based on the substrate-binding analysis, we identified three key residues involved in AAC(2′)-Ia's aminoglycoside specificity; Asp37, Glu149, and Trp178, and one additional residue, Asp176, required for plazomicin specificity (Fig. 4e). Asp32 and Ser114 are also important for substrate binding in all four structures; however, they were excluded from the conservation analysis since they interact with aminoglycosides using their backbone atoms (Fig. 4e). Although Trp178 also employs its backbone oxygen to interact with aminoglycosides, this is an unusual interaction. Trp178 is the C-terminal residue, and it is the carboxyl group that forms a hydrogen bond and charge interaction with aminoglycosides. Ordinarily, protein termini are inherently flexible, but in AAC(2′)-Ia, the Trp178 side-chain forms hydrogen bond and van der Waals interactions with the rest of the enzyme, pinning the terminal carboxyl group in a specific location and orientation. Therefore, our sequence analysis includes an inherent assessment of Trp178′s residue conservation simultaneously with its positioning as the last residue in the sequence. It is The box for the residues critical for plazomicin hydrogen bonding is colored in purple. www.nature.com/scientificreports/ worthwhile noting that interactions between the enzyme's carboxy-terminus and aminoglycosides have also been observed in other AMEs 12,19,31 . Sequence alignment of all 61 unique sequences (5 query sequences and 56 identified homologues) shows that the four residues involved in aminoglycoside binding are highly conserved (Fig. 5a). Asp37, Glu149, Trp178, and Asp176 are conserved 74, 88, 95, and 92% of the time, respectively, where conservation percentages for Glu149 and Asp176 include instances of both aspartic and glutamic acid (Fig. 5a). Interestingly, although Asp32 utilizes its backbone oxygen for binding, it is still conserved 75% of the time.
The predicted ability of the 61 enzymes to bind to either aminoglycosides or next-generation aminoglycoside plazomicin was assessed based on the number of conserved binding residues (Fig. 5b,c). Note, the prediction of aminoglycoside binding is based on enzymes whose substrate specificity has been described [AAC(2′)-Ia-e], however, the prediction of plazomicin binding is based solely on data from AAC(2′)-Ia. The assembly of enzymes were classified as either non-binders, unlikely binders, likely binders, or binders of aminoglycosides if 0, 1, 2, or 3 of the binding residues (Asp37, Glu149, Trp178) were observed in a sequence, respectively. The same classification was made for plazomicin if 0-1, 2, 3, or 4 of the binding residues (Asp37, Glu149, Trp178, Asp176) were observed in a sequence, respectively (Fig. 5b). For aminoglycosides, it was assessed that 2, 3, 29, and 66% of enzymes would be non-binders, unlikely binders, likely binders, and binders, respectively, reflective of the extensive conservation observed in the substrate binding site (Fig. 5a,b). Similarly, for plazomicin, it was predicted that 3, 3, 30, and 64% of enzymes would follow the same binding pattern, respectively (Fig. 5b). It is meaningful to note that only three enzymes change their classification on their ability to bind either aminoglycosides or next-generation aminoglycoside plazomicin (Fig. 5c). Sequence alignment of a subset of sequences from each of the binding classifications are provided in Supplementary Fig. S2 online.
The majority of the sequences found are chromosomally encoded, partly reflecting the contents of sequence databases; however, three of the sequences found are plasmid-encoded. These three sequences are found in the following bacterial species: Mycolicibacterium arabiense, Deinococcus wulumuqiensis, and Deinococcus sp. NW-56 (RefSeq Reference: WP_163924889.1, WP_114673790.1, WP_104992197.1) (Fig. 5c) 32,33 . Each of these sequences shares a 33.5, 42.0, and 43.5% sequence identity, and an 84, 98, and 99% sequence coverage with AAC(2′)-Ia from P. stuartii, respectively, where all four binding residues are conserved. A sequence alignment of all three sequences is provided in Supplementary Fig. S2 online. Models of these three enzymes show their aminoglycoside binding pocket can readily bind plazomicin (Fig. 6). To test this, we expressed and purified the D. wulumuqiensis homologue and examined its kinetic parameters against plazomicin. As predicted, AAC(2′)-I www.nature.com/scientificreports/ from D. wulumuqiensis is found to have very similar kinetic parameters against plazomicin compared with AAC(2′)-Ia from P. stuartii (Table 2).

Discussion
Plazomicin has shown activity against methicillin-resistant Staphylococcus aureus and multi-drug resistant Escherichia coli, Klebsiella pneumonia, and Enterobacter spp., and it is currently approved for the treatment of complicated urinary tract infections and pyelonephritis 34,35 . Plazomicin's effectiveness stems from the incorporation of two chemical groups at the N-1 and N-6′ positions of its aminoglycoside precursor, sisomicin 36 . It is understood that elevated activity against plazomicin is exclusive to AAC(2′)-Ia from P. stuartii, whereas van der Waals strain prevents clinically widespread resistance factors such as ANT(2′′), APH(2′′), and AAC(6′) from binding this antibiotic 11,36 . We provide here a detailed structural analysis of an aminoglycoside modifying enzyme that can alter an aminoglycoside with several different semi-synthetic additions. Our investigation presents an understanding of why AAC(2′)-Ia has the ability to accommodate the N-1 (S)-HABA or ethyl groups and the N-6′ hydroxy-ethyl group. Comparison of three crystal structures presented here, together with our recently reported plazomicin complex structure 18 , allows us to inspect structural differences in active site binding at both these positions. First, at the N-1 binding site, the accommodation of the (S)-HABA or ethyl groups is due to the flexibility of either residue Glu148 or Glu149. Our structures show that Glu148 can adopt two conformations (Fig. 7). The difference in the positioning of this residue is dependent on how the enzyme harbours the N-1 expansion. In the tobramycin-bound structure, there is no chemical addition at the N-1 amine, and therefore Glu148 can adopt either conformation (Fig. 7a). In the netilmicin bound structure, the ethyl addition at N-1 sits perpendicular to the aminoglycoside plane, requiring Glu148 to flip away from the active site (Fig. 7b). The plazomicin-bound structure sees the N-1 (S)-HABA adopting a similar conformation to the ethyl addition of netilmicin, where the (S)-HABA group first protrudes perpendicularly to the aminoglycoside plane, and then proceeds in a downward fashion (Fig. 7b,d). The amikacin-bound structure shows that the enzyme is also capable of adapting the (S)-HABA moiety in a second conformation. This second conformation, as displayed by amikacin, exhibits that the  www.nature.com/scientificreports/ N-1 tail can also sit parallel to the aminoglycoside plane (Fig. 7c). The adoption of this conformation is based on the movement of residue Glu149 (Fig. 7c). As opposed to the other three structures, this residue flips away from the aminoglycoside-binding site to accommodate this bulky substituent. Second, the N-6′ binding site consists of two residues, Asp32 and 37. With respect to aminoglycoside binding, accommodation of this chemical extension requires no conformational change when comparing binding to aminoglycosides void of this moiety. Moreover, interaction with these two residues is important for prime-ring binding in all four aminoglycoside-bound structures. It is noteworthy that the N-6′ hydrogen bond donor moiety of plazomicin has been implicated in being crucial for specificity for the bacterial ribosome over the eukaryotic homologue 18,37,38 .
Our analysis in Fig. 5 outlines the conservation of residues in the aminoglycoside/ plazomicin binding pocket across AAC(2′)-I enzymes. These residues are highly conserved, and our analysis suggests that 95% of the homologues that have been identified would be likely or capable of binding aminoglycosides (Fig. 5b,c). From these identified homologues, 98% of them are predicted to retain their ability to bind plazomicin (Fig. 5b,c). This result is troublesome as it reveals that plazomicin resistance is likely not isolated to a single bacterial species. Moreover, based on binding residue conservation, it is likely that the ability to chemically detoxify plazomicin is an innate feature of AAC(2′)-I enzymes.
Our analysis also provides an understanding of the evolutionary pathway for the AAC(2′)-I enzyme subtypes. Our survey of public sequence databases queried against five chromosomally encoded sequences from P. stuartii (AAC(2′)-Ia), M. fortuitum (AAC(2′)-Ib), M. tuberculosis (AAC(2′)-Ic), M. smegmatis (AAC(2′)-Id), and M. leprae (AAC(2′)-Ie) have allowed us to identify homologues from other species that are aminoglycoside and plazomicin binders [39][40][41][42] . While the results from this investigation show that the majority of homologues remain chromosomally encoded, three homologues were found to be plasmid-encoded (Fig. 5c). Additionally, these sequences have high similarity and identity to AAC(2′)-Ia, with all plazomicin binding residues being conserved (Fig. 6), where the D. wulumuqiensis homologue of AAC(2′)-I was found to have comparable kinetic parameters to AAC(2′)-Ia from P. stuartii against plazomicin ( Table 2). This finding is cause for alarm as the ability of transposable elements to disseminate quickly allows resistance to spread in pathogens of medical interest 6 . Moreover, this reinforces the urgency of preserving plazomicin as a viable treatment option. Currently, plazomicin is on the WHO's list of essential medicines, and is part of the reserve group of antibiotics, for use only against infections that are suspected to be caused by multidrug-resistant organisms. Although resistance is not yet widespread, the identification of sequences which are likely capable of binding plazomicin on mobile elements indicates that the spread of resistance is no longer theoretical, it is inevitable. This result, combined with the ability of AAC(2′)-Ia to bind N-1-substituted aminoglycosides in different conformations, enforces the need for next-generation aminoglycosides with a novel or additional chemical substituent in order to curb resistance to antibiotics of last resort.

Methods
Cloning. AAC(2′)-Ia from Providencia stuartii construct with non-cleavable C-terminal HIS-tag. The AAC(2′)-Ia gene from Providencia stuartii was synthesized and subcloned into the pET-15b expression vector between the NdeI and XhoI restriction sites with a non-cleavable C-terminal HIS-tag. From here forward in the methods section, we refer to this construct as AAC(2′)-Ia HIS , where HIS designates the non-cleavable HIS-tag. The DNA sequence was verified using the BioBasic Inc. gene synthesis service. The resulting vector was used to transform E. coli BL21(DE3) cells.

AAC(2′)-Ia from Providencia stuartii construct with cleavable N-terminal HIS-tag.
A second AAC(2′)-Ia construct from Providencia stuartii was synthesized and subcloned into the pET-15b expression vector between the NdeI and BamHI restriction sites with an N-terminal HIS-tag followed by a thrombin cleavage site. From here forward in the methods section, we simply refer to this construct as AAC(2′)-Ia. The DNA sequence was verified using the BioBasic Inc. gene synthesis service. The resulting vector was used to transform E. coli BL21(DE3) cells.

AAC(2′)-I from Deinococcus wulumuqiensis. The AAC(2′)-I homologue from
Deinococcus wulumuqiensis (Ref-Seq reference: WP_114673790.1) was synthesized and subcloned into the pET-15b expression vector between NdeI and BamHI restrictions sites with an N-terminal HIS-tag. From here forward in the methods section, we refer to this construct as AAC(2′)-I. The DNA sequence was verified using the BioBasic Inc. gene synthesis service. The resulting vector was used to transform LOBSTR E. coli cells 43 . Expression. Protein expression of the P. stuartii and D. wulumuqiensis constructs was carried out using the Studier method for auto-induction, as previously described 11,44 . Cells were harvested by centrifugation at 6000 g for 15 min at 4 °C and resuspended in 40 mL of lysis buffer. Lysis buffer for AAC(2′)-Ia HIS and AAC(2′)-Ia constructs from P. stuartii consisted of 50 mM TRIS-HCl, pH 8.0, 200 mM NaCl, 10 mM β-mercaptoethanol, 10% (v/v) glycerol, and one EDTA-free protease inhibitor tablet (Roche); while lysis buffer for AAC(2′)-I from D. wulumuqiensis consisted of 50 mM bicine, pH 9.0, 300 mM NaCl, and 10 mM imidazole. Cells were lysed by sonication, and cell debris was subsequently removed by centrifugation at 50,000 g for 30 min at 4 °C. The supernatant was further clarified by filtration through a 0.22 μm syringe-driven filter. HIS  Data collection. Diffraction data for optimized crystals of the four complexes were collected on a Bruker D8 Discovery consisting of a METALJET source (liquid gallium) coupled with a PHOTON II CPAD detector mounted on a KAPPA goniometer. A ten-fold data collection strategy was calculated for all data sets using the PROTEUM3 software suite (Bruker).

Purification. Affinity purification of AAC(2′)-Ia
Structure solution and refinement. Datasets for all structures were processed using the xia2 pipeline 45 , 48 ]. The structure of AAC(2′)-Ia HIS in complex with CoA was solved by molecular replacement using Phaser 49 , using a previously solved structure as the search model (PDB ID: 5US1) 30 . The data collection and refinement statistics for this structure are listed in Supplementary Table S1 online. The structure of AAC(2′)-Ia in complex with acetylated tobramycin and CoA was solved by molecular replacement using Phaser, with the CoA complex of AAC(2′)-Ia HIS used as the search model. The structure of AAC(2′)-Ia in complex with amikacin and acetyl-CoA was solved using molecular replacement with the acetylated tobramycin-CoA complex of AAC(2′)-Ia as the search model using Phaser. Finally, the structure in complex with acetylated netilmicin and CoA was determined using Fourier synthesis performed by phenix.refine 50 using the acetylated tobramycin-CoA complex stripped of all non-protein atoms. All structures were refined by iterative cycles of reciprocal-space refinement with phenix.refine and real-space refinement and model building in Coot 51 . The ligand restraints for CoA, acetyl-CoA, acetylated antibiotics, and amikacin were generated using eLBOW 52 . The data collection and final refinement statistics of the three aminoglycoside-bound models are listed in Table 1.
Phylogenetic Analysis of Aminoglycoside 2′-Acetyltransferases. AAC(2′) sequences were identified using PSI-BLAST (Position-Specific Iterated BLAST) 53 , with five separate searches using AAC(2′)-Ia, b, c, d, and e as the respective query sequences (GenBank/RefSeq reference: AAA03550, AAC44793, ACT23293, WP_011726942, CAC32082) [39][40][41][42] . The search was run using the reference proteins database (RefSeq), excluding models and uncultured sequences 54 . Search parameters included 1000 maximum target sequences with an expect threshold of 10. BLOSUM62 was used as the scoring matrix. The PSI-BLAST threshold was set at 0.005 www.nature.com/scientificreports/ and was run for five iterations for each query sequence. Final sequences for the phylogenetic tree were chosen from each search list based on sequence identity (> 23%), clinical prevalence, and relative e-value. The alignment of the final 61 sequences was generated in NGPhylogeny.fr using the MAFFT L-INS-I method 55,56 . NGPhylogeny was subsequently used to generate a maximum-likelihood phylogenetic tree using the PhyML algorithm 55,57 . The final phylogenetic tree was designed using iTOL (Interactive Tree of Life) 58 . Plasmid-encoded sequences were modelled using the SWISS-MODEL server [59][60][61][62] , with the AAC(2′)-Ia-acetylated plazomicin complex stripped of its ligands and water molecules used as the template.
Plazomicin Synthesis. Synthesis of plazomicin was performed starting from commercially available sisomicin sulfate as recently reported 63 in the modified version of the original report by Moser 15 .
stuartii against a panel of aminoglycosides (tobramycin, gentamicin, netilmicin and plazomicin) were obtained using the ThermoFisher NanoDrop One C Spectrometer. The acetylation of aminoglycosides was measured by a coupled assay where the formation of pyridine-4-thiolate can be detected at 324 nm 64,65 . The assays were performed in a 0.8 mL quartz cuvette (pathlength 1 cm), in a buffer containing 25 mM MES, pH 5.5, 100 mM NaCl, 500 µM 4,4′-dipyridyl disulfide (Aldrithiol-4, Sigma-Aldrich), 150 µM acetyl-CoA, and varying aminoglycoside concentrations (2.5 to 160 mM). The reaction was initiated by the addition of enzyme (0.5 µM, final concentration), where UV absorbance was measured over 10 min at 22 °C. Assays were run in triplicate, and data analysis was performed using the GraphPad5 software.
Kinetic assay of AAC(2′)-I from Deinococcus wulumuqiensis. The kinetic properties of AAC(2′)-I from D. wulumuqiensis against plazomicin were measured using the same assay as AAC(2′)-Ia from P. stuartii, with minor adjustments to the protocol. The assays were performed in the same buffer, except at pH 6.0. The aminoglycoside concentration range was adjusted (6.26 to 100 µM). The final concentration of the enzyme in the reaction mix was 0.14 µM.