Structural and biochemical analyses of an aminoglycoside 2′-N-acetyltransferase from Mycolicibacterium smegmatis

The expression of aminoglycoside-modifying enzymes represents a survival strategy of antibiotic-resistant bacteria. Aminoglycoside 2′-N-acetyltransferase [AAC(2′)] neutralizes aminoglycoside drugs by acetylation of their 2′ amino groups in an acetyl coenzyme A (CoA)-dependent manner. To understand the structural features and molecular mechanism underlying AAC(2′) activity, we overexpressed, purified, and crystallized AAC(2′) from Mycolicibacterium smegmatis [AAC(2′)-Id] and determined the crystal structures of its apo-form and ternary complexes with CoA and four different aminoglycosides (gentamicin, sisomicin, neomycin, and paromomycin). These AAC(2′)-Id structures unraveled the binding modes of different aminoglycosides, explaining the broad substrate specificity of the enzyme. Comparative structural analysis showed that the α4-helix and β8–β9 loop region undergo major conformational changes upon CoA and substrate binding. Additionally, structural comparison between the present paromomycin-bound AAC(2′)-Id structure and the previously reported paromomycin-bound AAC(6′)-Ib and 30S ribosome structures revealed the structural features of paromomycin that are responsible for its antibiotic activity and AAC binding. Taken together, these results provide useful information for designing AAC(2′) inhibitors and for the chemical modification of aminoglycosides.


Substrate specificity of AAC(2′)-Id.
To determine the substrate specificity of AAC(2′)-Id, we performed steady-state kinetic parameter measurements using acetyl-CoA and various aminoglycoside substrates. The results showed that among the tested substrates, gentamicin exhibited the highest binding affinity for AAC(2′)-Id, with dissociation constants (K m ) of (2.06 ± 0.36) × 10. However, regarding the specific activity (k cat / K m ), tobramycin performed best due to its relatively high turnover rate constant (k cat = 3.34 ± 0.10 s −1 ). Col-  www.nature.com/scientificreports/ lectively, the kinetic results indicate that AAC(2′)-Id has a very broad substrate-binding specificity, similar to AAC(2′)-Ic 9 . AAC(2′)-Id was not active on streptomycin because the antibiotic does not contain 2′ amino groups in its chemical structure, indicating that AAC(2′)-Id exhibits strict regioselectivity.

Substrate-and CoA-binding residues of AAC(2′)-Id.
To understand the substrate binding mechanism of AAC(2′)-Id, we determined the crystal structures of AAC(2′)-Id bound to four different aminoglycoside substrates ( Fig. 2 and Supplementary Fig. S2). Structural comparison between the apo-and the substrate-CoA-bound AAC(2′)-Id structures revealed significant conformational differences in the α1-α2 loop, α4-helix, and β8-β9 loop region between the structures. The α1-α2 loop is the most flexible region in the structure of AAC(2′)-Id and thus misses some residues. This loop may move and cover the substrate-binding site in the apostructure, whereas it exhibits a more open conformation in the substrate-bound AAC(2′)-Id structures. In addition, residue Arg179 located in the β8-β9 loop region also undergoes a dramatic conformational change upon substrate binding. In the apo-AAC(2′)-Id structure, Arg179 forms a salt bridge with Asp208 and Asp177, as well as hydrogen bonds with the carbonyl oxygen atom of Trp210. The salt bridge, however, is lost in all substratebound structures as Arg179 flips away from Asp208 and Asp177. Thus, the α1-α2 and β8-β9 loop regions may act as gatekeepers and thereby play a role in substrate recognition, substrate binding, product release, and solvent exclusion (Fig. 3). Gentamicin contains three ring structures referred to as the prime ring (ring I), central 2-deoxystreptamine core (ring II), and double prime ring (ring III). In the gentamicin-CoA-AAC(2′)-Id complex structure, the interactions between ring I of gentamicin and AAC(2′)-Id are hydrophobic, except for one hydrogen bond between Ser141 and the amino group of ring I (Fig. 4A). Ring II forms hydrogen bonds with Asp177 and Trp210, and www.nature.com/scientificreports/ ring III forms hydrophobic interactions with Ala142, Asp144, and Glu176. Sisomicin is structurally similar to gentamicin but has a unique unsaturated diamino sugar at the ring I position (Fig. 4B). In the sisomicin-CoA-AAC(2′)-Id complex structure, the overall interaction mode of sisomicin is identical to that of gentamicin, except for an additional hydrogen bond formed between the main chain carbonyl group of Asp54 and the second amino group on ring I of sisomicin (Fig. 4B). The chemical structures of both neomycin and paromomycin contain a different linkage at ring II and an additional hexose sugar ring (ring IV) compared with those of gentamicin and sisomicin. Paromomycin has a hydroxyl group at position 6′ of ring I, whereas neomycin bears an amino group at the corresponding position. In the neomycin-CoA-AAC(2′)-Id complex structure, the 6′-amino group of ring I forms direct hydrogen bonds with Asp54 and Asp59 (Fig. 4C). In contrast, the 6′-hydroxyl group of ring I of paromomycin exhibits calcium ion-mediated interactions with Asp54 and Asp59 (Fig. 4D). Compared to neomycin, paromomycin forms different interaction networks at the positions of oxygen atom of C61, leading to metal ion (calcium)-mediated interactions with the enzyme. This interaction may explain the higher binding affinity of paromomycin than neomycin in our activity assay. Taken together, the broad substrate specificity of AAC(2′)-Id could be explained by the fact that most of its interactions with aminoglycoside antibiotics are hydrophobic, except for several common hydrogen bonds and some flexible interactions of the α1-α2 loop, α4-helix, and β8-β9 loop region. In addition, metal ion-mediated interaction and side chain reorientations upon substrate binding could also contribute to the plasticity and broad substrate specificity of this enzyme (Fig. 4). Structural comparison of the apo-and the four different aminoglycoside substrate-bound AAC(2′)-Id structures revealed that the ring II structure of each substrate exhibits common core interactions; the two nitrogen atoms of ring II form hydrogen bonds with Asp177 (or Asp208) and the carbonyl oxygen atom of Trp210. As www.nature.com/scientificreports/ described earlier, these negatively charged residues (Asp177 and Asp208) form a salt bridge with Arg179 in the apo-structure. Chain A of the gentamicin-CoA-AAC(2′)-Id complex structure contains both a CoA and a gentamicin molecule, whereas chain B contains only a CoA molecule. Thus, the structural comparison of apo-AAC(2′)-Id with chain A and chain B of gentamicin-CoA-AAC(2′)-Id enabled us to identify structural changes induced by the sequential binding of CoA and gentamycin (Fig. 5). Structural superposition revealed that CoA binding induces only small conformational changes in the α4-helix region (Fig. 5A); however, subsequent binding of gentamicin causes more dramatic structural changes in the α4-helix and β8-β9 loop regions (Fig. 5B). Moreover, the binding of gentamicin leads to closer contact between CoA and the α4-helix, thus resulting in tighter binding of the coenzyme. In summary, the binding of CoA and gentamicin induces a closing motion of the α4-helix and an opening motion of the β8-β9 loop region. As the catalytically important residue Tyr150 and the catalytic residue Ser143 are located on the α4-helix and the β5-α4 loop, respectively, the structural change of the α4-helix observed here is closely related to the enzymatic reaction mechanism of AAC(2′)-Id. In our activity assay, the activity of the Tyr150Ala mutant was remarkably lower than that of wild-type AAC(2′)-Id (Table 3). Previous structural and activity assay studies on AAC(6′)-Ii showed that AAC(6′)-Ii also has a similar Tyr147 residue near the CoA binding site, similarly to the AAC(2′)-Id structure. The authors suggested that Tyr147 is important for making correct orientation between aminoglycoside substrates and CoA to transfer efficiently to the 6′-amino group of aminoglycosides 13 . It is also suspected that the Tyr150 residue in AAC(2′)-Id may have a similar function to Tyr147 in AAC(6′)-Ii. www.nature.com/scientificreports/ Notably, structural analysis of four different aminoglycoside substrate-bound AAC(2′)-Id structures revealed that the binding of all aminoglycosides to AAC(2′)-Id requires both α1-α2 and β8-β9 loop opening. However, three ring-containing aminoglycosides (4,6-disubstituted aminoglycosides: gentamicin and sisomicin) bind toward the β8-β9 loop region and make several interactions with this loop, whereas four ring-containing aminoglycosides (4,5-disubstituted aminoglycosides: neomycin and paromomycin) bind toward the α1-α2 loop region and make several interactions with it. Probably, this major binding mode difference between three ringcontaining aminoglycosides and four ring-containing aminoglycosides results in the different binding affinity and catalytic efficiency among various aminoglycosides (Table 3). In our activity assay data, AAC(2′)-Id showed a stronger binding affinity and more catalytic efficiency to three ring-containing aminoglycosides (gentamicin and sisomicin) than four ring-containing aminoglycosides (neomycin and paromomycin). It is thought that three ring-containing-aminoglycosides form additional hydrophobic interactions with the β8-β9 loop as well as the β5-α4 loop. Thus, three ring-containing aminoglycosides may be able to bind to AAC(2′)-Id more favorably.
Comparison analysis between gentamicin and sisomicin complex structures indicated that interaction between gentamicin and AAC(2′)-Id is stronger than that between AAC(2′)-Id and sisomicin. Unlike sisomicin, gentamicin contains methyl groups connected to the 6′ and amine group in ring I. The methyl group of 6′ forms a hydrophobic interaction with F42 and F55 (Fig. 6), and the other methyl group connected to the amine group interacts with the D59 backbone and probably influence the shift of the 45-53 loop region toward gentamicin and cover the active site. Consistent with this observation, the turnover rate of AAC(2′)-Id for gentamicin is slower than that for sisomicin. Paromomycin has the same chemical structure as neomycin, except that an oxygen atom is linked to C61 of paromomycin, while a nitrogen atom is linked to C61 of neomycin. The nitrogen atom linked to C61 of neomycin makes direct hydrogen bonds with the side chain OD2 of Asp59 and the backbone carbonyl group of Asp54 residue, whereas the oxygen atom linked to C61 of paromomycin forms metal ion interaction. This difference in interaction might explain why AAC(2′)-Id has ten times higher catalytic efficiency for paromomycin than for neomycin.

Structural comparison of AAC(2′)-Id with other AAC enzymes.
Comparison of the structures of AAC(2′)-Id, AAC(2′)-Ic 9 , and AAC(2′)-Ia 11 revealed remarkable structural and sequential differences in the α1-α2, β3-β4, β8-β9, and β9-β19 loop regions between these enzymes. The α1-α2 and β8-β9 loop regions are located near the active site and speculated to be involved in the initial substrate binding of each enzyme. Thus, the difference in substrate specificity between the examined AAC(2′) enzymes may be due to structural and amino acid residue differences in the α1-α2 and β8-β9 loop regions (Fig. 7).
Next, the regioselectivity mechanism of AAC enzymes was investigated by structural comparison of the sisomicin-and CoA-bound AAC(3)-VIa (PDB code 6BC3) 14 , neomycin-and CoA-bound AAC(3)-IIIb (PDB code 6MB9) 15 , and paromomycin-and acetyl-CoA-bound AAC(6′)-Ib structures (PDB code 2VQY) 16 . Sequence identities among AAC enzymes are generally low (~ 10%); however, they all contain an acetyl-CoA and an aminoglycoside binding site. Structural comparison showed that aminoglycoside substrates bind with different conformations to each class of AACs, exhibiting different positions and rotation angles. The transfer of the acetyl group from acetyl-CoA to the aminoglycoside substrate requires optimal interaction distances and planar  In the neomycin-bound AAC(2′)-Id structure, ring I of neomycin is most deeply buried in the enzyme's active site, whereas ring II is the most buried region in neomycin-bound AAC(3)-IIIb structure. In the sisomicin-bound AAC(3)-VIa structure, rings II and III of the antibiotic are the major interaction regions. In the case of paromomycin, rings I and II make multiple interactions with AAC(2′)-Id, while rings III and IV exhibit only a few interactions with the enzyme. However, paromomycin adopts a folded, L-shaped conformation in the structure of AAC(6′)-Ib, and all rings (I, II, III, and IV) exhibit strong interactions with the enzyme (Fig. 8).
Aminoglycosides bind to the bacterial 30S ribosome and thereby inhibit protein synthesis. Previous studies have described the crystal structures of the 30S subunit complexed with the antibiotics paromomycin, streptomycin, and spectinomycin 2 . Thus, it is worthwhile to investigate the interaction modes of paromomycin with the 30S ribosome and AAC enzymes. The present work shows that paromomycin exhibits different binding modes, depending on the protein to which it binds. These results provide novel directions in the design of chemically modified variants of paromomycin that exhibit antibiotic activity and overcome antibiotic resistance mediated by AAC enzymes. In detail, the O31 oxygen atom of ring I and the O23 oxygen atom of ring III are essential for the direct interaction between paromomycin and AAC enzymes. These oxygen atoms are, however, not involved in the binding of paromomycin to the 30S ribosome (Fig. 9). Therefore, chemical modifications of these oxygen atoms may inhibit the binding of paromomycin to AAC enzymes, while preserving that to the 30S ribosome for antibiotic activity.
In this study, we presented the first crystal structures of AAC(2′)-Id in its apo-form and AAC(2′)-Id complexed with coenzyme A and different aminoglycosides (neomycin, paromomycin, sisomicin, and gentamicin). Structural comparison of these structures enabled us to identify important residues for substrate binding and specificity. In addition, the present structures, together with previously solved aminoglycoside-bound structures (AAC enzymes and 30S ribosome), provide essential information for the design of novel AAC inhibitors and aminoglycoside derivatives. Previous studies have also shown that several aminoglycoside derivatives show increased activity against pathogens 11,17,18 .   X-ray diffraction data of all crystals were collected on the beamlines 5C and 7A at the Pohang Accelerator Laboratory (PAL, Pohang, Republic of Korea) using a CCD EIGER 9 M detector (Dectris, Baden, Switzerland) and a Quantum 270 CCD detector (ADSC, USA), respectively 20 . All data were indexed, integrated, and scaled using HKL2000 21 . Crystallographic data statistics are summarized in Table 1.

Methods
Structure determination and refinement. The apo-form structure of AAC(2′)-Id was solved by molecular replacement with MOLREP from the CCP4i suite 22 using the apo-AAC(2′)-Ic structure (PDB code 1M44) as the search model. The ternary complex structures were solved by molecular replacement using the www.nature.com/scientificreports/ apo-AAC(2′)-Id structure as the search model. Manual model building was performed in WinCoot 23 , and each model was refined using REFMAC5 24 and Phenix 25 . All structures were deposited in the PDB (accession codes 7CRM, 7CS1, 7CS0, 7CSI, and 7CSJ for the apo-, gentamicin-, sisomicin-, neomycin-, and paromomycin-bound structures, respectively). All figures were prepared with PyMOL 26 .
Enzyme activity assay. Kinetic assays were performed on AAC(2′)-Id using acetyl-CoA as the coenzyme, eight different aminoglycosides as substrates, and 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) as an indicator. All assays were performed in 96-well plates with a final reaction volume of 200 μL at 25 °C. The reaction mixtures contained 50 mM MES (pH 6.5), 300 μM acetyl-CoA, 200 μM DTNB, and varying concentrations of aminoglycoside. The reaction was initiated by the addition of the enzyme, and the initial acetyltransferase rate of acetyl-CoA was determined by an increase in the absorbance at 412 nm, using an extinction coefficient (TNB) of 14,150 M −1 cm −1 . The Michaelis-Menten parameters were obtained by fitting the initial velocity data using OriginLab software (version 8.0, Northampton, MA, USA).