Structural and Biochemical Characterization of Acinetobacter spp

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Structural and biochemical characterization of Acinetobacter spp. aminoglycoside acetyltransferases highlights functional and evolutionary variation among antibiotic resistance enzymes Peter J Stogios, Misty Lee Kuhn, Elena Evdokimova, Melissa Law, Patrice Courvalin, and Alexei Savchenko ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.6b00058 • Publication Date (Web): 27 Oct 2016 Downloaded from http://pubs.acs.org on October 28, 2016

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ACS Infectious Diseases

Structural and biochemical characterization of Acinetobacter spp. aminoglycoside acetyltransferases highlights functional and evolutionary variation among antibiotic resistance enzymes

Peter J. Stogios1,2*, Misty L. Kuhn2,3,4*,%, Elena Evdokimova1,2, Melissa Law4, Patrice Courvalin5 and Alexei Savchenko1,2,# 1

Department of Chemical Engineering and Applied Chemistry, 200 College St, University of

Toronto, Toronto, Ontario, M5G 1L6, Canada. 2Center for Structural Genomics of Infectious Diseases (CSGID). 3Department of Pharmacology and Cellular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. 4Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132. 5Institut Pasteur, Unité des Agents Antibactériens, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.

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ABSTRACT Modification of aminoglycosides by N-acetyltransferases (AACs) is one of the major mechanisms of resistance to these antibiotics in human bacterial pathogens. More than fifty enzymes belonging to the AAC(6’) subfamily have been identified in Gram-negative and Grampositive clinical isolates. Our understanding of the molecular function and evolutionary origin of these resistance enzymes remains incomplete. Here we report the structural and enzymatic characterization of AAC(6’)-Ig and AAC(6’)-Ih from Acinetobacter spp. The crystal structure of AAC(6’)-Ig in complex with tobramycin revealed a large substrate-binding cleft remaining partially unoccupied by the substrate, which is in stark contrast with the previously characterized AAC(6’)-Ib enzyme. Enzymatic analysis indicated that AAC(6’)-Ig and -Ih possess a broad specificity against aminoglycosides but with significantly lower turnover rates as compared to other AAC(6’) enzymes. Structure- and function-informed phylogenetic analysis of AAC(6’) enzymes led to identification of at least three distinct subfamilies varying in oligomeric state, active site composition and drug recognition mode. Our data support the concept of AAC(6’) functionality originating through convergent evolution from diverse Gcn5-related-Nacetyltransferase (GNAT) ancestral enzymes, with AAC(6’)-Ig/Ih representing enzymes that may still retain ancestral non-resistance functions in the cell as provided by their particular active site properties.

Keywords: Acinetobacter, antibiotic resistance, aminoglycoside, crystal structure, Gcn5-related N-acetyltransferase

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INTRODUCTION

Aminoglycosides (AGs) are potent antibiotics against both Gram-positive and Gramnegative bacteria and their bactericidal activity is based on the binding to the 30S subunit of the ribosome, leading to protein mistranslation1. However, as with all drug classes, resistance to AGs along with their toxicity has hampered their use in the clinic. Resistance to these antibiotics is conferred primarily through AG-modifying enzymes (AMEs) that suppress their ribosome binding. The prevalence of AG resistance and discovery of new AMEs has continued unabated and there are now upwards of 100 known AMEs1. The dissemination of AMEs in pathogens led to the gradual removal of AGs from the front-line of the anti-bacterial arsenal2. However, diminishing output of antibiotic discovery pipelines, particularly for Gram-negative pathogens and the inevitable rise of resistance to other antibiotics are leading to a re-evaluation of the role of this class in therapy. Studies of the molecular mechanisms of AMEs inform on their inactivation efficiency and substrate specificities. These studies also pave the way to the rational design of less resistance-prone AGs and AME inhibitors that can be used as antibiotic adjuvants3-6. AMEs are classified

by

the

reaction

catalyzed:

O-phosphotransferases/APHs),

O-

nucleotidylyltransferases/ANTs) and N-acetyltransferases/AACs), the latter being the most widespread in clinical isolates1. N-acetylation at the 6’ position of the AG scaffold is mediated by AAC(6’) enzymes which are divided into two types based on substrate specificity: AAC(6’)-I enzymes acetylate amikacin but not gentamicin C1, while AAC(6’)-II acetylate all three types of gentamicin but not amikacin. There are more than fifty AAC(6’) enzymes identified in clinical isolates1 with AAC(6’)-Ib present in over 70% of Gram-negative clinical isolates having an 3 ACS Paragon Plus Environment


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AAC(6’)-I activity7. One of the variants called AAC(6’)-Ib-cr, has also evolved activity against fluoroquinolones via key active site mutations8. Genes encoding AAC(6’) are typically located in mobile genetic elements or resistance cassettes, reflecting their selection in response to the use of AGs. However, many bacterial species also carry aac(6’) genes in the chromosome, e.g. aac(6’)-Ic in Serratia marcescens, aac(6’)-Ii in Enterococcus spp.9 and aac(6’)-Iy in Salmonella enterica and Salmonella enteritidis10. AAC(6’)-Ig from Acinetobacter haemolyticus and AAC(6’)-Ih from Acinetobacter gyllenbergii are among eleven enzymes ancestral to the Acinetobacter genus (i.e. AAC(6’)-Ij, Ik, Ir, Is, It, Iu, Iv, Iw, Ix)11-13.

AAC(6’)-Ih was initially identified in a non-conjugative

Acinetobacter baumannii plasmid14 and later found to originate from Acinetobacter gyllenbergii12. These enzymes share significant sequence similarity, suggesting that they represent a set of functional paralogs11. Previous studies of AAC(6’)-Ig and –Ih focused on the identification of their potential to confer resistance to a small range of aminoglycosides (amikacin, gentamicin, tobramycin, netilmicin)12,14,15. The full range of acetylation of AGs and of other small molecules by AAC(6’) remains unexplored, and the molecular determinants of their activity have not been fully understood. Structural characterization of representatives of the AAC(6’)-I family demonstrated that these enzymes share a GCN5-N-acetyltransferase (GNAT) fold first described for the yeast GCN5 transcription factor16-23. Despite the structural conservation of the core GNAT fold, AAC(6’) enzymes demonstrate significant sequence variation; for example, AAC(6’)-Ig and Ih share only 15% sequence identity with AAC(6’)-Ib. Such a variation among members of this


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important AME family incited us to carry out additional structural and molecular studies of uncharacterized enzymes. We focused on the Acinetobacter-specific group of AAC(6’) and determined the crystal structures of the AAC(6’)-Ig and -Ih enzymes, including the structure of AAC(6’)-Ig in complex with tobramycin. We conducted extensive kinetic analysis of these enzymes against a panel of AG substrates.

Using these structural and functional data we propose a structure-based

classification for AAC(6’)-I family that postulates multiple modes of drug recognition, distinct substrate turnover kinetics, and a convergent evolutionary past.

Finally, our results were

consistent with the notion that the AAC(6’)-Ig and -Ih enzymes acetylate non-AG substrates.

RESULTS AND DISCUSSION

AAC(6’)-Ig and -Ih demonstrate activity against a broad range of aminoglycosides To characterize the substrate range of AAC(6’)-Ig and –Ih enzymes, we tested their ability to acetylate a diverse panel of 94 small molecule substrates containing amine groups using a previously described acetyltransferase activity assay24. Our results (Supporting Information Figure S1) showed acetylation of AG substrates but no activity against other small molecules except for thialysine and indicated that at least when tested against the small molecules in our screen, AAC(6’)-Ig and -Ih possess specificity for AGs. We further studied the activity of the two enzymes by carrying out a full kinetic characterization against an extensive panel of AG substrates using previously described procedures25,26. The results (Table 1) show that both enzymes are active against a broad range of AGs across the two classes, 4,6- and 4,5-disubstituted deoxystreptamines.

The enzymes



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exhibited some selectivity as both displayed activity toward amikacin, dibekacin, gentamicin, isepamicin, kanamycin B, 2’-N-ethyl netilmicin, sisomicin, tobramycin, butirosin A, neomycin C, and ribostamycin and did not show activity toward streptomycin, apramycin, hygromycin B, G418, paromomycin, and lividomycin. These results confirm the substrate profile and site specificity characteristics for AAC(6’)-I enzymes27. At higher concentrations of AGs, both –Ig and –Ih enzymes exhibited substrate inhibition for amikacin, isepamicin, kanamycin B, and butirosin A. The –Ih enzyme also showed substrate inhibition for sisomicin and neomycin C. Substrate activation was observed for all other substrates. The apparent pattern of substrate inhibition was noncompetitive relative to aminoglycoside substrate (see Supplementary Information for proposed model of inhibition). Additionally, both enzymes exhibited positive cooperativity toward many AGs as indicated by their respective Hill numbers (n>1) (Table 1). Observed substrate inhibition and activation of AAC(6’)-Ig and –Ih enzymes appears to be a common characteristic of aminoglycoside acetyltransferases 28-30. According to our enzymatic analysis, the AAC(6’)-Ig and -Ih enzymes demonstrated similar catalytic efficiencies that ranged from 1.84 x 103 demonstrated by AAC(6’)-Ih for gentamicin to 7.74 x 103 M-1s-1 demonstrated by AAC(6’)-Ig for sisomicin (Table 1, raw data and fitted curves shown in Figure S2). Of all AGs tested, -Ig displayed the highest catalytic efficiency toward sisomicin, neomycin C, isepamicin, and amikacin, and –Ih toward tobramycin, neomycin C, and ribostamycin. Both enzymes were least efficient in in vitro acetylation of gentamicin as compared to the other AGs. Notably, the analysis of in vitro kinetic parameters of these two enzymes did not reveal the trends previously observed for clinical isolates of A. haemolyticus and A. baumannii harboring aac(6’)-Ig or aac(6’)-Ih14,15. According to these previous studies, in vivo expression of


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both enzymes conferred low level (0.5 – 4 µg/mL) resistance to tobramycin and gentamicin, and higher level resistance to amikacin and netilmicin, with aac(6’)-Ih conferring much higher resistance to netilmicin as compared to aac(6’)-Ig according to the MIC values (128 versus 16). Conversely, in our in vitro assays the catalytic efficiency of AAC(6’)-Ih for netilmicin was actually lower than that of AAC(6’)-Ig. The in vitro catalytic efficiency of AAC(6’)-Ig was higher for amikacin (7.44 x 103 M-1 s-1) and netilmicin (5.30 x 103 M-1 s-1) than for tobramycin (3.86 x 103 M-1 s-1) and gentamicin (3.13 x 103 M-1 s-1), which is in line with reported MIC values. However, AAC(6’)-Ih displayed higher catalytic efficiencies for amikacin (4.98 x 103 M1 -1

s ) and tobramycin (7.59 x 103 M-1s-1) and lower efficiencies for netilmicin (2.00 x 103 M-1s-1)

and gentamicin (1.84 x 103 M-1s-1). The discrepancy between the observed in vitro kinetic parameters and previously reported in vivo resistance data suggests that additional factors such as different cell envelope penetration of specific AG molecules and possible presence of competing alternative substrates are playing active roles in shaping the actual level of resistance conferred by AAC(6’)-Ih and –Ig enzymes in clinical isolates. However, in this study we focused on analysis of biochemical properties of these enzymes in comparison with other representatives of AAC(6’) enzyme family. Next, we compared these kinetic values to those of previously characterized AAC(6’)-Iy, -Ib, -Ie and -Ii29-32. For this analysis we focused on activity against kanamycin as this AG features the basic 4,6-disubstituted deoxystreptamine scaffold with no substituents at positions 1 or 6’ (Table 2). All AAC(6’) enzymes exhibited similar micromolar apparent affinity for substrate but both AAC(6’)-Ig and -Ih enzymes showed significantly lower turnover rates. Similarly, the catalytic efficiencies of the AAC(6’)-Ig and -Ih enzymes were much lower than those established for the -Ii, -Ie and -Ib enzymes by 91-, 90- and 385-fold for AAC(6’)-Ig and



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114-, 112- and 481-fold for AAC(6’)-Ih. With respect to AAC(6’)-Iy, only the vmax value was reported30. AAC(6’)-Iy was shown to be two-fold less efficient than AAC(6’)-Ib18. Overall, comparative analysis of kinetic parameters indicates that while AAC(6’)-Ig and -Ih show broad specificity against AGs and that the apparent affinity of these enzymes for their substrates is similar to that of other AAC(6’)-I, the two Acinetobacter enzymes catalyze AG acetylation with significantly lower substrate turnover rates.

AAC(6’)-Ig and -Ih enzymes are dimers and adopt the GNAT fold In order to understand the molecular basis for the difference in catalytic properties of AAC(6’)-Ig and -Ih relative to other AAC(6’)-I enzymes, we determined their crystal structures. We solved the structures by the Molecular Replacement (MR) method using the structure of AAC(6’)-Iy as a search model and the X-ray crystallographic statistics for both structures are summarized in Table 3. The crystal structures of both enzymes were comprised of two polypeptides forming a dimer in their asymmetric units (Figure 1b). The enzymes adopt essentially the same structure and the AAC(6’)-Ig and -Ih dimers superimpose with RMSD of 0.87 Å over 246 Cα atoms. Each chain in both enzymes adopt the GNAT fold16 which was observed in every structurallycharacterized AAC enzyme; this fold is based on a central 6-stranded mixed β-sheet with two αhelices present between β1 and β2, a third α-helix between β4 and β5, and a fourth α-helix between β5 and β6. Both dimers feature domain-swapping with the C-terminal β-strand (β6) interdigitated between the β5 and β6 strands of the partner chain. The other major component of the dimer interface in both enzymes is formed by the β3-β4 loop corresponding to residues 63-75 of AAC(6’)-Ih and residues 62-74 of AAC(6’)-Ig, which pack against the partner chain and the


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domain-swapped region. Overall, the dimer interface in each structure accounts for over 2000 Å2 of solvent-occluded surface, which is consistent with our size-exclusion chromatography results showing that both AAC(6’)-Ig and -Ih are predominantly dimeric in solution (data not shown). Both AAC(6’)-Ig and –Ih structures contained two symmetry-related deep, negativelycharged clefts (~1400 Å3 in volume) that are formed by the core β-sheet of the GNAT fold (β3, β4, β5) and lined by the α1-α2 hairpin on one face and the β3’-β4’ loop from the partner chain on the other face (Figure 1c). Owing to previous studies of GNAT enzymes, this central pocket corresponds to the active sites of these AAC(6’)-I enzymes.

Tobramycin occupies only a small portion of the AAC(6’)-Ig substrate binding site Next we determined the structure of the AAC(6’)-Ig enzyme in complex with tobramycin, using the apo structure as search model. The crystallographic statistics are summarized in Table 3. The conformation of the AAC(6’)-Ig dimer in complex with tobramycin was essentially unchanged compared to the apoenzyme structure (RMSD of 0.67 Å over 238 matching Cα atoms). In accordance with the central cleft harboring the enzyme’s active center, each negatively-charged pocket of the AAC(6’)-Ig dimer contained well-defined additional electron density corresponding to a tobramycin molecule (Figures 1c, 2a). Tobramycin is held in place by stacking interactions on either face of the central 2-deoxystreptamine ring (ring II) involving Trp22 in the α1-α2 loop and Tyr65’ from the β3’-β4’ loop (Figure 2a). Notably, both these structural elements are also involved in enzyme’s dimerization interface. Ring I of the substrate appears to be involved in more contacts with the protein compared to ring III and the



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6’-amine. Altogether, the observed orientation of the tobramycin molecule in the active site is consistent with the regiospecificity of the AAC(6’)-I family. The crystal structure of the AAC(6’)-Ig•tobramycin complex allowed rationalization of the ability of this enzyme to acetylate the 1-N-substituted AG substrates such as amikacin, isepamicin and butirosin.

Modeling of 1-N-2-hydroxybutylylamine substitution onto the

tobramycin molecule bound in the enzyme’s active site produced no steric clashes, and showed that this drug can be accommodated into a pocket with Ala42 at its base (Figure 2b). While the analogous position in the AAC(6’)-Ih crystal structure is occupied by a larger residue (Arg42, Figure 2b) this pocket would still be able to accommodate 1-N-substituted AG substrates, consistent with this enzyme demonstrating comparable activity against amikacin, isepamicin and butirosin (Table 1). Analysis of the AAC(6’)-Ig•tobramycin complex structure provides a possible explanation of this enzyme’s activity against 4,5-disubstituted AGs (ribostamycin, butirosin and neomycin). The 5-group of tobramycin faces away from the active site and is exposed to solvent (Figure 2b) and thus the ribose rings of these substrates would not interfere with binding. Since we were unable to obtain an AAC(6’)-Ig or -Ih crystal structure in complex with acetyl-CoA or CoA, we modeled the binding of this acetyl group donor using the homologous AAC(6’)-Iy•ribostamycin•CoA complex structure (Supporting Information Figure S3). Analysis of the AAC(6’)-Ig•tobramycin•CoA complex model suggests that a significant portion of the electronegative central cleft of AAC(6’)-Ig remains unoccupied after binding of both cosubstrates (Supporting Information Figure S3 and Figure 1c). The unoccupied regions account for 410 and 480 Å3 in volume in the AAC(6’)-Ig and -Ih structures, respectively, and suggest that these enzymes may be able to accommodate substrates significantly larger than aminoglycosides.


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This unoccupied region may also represent the site of noncompetitive inhibition by aminoglycosides, but that site is currently unknown.

Comparative analysis of AAC(6’)-Ig, -Ih and -Iy suggests that these enzymes are orthologs Analysis of structural homologs of AAC(6’)-Ig and -Ih enzymes revealed four distinct groups (Table 4). The closest homologous group (Group A) was represented by the AAC(6’)-Iy structure, which superimposed with RMSD values of 1.3 and 1.3 Å with -Ig and -Ih, respectively. The next group (Group B) included AAC(6’)-Ii; this structure superimposed with those of–Ig and –Ih with RMSD values between 1.8 and 2.6 Å. Group C comprised more distant structural homologs, including AAC(6’)-Ib and -Ie. Finally, Group D consisted of the most distant homolog, AAC(2’)-Ic from Mycobacterium tuberculosis. Interestingly, the distribution of structural similarity between AAC(6’)-I enzymes correlated with their catalytic efficiencies towards AGs: AAC(6’)-Ig, -Ih and -Iy are the least efficient, followed by -Ii, -Ie and finally -Ib, which is the most efficient AAC(6’)-I. The AAC(6’)-Ig, -Ih and -Iy enzymes were also much less efficient than AAC(2’)-Ic, which showed a kcat/Km ratio of 0.38 x 106 M-1 s-1 against kanamycin B33. Thus, we reasoned that the difference in activity of AAC(6’)-Ig and -Ih could be rationalized via a detailed comparative analysis of the structures of AAC(6’)-I enzymes. To this end, we conducted separate structural comparisons of the AAC(6’)-Ig and -Ih versus the closest group A (i.e. versus -Iy) and group C (i.e. versus -Ib and –Ie); we did not compare -Ig and -Ih with group C as no member of this group has been crystallized in complex with an AG, and group D is only distantly similar. AAC(6’)-Ig, -Ih and -Iy adopt very similar dimeric domain-swapped structures including in the conformations of the key α1-α2 and β3-β4 loops that participate in the dimerization



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interfaces and active sites of these enzymes (Figure 3a). AAC(6’)-Iy also contains a large, electronegative active site cleft that was not occupied by either AG or CoA23, a feature consistent with the -Ig and -Ih enzymes (Figure 3b). We conducted a deeper comparison of the AG binding modes of the AAC(6’)-Ig and -Iy. Rings I and II of tobramycin and ribostamycin adopt the same spatial position when bound to the active sites of these two enzymes (Figure 3c). Likewise, the -Ig and -Iy enzymes provide identical interactions with these regions of the AG and involve nine amino acids, from the -Ig and -Iy enzymes, respectively: Trp22/Trp22 (from the key α1-α2 loop), Asp24/His25 (α1-α2 loop), Glu32/Asp33, Tyr65/Tyr66 (from the key β3-β4 loop), Asn67/Asn68 (β3-β4 loop), Glu78/Glu79, Asp114/Asp115, the backbone of Ala115/Thr116 and Glu135/Glu136 from Ig/Iy, respectively. Overall, this comparative analysis is consistent with a grouping of AAC(6’)-Ig, -Ih and -Iy into a subclass of AAC(6’)-I with a conserved overall structure and AG binding mode and similar catalytic efficiency. The fact that the large active site clefts of AAC(6’)-Ig, -Ih and -Iy remain only partially occupied by AG substrates reflects previous conjectures that these enzymes may acetylate an asyet unknown substrate in their natural biological role27,34. The aac(6’)-Iy gene is cryptic in Salmonella and was activated as an AG resistance enzyme following a large chromosomal deletion that brought the gene downstream from a strong promoter10. The AAC(6’)-Iy crystal structure revealed that its large cleft can accommodate a peptide23 and that this protein acetylates histone peptides. The aac(6’)-Iy gene is located close to open reading frames with predicted roles in carbohydrate metabolism; this lead to the suggestion that the native role of AAC(6’)-Iy is in sugar metabolism10. Combining these observations with structural analysis of the AAC(6’)Iy enzyme, we hypothesized that the large active site clefts of group A AAC(6’)-I enzymes may


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be evolved toward binding of substrates larger than aminoglycosides, such as polypeptide chains. To test this hypothesis and taking into account the predominantly anionic charge of the active sites of the -Ig and -Ih enzymes, we tested a small subset of readily available cationic tri- and tetra-peptides (i.e. KKK, VKKR, KKGE; see Methods) as possible substrates for these enzymes but we did not observe the formation of any acetylated products under our experimental conditions. This result however does not rule out polypeptides as possible substrates for these enzymes since protein acetyltransferases often demonstrate high specificity toward certain peptide length or sequence16. The exact chemical nature of native substrates for AAC(6’)–Ig and –Ih thus remain to be determined.

AAC(6’)-Ig dramatically differs in structure from the AAC(6’)-Ib and AAC(6’)-Ie enzymes According to our analysis AAC(6’)-Ig demonstrated significantly lower activity compared to the previously-characterized -Ib and -Ie enzymes. In order to identify the molecular features potentially responsible for such drastic differences we performed detailed comparative structural analysis of the group C AAC(6’)-Ib and -Ie versus -Ig. The AAC(6’)-Ib and -Ie structures superimpose with a single chain of AAC(6’)-Ig with RMSD of 3.0 Å and 3.0 Å over 133 and 136 matching Cα, respectively, confirming conservation of the GNAT fold (Figure 4a). The oligomeric states of these enzymes however dramatically differ: the AAC(6’)-Ig enzyme forms a stable dimer while AAC(6’)-Ib as well as the AAC(6’)-Ie enzymes are primarily monomeric. Accordingly, the latter enzymes lack the structural elements involved in dimerization of AAC(6’)-Ig. Specifically, this difference in oligomeric state could be traced to five structural features (Figure 4b): i) -Ib and -Ie are not domain-swapped; the sequence and conformation of the ii) α1-α2 loop and iii) β3-β4 loop; iv) the composition of aromatic



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residues in the AG binding site; and v) -Ib has a large proportion of aromatic residues in the AG binding site (four tryptophans and one tyrosine), while -Ig and -Ii have only two (Figure 4c). More importantly, we noticed significant differences between substrate binding sites of the AAC(6’)-Ig relative to the AAC(6’)-Ib and AAC(6’)-Ie enzymes. The antibiotic binding sites in AAC(6’)-Ib and -Ie (Figure 4c) are much smaller in volume than that of AAC(6’)-Ig, which is of a more extended nature. The AAC(6’)-Ib and AAC(6’)-Ie enzymes also provide stacking interactions with the drugs not seen in the AAC(6’)-Ig•tobramycin complex structure (i.e. Trp49 and Trp102 of the AAC(6’)–Ib enzyme stacks with ring I of kanamycin, Figure 4c) suggesting that these structural differences underpin the higher catalytic efficiency of the AAC(6’)-Ib and AAC(6’)-Ie enzymes compared to AAC(6’)-Ig and -Ih.

The Acinetobacter-specific class of AAC(6’)-I likely adopts the structure and function of AAC(6’)-Ig and -Ih Taking advantage of the crystal structures of AAC(6’)-Ig and -Ih, we extended our analysis to the eleven Acinetobacter-specific AAC(6’) proteins.

Significant sequence

conservation (Supporting Information Figure S4) between these enzymes and AAC(6’)-Ig and Ih suggests that they share overall structural architecture including dimerization, active site cleft and specific elements involved in AG substrate binding. The amino acids which interact with the drug in the AAC(6’)-Ig•tobramycin complex are conserved (Supporting Information Figure S3), implying the same binding mode for AGs. Finally, 3D models showed that these enzymes contain deep clefts akin to those in the active site clefts of AAC(6’)-Ig and -Ih (not shown), and that the sequence of the unoccupied regions of these clefts are also highly conserved.


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Altogether, this analysis suggests a common mechanism of AG binding and cellular functions across this family of Acinetobacter-specific AAC(6’)-I.

Structure- and function-based classification of AAC(6’)-I enzymes into three subclasses that separately evolved AAC(6’) functionality Our combined structural and functional comparative analysis of AAC(6’)-I family representatives is consistent with early sequence-based proposed categorization of this family into three distinct subfamilies35-38 by providing support from 3D structural and enzymological data. The first one (subfamily A) encompasses the structurally-characterized AAC(6’)-Ig, -Ih and -Iy and the other Acinetobacter-specific enzymes -Ig, -Ij, -Ik, -Ir, -Is, -It, -Iu, -Iv, -Iw and -Ix. According to our analysis, representatives of this group are dimeric enzymes containing large active site clefts only partially occupied by AGs substrates, and consequently these enzymes have relatively low AG turnover rates compared to the other groups of AAC(6’). Notably, this group likely possesses acetylation activity against non-AG substrates that could be accommodated into their extended active site clefts. Subfamily C is the most distantly related to group A and contains AAC(6’)-Ib and -Ie; these two enzymes are monomeric due to their lack of domain swapping and thus contain small active sites which appear to better suited to accommodate the chemical scaffold of AG substrates; accordingly, this group’s representatives demonstrate much higher AG acetylation efficiency.



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The third group (subfamily B) shows intermediate enzymatic efficiency toward AG substrates; it is represented by the dimeric AAC(6’)-Ii which demonstrates catalytic properties similar to those of AAC(6’)-Ie. Given the low sequence identity and vastly different active site architectures of these three subfamilies of AAC(6’)-I, the presence of these distinct groups may be due to convergent evolution from different ancestral proteins as multiple paths towards resistance against AGs35. These groups may have evolved from different housekeeping cellular functions, and our results suggest that the subfamily A representatives evolved from peptide, or protein acetyltransferases. As we showed for the nucleotidylyltransferase and peptidase folds and their roles in antibiotic resistance39,40, the observation of the recruitment of the GNAT fold for the evolution of multiple modes of AG acetylation is yet another example of a versatile reservoir for the evolution of antibiotic resistance enzymes. Since the GNAT fold’s adaptation towards at least the three observed modes of AG recognition, the subsequent dissemination of AAC(6’) enzymes has jeopardized the effectiveness of AGs for clinical use and illustrates the daunting task facing efforts in development of resistance-proof AGs.

METHODS

Cloning of the aac(6’)-Ig and aac(6’)-Ih genes and protein purification


Page 17 of 41

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The aac(6’)-Ig and the -Ih sequences were PCR amplified from A. haemolyticus BM2685 genomic DNA and A. baumannii plasmid pAT79 DNA, respectively, and cloned by ligaseindependent cloning in the p15TV-LIC vector which provided a N-terminal His6-tag fusion followed by a TEV protease cleavage site. Proteins were expressed natively following the protocol detailed in reference39.

Kinetic analysis The acetylation activity of AAC(6’)-Ig and -Ih was detected using the absorbance of 2nitro-5-thiobenzoate anion generated from 5,5’-Dithiobis(2-nitrobenzoic acid) that reacts to form a mixed disulfide with the product CoA of the acetylation reaction between the enzymes, acetylCoA and AGs or peptides. The full protocol for the assay is described in reference24. The reaction was performed under the following conditions: 50 mM Tris HCl buffer pH 8.0, 0.5 mM acetyl-CoA, varying concentrations of AGs from 0-1 mM, at 37 degrees C for 10 min. Substrate saturation curves showed substrate inhibition or substrate activation at higher concentrations of AG. Therefore, we determined the kinetic parameters for all substrates using nonlinear regression with the Levenberg-Marquardt algorithm in Origin 2016 by fitting data that corresponded to activity achieved prior to inhibition or activation using the Hill equation (equation 1) (Table 1). The BiHill equation (equation 2; derivation and model in Supporting Information Figure S5) was used to estimate the I0.5 (the concentration of inhibitor that gives half the maximal velocity) for AGs that displayed significant substrate inhibition (Table 1). The I0.5 was estimated from initial parameters, which were estimated automatically in Origin using dataset-specific parameter estimates from raw data. The I0.5 was then held constant as vmax, S0.5, nS (activation Hill coefficient), and nI (inhibitory Hill coefficient) were determined. vmax was



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converted to kcat using the molecular weights of the monomer as 16,448 Da and 16,709 Da for Ig and -Ih, respectively. (equation 1) (equation 2)

=

=

.

.

.

The following peptides were purchased from Bachem as the acetate salts and dissolved in water: H-Lys-Lys-Lys-OH, H-Val-Lys-Lys-Arg-OH, and H-Lys-Lys-Gly-Glu-OH. All peptides were assayed for activity as described above except that the concentration of peptide was held constant at 2.5 mM and no AG was included in the reaction. The concentration of the –Ig and –Ih enzymes were 0.45 µM and 0.57 µM for acetylation of AGs, and 2.29 µM and 2.87 µM for acetylation test against peptides, respectively.

Crystallization of AAC(6’)-Ig and AAC(6’)-Ih All crystals were grown at room temperature using the sitting-drop method.

The

apoenzyme form of AAC(6’)-Ig (residues 1-143, His6-tag removed, leaving a residual glycine residue from TEV cleavage site) was crystallized from 2 µL of a 6 mg/mL protein solution mixed with 2 µL of 0.2 M magnesium chloride, 0.1 M bis-Tris pH 5.5, 25% (w/v) PEG3350. The apoenzyme form of AAC(6’)-Ih (residues 1-146, His6-tag removed, leaving a residual glycine residue from TEV cleavage site) was crystallized from 0.5 µL of a 4 mg/mL protein solution mixed with 0.5 µL of 0.1 M bis-Tris pH 6.5, 20% (w/v) PEG5000 MME. For the AAC(6’)-Ig•tobramycin crystal, 6 mg/mL protein was mixed with 5 mM tobramycin and 0.5 µL of this solution was mixed with 0.5 µL of 0.05 M magnesium chloride, 0.05 M tri-sodium citrate dihydrate pH 4.6 and 25% (w/v) PEG3350. All crystals were cryoprotected with paratone oil.


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X-ray diffraction data for the AAC(6’)-Ig apoenzyme crystal were collected at 100 K at the Structural Genomics Consortium (Toronto) using a Rigaku FR-E Superbright rotating anode fitted with a Rigaku Saturn A200 CCD. X-ray diffraction data for the AAC(6’)-Ig•tobramycin complex and for the AAC(6’)-Ih apoenzyme were collected at 100 K on an in-house Rigaku Micromax 007-HF rotating anode fitted with a Rigaku RAXIS IV image plate detector. All xray data were reduced using HKL-300041.

The AAC(6’)-Ig and AAC(6’)-Ih apoenzyme

structures were solved by Molecular Replacement using a Phyre2-generated42 model of the sequence of these enzymes onto the structure of AAC(6’)-Iy (PDB 1S3Z, reference

23

) as the

search model using Phenix.phaser43. Structure refinement was performed using Phenix.refine plus manual building with Coot44. The crystal structure of the AAC(6’)-Ig•tobramycin complex was solved by Molecular Replacement using the AAC(6’)-Ig apoenzyme structure.

The

presence of tobramycin molecules was validated using omit maps: all atoms of the ligand plus other atoms within 5 Å of the ligand were deleted, followed by simulated annealing (Cartesian) using Phenix.refine with default parameters, followed by model building into residual Fobs – Fcalc density. All amino acids from the sequence of AAC(6’)-Ig and -Ih were visible in each structure. For all structures, B-factors were refined as anisotropic for protein atoms and isotropic for nonprotein atoms and TLS parameterization was included in the refinement. Average B-factor and bond angle/length RMSD values were calculated using Phenix. All geometry was verified using the Phenix, Coot and wwPDB validation tools. The structures have good backbone geometry with the following percentage of residues in the favored and allowed regions, respectively, of the Ramachandran plot: AAC(6’)-Ig apoenzyme: 97.6%, 2.4%; AAC(6’)-Ig•tobramycin complex: 97.4%, 2.6%; AAC(6’)-Ih apoenzyme: 98.0%, 2.0%.

The structure factors and atomic



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coordinates for the AAC(6’)-Ig (apoenzyme), AAC(6’)-Ig•tobramycin complex and AAC(6’)-Ih (apoenzyme) have been deposited in the Protein Data Bank with the identifiers 4F0Y, 4EVY and 4E8O, respectively.

Structure analysis and homology modeling Structure similarity searches of the Protein Data Bank were performed using the Dali server45. Interactions between AAC(6’)-Ig and tobramycin were identified in Coot. Proteinprotein interfaces were determined using the PDBePISA server46. Electrostatic potential surfaces were generated using Chimera47. The active site cleft volumes were calculated using the CASTp server48. Homology models of the Acinetobacter spp. AAC(6’)-Ij, -Ik, -Ir, -Is, -It, -Iu, -Iv, -Iw and -Ix enzymes were generated using the Phyre2 server and the structures of AAC(6’)-Ig or -Ih. The model of the AAC(6’)-Ig•tobramycin•CoA complex was generated by aligning the AAC(6’)-Ig•tobramycin and AAC(6’)-Iy•ribostamycin•CoA complexes and placing the coordinates from the CoA molecule of the latter complex into the former complex.

FIGURES


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Figure 1. Crystal structures of AAC(6’)-Ig and AAC(6’)-Ih. a) Chemical structures of a representative 4,6-disubstituted AG (tobramycin) and a 4,5disubstituted AG (ribostamycin). The enzymatic reaction catalyzed by AAC(6’) enzymes and the added N-acetyl group at the 6’ position are shown. b) Cartoon representations of the AAC(6’)-Ig (left) and -Ih (right) dimers, each chain coloured differently. Termini and secondary structure elements are labeled.



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c) Electrostatic surface representation of AAC(6’)-Ig•tobramycin complex (left) and –Ih apoenzyme (right). Scale bar is indicated. Dashed boxed region with asterisk indicates putative acetyl-CoA binding sites.

Tobramycin bound to AAC(6’)-Ig is shown as cyan sticks and

acetylation site (6’-amine) is circled. The unoccupied region of the AAC(6’)-Ig active site cleft is indicated by a solid box.


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Figure 2. Recognition of aminoglycosides by AAC(6’)-Ig and -Ih. a) Interactions between AAC(6’)-Ig and tobramycin. The electron density for tobramycin is a simulated annealing Fobs – Fcalc omit map contoured at 2.0 σ. b) Binding pocket for 1-N-2-hydroxybutyrlylamine substituents on aminoglycosides in AAC(6’)Ig (left) and –Ih (right). Asterisk indicates space that would accommodate such a substituent. Tobramycin, residues Ala42 and Arg42 are shown in sticks.



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Figure 3. Comparison of the structures of AAC(6’)-Ig, -Ih and -Iy. a) Overall comparison. Key loops involved in dimerization and AG binding are labeled with boxes. b) Details of AAC(6’)-Ig and -Iy showing unoccupied active site clefts. Chain A from each dimer is shown in surface representations and chain B as cartoon.

Tobramycin (left) and 24


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ribostamycin (right) are shown as sticks and CoA as thin lines. Empty/unoccupied regions are boxed in white. Key secondary structure elements involved in AG binding and dimerization are labeled and coloured (light blue and yellow for α1-α2 and β3’- β4’ loops, respectively). c) Detailed comparison of AAC(6’)-Ig and -Iy aminoglycoside binding sites. Tobramycin is shown in black sticks, ribostamycin in grey sticks.



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Figure 4. Comparison of the structures of AAC(6’)-Ig, -Ib and -Ie. a) Overall comparison showing conservation of GNAT fold of chain A of –Ig dimer and the monomers of -Ib and -Ie. b) Details of AAC(6’)-Ig, -Ib and -Ie showing AG binding. For –Ig, chain A is shown in surface representations and chain B as cartoon; for –Ib and –Ie the monomers are shown in surface. Tobramycin (left), kanamycin (center and right) are shown as sticks and CoA as thin lines. Empty/unoccupied region in AAC(6’)-Ig is boxed in white. Key secondary structure elements involved in AG binding are labeled and coloured (light blue and yellow for α1-α2 and β3-β4/β3’26 ACS Paragon Plus Environment

Page 27 of 41

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β4’ loops, respectively). c) Detailed comparison of AAC(6’)-Ig, -Ib and -Ie AG binding sites. Tobramycin is shown in black sticks, kanamycin in grey sticks. Note that the composition of the AG binding site AAC(6’)-Ig differs dramatically from those of the two other enzymes. AAC(6’)-Ie and -Ib form much more intimate contacts with kanamycin and contain more aromatic residues than –Ig. AAC(6’)-Ie was crystallized in complex with sulfinic-CoA (PDB 4QC6).



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Page 28 of 41

Table 1. Kinetic parameters and MIC values of AAC(6’)-Ig and AAC(6’)-Ih for aminoglycoside acetylation. The substrate concentration range used for fitting each model is shown (See Materials and Methods for more information). Substrate saturation curves displayed substrate inhibitiona or activationb at concentrations that exceeded the range used for determining kinetic parameters from fitting the Hill equation. If substrate inhibition was observed, kinetic parameters were estimated from fitting the BiHill equation that takes into account substrate inhibition. S0.5 and I0.5 are defined as the concentration of substrate or inhibitor at half the maximal velocity, n is the Hill number calculated from the fitting of the Hill equation, nS and nI correspond to the activation and inhibition Hill coefficients from the BiHill equation, respectively. Hill equation % Aminoglycoside

AAC (6’)-

MIC (µg/mL) **

kcat (s-1)

BiHill equation # kcat/S0.5 (M-1s-1) x 103

S0.5 (µM)

n

[S] range (µM)

kcat (s1 )

kcat/S0.5 (M-1s-1) x 103

I0.5 (µM)&

0.382 0.407

40.0 57.1

± ±

4.3 11.0

9.55 7.13

254 220

2.02 1.84

± ±

0.32 0.35

0.92 1.51

± ±

0.12 0.20

0-1000 0-1000

1.56 1.17 0.352 0.100

168 186 51.4 15.2

± ± ± ±

38 22 9.9 3.6

9.29 6.29 6.85 6.58

480 665 150 53.8

1.42 1.40 1.62 2.21

± ± ± ±

0.17 0.09 0.21 0.71

0.73 0.80 1.27 0.93

± ± ± ±

0.16 0.12 0.29 0.24

0-1000 0-1000 0-200 0-200

0.065

9.8

±

1.6

6.63

73.2

2.62

±

0.97

0.60

±

0.23

0-200

0.210 0.202

26.5 37.9

± ±

8.0 10.7

7.92 5.33

84.4 125

1.82 1.67

± ±

0.57 0.38

0.83 1.79

± ±

0.30 0.44

0-200 0-200

0.106

12.1

±

5.5

8.76

40

1.89

±

0.90

1.32

±

0.51

0-100

S0.5 (µM)

nS

[S] range (µM)

nI

4,6-disubstituted amikacin arbekacin dibekacin gentamicin* isepamicin kanamycin B 2’-N-ethyl netilmicin sisomicin tobramycin

Iga Iha Igb Ihb Igb Ihb Igb Ihb Iga Iha Iga Iha Igb Ihb Igb Iha Igb Ihb

8 32

0.5 1

16 128

8 4

0.328 0.496 0.077 0.021 0.127 0.040 0.055 0.016 0.779 0.620 0.181 0.060& 0.097 0.098& 0.130 0.051 0.085 0.022

44.1 99.6 13.9 5.9 25.9 9.8 17.6 8.7 104 111 29.0 12.0 18.3 49.0 16.8 10.5 22.0 2.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5.6 12.5 1.1 0.8 2.5 1.4 2.7 3.1 15 13 3.4 1.1 1.5 5.9 2.0 0.8 2.9 1.0

7.44 4.98 5.54 3.56 4.90 4.08 3.13 1.84 7.49 5.59 6.24 5.00 5.30 2.00 7.74 4.86 3.86 7.59

1.55 1.26 2.19 2.5& 1.26 1.42 2.05 1.84 1.45 1.48 1.93 2.30 2.28 1.39 2.04 1.93 1.21 2.12

± ± ±

0.15 0.05 0.29

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.09 0.20 0.51 1.15 0.16 0.13 0.32 0.42 0.35 0.22 0.43 0.19 0.11 1.44

0-100 0-100 0-50 0-50 0-100 0-50 0-100 0-50 0-400 0-400 0-100 0-30 0-100 0-200 0-200 0-30 0-100 0-50

0.107& 0.113& 0.057 0.060& 0.072 0.044

17.3 21.6 7.4 7.9 11.8 6.2

± ± ± ± ± ±

2.8 2.4 0.5 1.0 1.7 1.5

6.18 5.23 7.70 7.59 6.10 7.10

1.77 2.03 1.43 2.05 1.96 1.92

± ± ± ± ± ±

0.52 0.45 0.11 0.5 0.43 0.82

0-100 0-100 0-50 0-30 0-50 0-100

4,5-disubstituted butirosin A neomycin C ribostamycin

Iga Iha Igb Iha Igb Ihb

*

commercially available gentamicin is a mix of gentamicins C1, C1a and C2. MIC values for amikacin, 2’-N-ethyl-netilmicin, tobramycin and gentamicin versus Acinetobacter haemolyticus BM2685 harboring aac(6’)-Ig and Acinetobacter baumannii BM2686 harboring aac(6’)-Ih were measured in references14,15. No MIC values have been reported for all other antibiotics displayed in the table. & fixed parameter during model fitting in Origin. See Materials and Methods. % equation 1 and #equation 2 are shown in Materials and Methods. **


Page 29 of 41

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 41

Table 2 – Comparison of kinetic parameters of AAC(6’)-I enzymes for acetylation of kanamycin AAC(6’)Enzyme

kcat (s1 )

Fold change vs. AAC(6’)-Ig

Apparent affinity for substrate


Catalytic efficiency


Ref.

1

This study

0.80

This study

**

**

30

(M-1 s-1)

(µM) Ig

0.181*

1

29.0

1

Ih

0.060*

0.33

12.0

0.41

Iy

**

**

105*

6.24 x 103

5.00 x 103

3.62 Ii

1.08*

18.9

0.65

570 x 103

91.3

29

31

1.07

560 x 103

89.7

32

1.0

0.03

2.4 x 106

385

31

5.97 Ie

1.7 9.39

Ib

2.4 13.3

*tested against kanamycin B. No asterisk = tested against kanamycin A. ** = values could not be calculated due to original presentation as Vmax.

Table 3 – X-ray crystallographic statistics PDB Code Data collection Space group Cell dimensions a, b, c, Å α, β, γ, ° Resolution, Å Rsyma I / σ(I) Completeness, % Redundancy Refinement Resolution, Å No. of unique reflections: working, test R-factor/free R-factorc No. of refined molecules Protein Tobramycin

AAC(6’)-Ig

AAC(6’)-Ig•tobramycin

AAC(6’)-Ih

4F0Y

4EVY

4E8O

P1

C2

P1

38.0, 44.0, 46.6 83.2, 87.4, 72.7 25.0 – 2.56 0.039 (0.133)b 8.21 (2.10) 96.9 (94.6) 2.0 (1.9)

39.7, 43.8, 46.3 83.7, 87.5, 68.4 29.0 – 1.77 0.051 (0.658) 40.79 (2.91) 93.5 (87.3) 3.8 (3.8)

40.0, 46.0, 46.1 102.6, 97.1, 111.2 25.0 – 2.00 0.033 (0.151) 11.65 (2.05) 95.6 (92.0) 2.0 (1.9)

24.1 – 2.56 8893, 435

29.0 – 1.77 25205, 1245

23.5 – 2.14 15377, 770

18.4/24.0 (25.8/37.8)

16.9/21.3 (34.7/35.9)

18.8/22.8 (21.2/24.7)

2316 N/A 2

2370 64 N/A

2384 N/A N/A

atoms,


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Magnesium N/A 2 N/A Potassium 3 8 3 Chlorine 12 N/A N/A Glycerol 84 227 139 Water B-factors Protein 39.5 35.9 37.0 Tobramycin N/A 30.6 N/A Magnesium 47.5 N/A N/A Potassium N/A 30.8 N/A Chlorine 62.8 35.5 72.1 Glycerol 69.9 N/A N/A Water 26.5 39.5 40.5 r.m.s.d. Bond lengths, Å 0.004 0.012 0.004 0.655 1.356 0.805 Bond angles, ° a Rsym = ΣhΣi|Ii(h) - 〈I(h)〉/ΣhΣiIi(h), where Ii(h) and 〈I(h)〉 are the ith and mean measurement of the intensity of reflection h. b

Figures in parentheses indicate the values for the outer shells of the data.

c

R = Σ|Fpobs – Fpcalc|/ΣFpobs, where Fpobs and Fpcalc are the observed and calculated structure factor amplitudes, respectively.

Table 4 – Structural homologs of AAC(6’)-Ig and AAC(6’)-Ih Protein

PDB code(s)

RMSD (Å)

Sequence Identity (%)

AAC(6’)-Ig

AAC(6’)-Ih

AAC(6’)-Ig

AAC(6’)-Ih

1S3Z, 1S60, 2VBQ, 1S5K

1.3

1.3

40

43

Histone acetyltransferase HPA2 (Saccharomyces cerevisiae)

1QSM, 1QSO

2.1

2.1

12

12

Other acetyltransferases

3MGD, 3FYN, 2PDO, 2Q4V, 2DXQ, 2EUI, 3DSB, 4QVT, 2BEI, others

1.8-2.5

1.8-2.4

12-21

12-20

AAC(6’)-Ii (Enterococcus faecium)

1B87, 2A4N, 1N71

2.4-2.8

2.2-2.8

11-12

16-17

Group A AAC(6’)-Iy (Salmonella enterica) Group B



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Page 32 of 41

AAC(3)-

1BO4

2.7

2.6

19

17

BH1408 (Bacillus halodurans)

4F6A, 4E0A

2.7

2.7

10

10

BH1968 (Bacillus halodurans)

1Z4E

2.6

2.8

16

17

AAC(3)-Ib

4YFJ

2.8

2.8

17

16

AAC(6’)-Ib

2PRB, 2QIR, 1V0C

2.9-3.0

2.9-3.1

15

12-15

AAC(6’)-Ie

4QC6

3.0

2.9

11

13

Putative AAC(6’) (Legionella pneumophila)

3F5B

3.1

3.2

12

11

1M4I, 1M4D, 1M44, 1M4G, 1M4I

3.7

3.5-3.7

13

12

Group C

Group D AAC(2’)-Ic (Mycobacterium tuberculosis)

ABBREVIATIONS AAC = Aminoglycoside N-acetyltransferase; AAC(6’) = Aminoglycoside 6’-N-acetyltransferase; AG = aminoglycoside; AME = aminoglycoside-modifying enzyme; GNAT = Gcn5-related-Nacetyltransferase; MIC = minimum inhibitory concentration; PDB = Protein Databank.

AUTHOR INFORMATION Corresponding Author #

(A.S.) E-mail: [email protected].

%

M.L.K present address

Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132, USA. Author contribution


Page 33 of 41

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*

P.J.S. and M.L.K. equally contributed to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENT We thank V. Yim and O. Egorova for technical assistance, D. M.-Cherif and G. Gassner for helpful discussions, A. Dong for X-ray data collection and G. Minasov for structure refinement assistance. This project has been funded in whole or in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract Nos. HHSN272200700058C (September 1, 2007 to September 27, 2012) and HHSN272201200026C (starting September 1, 2012).

SUPPORTING INFORMATION One file includes: Table S1 - substrates for N-acetyltransferase screen; Figure S1 - N-

screening reaction and results; Figure S2 - fitted curves and raw data for kinetic characterization; Figure S3 - model of ternary complex of AAC(6’)-Ig; Figure S4 – multiple sequence alignment; Figure S5 – model and its derivation for substrate inhibition; reference page. This information is available free of charge via the Internet at http://pubs.acs.org/.

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a

tobramycin Page 37 of 41 Acetyl-CoA AAC(6’)

O

CoA

ribostamycin ACS Infectious Diseases Acetyl-CoA AAC(6’)

CoA

O

1 N 2 N 6’ 6’ 3 5 4’ O ring I O HO 4 HO H2N NH2 HO 4 2’ ring II 5 ring I 4 4 3 3 H2N H N 2 O O 1 NH2 6 1 NH2 O HO 5 HO 5 7 O OH ring II 6” O OH 8 O 9 4” ring III 2” 10 HO OH OH NH2 HO 11 b 12 AAC(6’)-Ig AAC(6’)-Ih 13 β3-β4 14 loop α2 β2 α2 β2 15 β1 α1 β1 C β3 β3 16 α1 β4 β4 17 N β5 α3 β5 N 18 C’ C’ α3 19 20 β6 β6 21 α1-α2 α4 α4 loop 22 23 AAC(6’)-Ig•tobramycin AAC(6’)-Ih c 24 25 26 27 28 29 30 31 * 32 * 33 ACS Paragon Plus Environment 34 tobramycin -10 +10 kB/ T 35

C

AAC(6’)-Ig•tobramycin Page 38 of 41 E78ACS Infectious Diseases

a

D114

6’

3

W22 (α1-α2 loop)

1

R18

2” 1 Cl 2 3” N67 3 β3-β4 loop 2’ Y65 β3-β4 loop 4 (chain B) (chain B) D24 tobramycin 5 (α1-α2 loop) E135 (chain B) 6 b 7AAC(6’)-Ig•tobramycin AAC(6’)-Ih 8 A42 9 R42 10 * 11 1 12 2” 13 4’ 3 ACS Paragon Plus Environment 5 3” 6’ 14 tobramycin 15 A115

-

E32

Page 39 of 41

α1-α2 loops


AAC(6’)-Iy•ribostamycin•CoA

chain A

chain B

chain A

chain B

pt y

1 2 3 4 5 6 7 8 9 10 b11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 c 28 29 30 31 32 33 34 35 36 37 38 39 40

AAC(6’)-Ig chain A/chain B ACS chain Infectious Diseases AAC(6’)-Ih A/chain B AAC(6’)-Iy chain A/chain B

β3-β4 loops

em

a

α1-α2 loop

α1-α2 loop

β3-β4 loop (chain B)


6’ 3

A115

N67 (β3-β4 loop chain B) E135’

β3-β4 loop (chain B)

E79

CoA W22 R18 (α1-α2 loop) 1 2” 5

Cl-

CoA

AAC(6’)-Iy•ribostamycin•CoA

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D114

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D115

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3” SO42N68 (β3-β4 loop Environment chain B) T116

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ACS Paragon Plus

Y65 (β3-β4 loop chain B)

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W22 (α1-α2 loop)

D24 (α1-α2 loop)

2’

Y66 (β3-β4 loop E136’ chain B)

2” 3”

H25 (α1-α2 loop)

a

AAC(6’)-Ig chain A/chain B Infectious AAC(6’)-IeACS monomer AAC(6’)-Ib monomer

Diseases

Page 40 of 41

em

pt y

1 2 3 4 5 6 b7 AAC(6’)-Ig•tobramycin AAC(6’)-Ib•kanamycin•acetyl-CoA AAC(6’)-Ie•kanamycin•CoA 8 9 chain A chain B 10 11 12 13 14 15 16 17 18 α1-α2 α1-α2 α1-α2 19 CoA β3-β4 β3-β4 loop β3-β4 loop loop CoA 20 loop (chain B) loop loop 21 c22 AAC(6’)-Ig•tobramycin AAC(6’)-Ib•kanamycin•acetyl-CoA AAC(6’)-Ie•kanamycin•CoA E78 23 Q91 Y93 W22 D99 24 R18 Q74 (α1-α2 loop) E73 25 D114 6’ 1 L82 CoA* D115 26 3 E32 2” W103 Y86 27 Y34 AcCoA 3” 5 Cl 28 A115 6’ N67 29 6’ 2’ D152 (β3-β4 loop) 30 W49 D24 W102 D136 Y65 31 G50 (β3-β4 loop) (α1-α2 loop) ACS Paragon Plus Environment 32 E135 G35 Q163 33 α1-α2 α1-α2 W48 F33 loop 34 loop -

For Table of Contents Use Only “Structural and biochemical characterization of Acinetobacter spp. Page 41 ofACS 41 Infectious Diseases aminoglycoside acetyltransferases highlights functional and

evolutionary variation among antibiotic resistance enzymes” by Peter J. Stogios, Misty L. Kuhn, Elena Evdokimova, Melissa Law, Patrice Courvalin and Alexei Savchenko

1 3 families of 2aminoglycoside resistance acetyltransferases antibiotics 3 4 6’NH2 N-acetyl O O HO5 HO NH2 NH2 Ig H2N O O 6H2N HO NH2 NH2 HO OH OH O O 7 O O 8 OH OH Ib ACS Plus Environment HO NHParagon HO NH2 2 9 10 ribosome binding

Ie

ribosome binding

Structural and Biochemical Characterization of Acinetobacter spp

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