RNA Chemical Labeling with Site-Specific, Relative Quantification by Mass Spectrometry for the Structural Study of a Neomycin-Sensing Riboswitch Aptamer Domain.

High-resolution mass spectrometry was used for the label-free, direct localization and relative quantification of CMC+ -modifications of a neomycin-sensing riboswitch aptamer domain in the absence and presence of the aminoglycoside ligands neomycin B, ribostamycin, and paromomycin. The chemical probing and MS data for the free riboswitch show high exposure to solvent of the uridine nucleobases U7, U8, U13, U14, U18 as part of the proposed internal and apical loops, but those of U10 and U21 as part of the proposed internal loop were found to be far less exposed than expected. Thus, our data are in better agreement with the proposed secondary structure of the riboswitch in complexes with aminoglycosides than with that of free RNA. For the riboswitch in complexes with neomycin B, ribostamycin, and paromomycin, we found highly similar CMC+ -modification patterns and excellent agreement with previous NMR studies. Differences between the chemical probing and MS data in the absence and presence of the aminoglycoside ligands were quantitative rather than qualitative (i. e., the same nucleobases were labeled, but to different extents) and can be rationalized by stabilization of both the proposed bulge and the apical loop by aminoglycoside binding. Our study shows that chemical probing and mass spectrometry can provide important structural information and complement other techniques such as NMR spectroscopy.

Molecular mechanisms for dynamic regulation of N1 riboswitch by aminoglycosides.

A synthetic riboswitch N1, inserted into the 5'-untranslated mRNA region of yeast, regulates gene expression upon binding ribostamycin and neomycin. Interestingly, a similar aminoglycoside, paromomycin, differing from neomycin by only one substituent (amino versus hydroxyl), also binds to the N1 riboswitch, but without affecting gene expression, despite NMR evidence that the N1 riboswitch binds all aminoglycosides in a similar way. Here, to explore the details of structural dynamics of the aminoglycoside-N1 riboswitch complexes, we applied all-atom molecular dynamics (MD) and temperature replica-exchange MD simulations in explicit solvent. Indeed, we found that ribostamycin and neomycin affect riboswitch dynamics similarly but paromomycin allows for more flexibility because its complex lacks the contact between the distinctive 6' hydroxyl group and the G9 phosphate. Instead, a transient hydrogen bond of 6'-OH with A17 is formed, which partially diminishes interactions between the bulge and apical loop of the riboswitch, likely contributing to riboswitch inactivity. In many ways, the paromomycin complex mimics the conformations, interactions, and Na+ distribution of the free riboswitch. The MD-derived interaction network helps understand why riboswitch activity depends on aminoglycoside type, whereas for another aminoglycoside-binding site, aminoacyl-tRNA site in 16S rRNA, activity is not discriminatory.

Linear self-assembly formation between gold nanoparticles and aminoglycoside antibiotics.

Ribostamycin is a broad-spectrum aminoglycoside antibiotic with a molecular weight of 454.5 g/mol. Under neutral pH conditions, ribostamycin is highly positive charged because it carries multiple amino groups in its structure. Negatively charged citrate ligand capped-gold nanoparticles (AuNPs) have been studied extensively for their interactions with a wide range of biomolecules including proteins, carbohydrates, and small drug compounds. These studies are aimed at developing new therapeutics and diagnostics by exploiting the unique properties of gold nanoparticles. Under this general aim, we studied the interaction between ribostamycin and AuNPs. Using a suite of analytical techniques including dynamic light scattering (DLS), UV-vis absorption spectroscopy, and dark field optical microscope imaging (DFM), we analyzed the mixture products of AuNPs with various sizes and ribostamycin under different concentrations. Our study revealed for the first time that ribostamycin has a tendency to self-assemble into linear oligomers at increased concentrations (above 250-500 µM). Such self-assembled oligomers then interact with negatively charged AuNPs to produce rod-like AuNP assemblies. Similar findings were observed from another structurally related aminoglycoside antibiotic, amikacin. It is technically challenging to detect and characterize oligomer formation of small molecules. It is especially challenging when the interactions that are holding the oligomers are not very strong. Through their interaction with gold nanoparticles that have exceptionally strong light scattering properties, we were able to observe the self-assembling of ribostamycin and amikacin in solution using various spectroscopic and microscopic techniques. This concentration-dependent self-assembling behavior of ribostamycin and amikacin may have direct relevance to the antibiotic effect of ribostamycin, amikacin and other structurally similar antibiotics.

Single lipoaminoglycoside promotes efficient intracellular antibody delivery: A comprehensive insight into the mechanism of action.

Delivery of biologically active proteins into cells is emerging as important strategy for many applications. Previous experiments have shown that lipoaminoglycosides were capable of delivery of the anti-cytokeratin8 antibody (anti-K8) but only when formulated with lipid helpers potentially leading to toxicity from excess lipids. Here, we optimized anti-K8 delivery with various lipoaminoglycosides in the absence of a lipid helper. Results led to the identification of the aminoglycoside lipid dioleyl phosphoramido ribostamycin (DOPRI) as a potent intracellular delivery system for anti-K8. Electron microscopy revealed that delivered anti-K8 molecules were bound to intermediate filaments in cells. Anti-K8 was bound to the surface of DOPRI vesicles without perturbing lipid organization. Macropinocytosis and caveolin mediated endocytosis contributed to anti-K8 internalization and to filament labeling with a major contribution being made by the caveolin pathway. The results showed that the unique properties of DOPRI were sufficient for efficient intracellular protein delivery without requiring lipid helpers.

The structure of RbmB from Streptomyces ribosidificus, an aminotransferase involved in the biosynthesis of ribostamycin.

Aminoglycoside antibiotics represent a classical group of antimicrobials first discovered in the 1940s. Due to their ototoxic and nephrotoxic side effects, they are typically only used against Gram negative bacteria which have become resistant to other therapeutics. One family of aminoglycosides includes such compounds as butirosin, ribostamycin, neomycin, and kanamycin, amongst others. The common theme in these antibiotics is that they are constructed around a chemically stable aminocyclitol unit referred to as 2-deoxystreptamine (2-DOS). Four enzymes are required for the in vivo production of 2-DOS. Here, we report the structure of RbmB from Streptomyces ribosidificus, which is a pyridoxal 5'-phosphate dependent enzyme that catalyzes two of the required steps in 2-DOS formation by functioning on distinct substrates. For this analysis, the structure of the external aldimine form of RbmB with 2-DOS was determined to 2.1 Å resolution. In addition, the structure of a similar enzyme, BtrR from Bacillus circulans, was also determined to 2.1 Å resolution in the same external aldimine form. These two structures represent the first detailed molecular descriptions of the active sites for those aminotransferases involved in 2-DOS production. Given the fact that the 2-DOS unit is widespread amongst aminoglycoside antibiotics, the data presented herein provide new molecular insight into the biosynthesis of these sugar-based drugs.

Abundance and distribution of antibiotic resistance genes in a full-scale anaerobic-aerobic system alternately treating ribostamycin, spiramycin and paromomycin production wastewater.

The occurrence of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs) has been intensively investigated for wastewater treatment systems treating single class of antibiotic in recent years. However, the impacts of alternately occurring antibiotics in antibiotic production wastewater on the behavior of ARGs in biological treatment systems were not well understood yet. Herein, techniques including high-capacity quantitative PCR and quantitative PCR (qPCR) were used to investigate the behavior of ARGs in an anaerobic-aerobic full-scale system. The system alternately treated three kinds of antibiotic production wastewater including ribostamycin, spiramycin and paromomycin, which referred to stages 1, 2 and 3. The aminoglycoside ARGs (52.1-79.3%) determined using high-capacity quantitative PCR were the most abundant species in all sludge samples of the three stages. The total relative abundances of macrolide-lincosamide-streptogramin (MLS) resistance genes and aminoglycoside resistance genes measured using qPCR were significantly higher (P < 0.05) in aerobic sludge than in sewage sludge. However, the comparison of ARGs acquired from three alternate stages revealed that MLS genes and the aminoglycoside ARGs did not vary significantly (P > 0.05) in both aerobic and anaerobic sludge samples. In aerobic sludge, one acetyltransferase gene (aacA4) and the other three nucleotidyltransferase genes (aadB, aadA and aadE) exhibited positive correlations with intI1 (r 2 = 0.83-0.94; P < 0.05), implying the significance of horizontal transfer in their proliferation. These results and facts will be helpful to understand the abundance and distribution of ARGs from antibiotic production wastewater treatment systems.

Potentiation of Aminoglycoside Activity in Pseudomonas aeruginosa by Targeting the AmgRS Envelope Stress-Responsive Two-Component System.

A screen for agents that potentiated the activity of paromomycin (PAR), a 4,5-linked aminoglycoside (AG), against wild-type Pseudomonas aeruginosa identified the RNA polymerase inhibitor rifampin (RIF). RIF potentiated additional 4,5-linked AGs, such as neomycin and ribostamycin, but not the clinically important 4,6-linked AGs amikacin and gentamicin. Potentiation was absent in a mutant lacking the AmgRS envelope stress response two-component system (TCS), which protects the organism from AG-generated membrane-damaging aberrant polypeptides and, thus, promotes AG resistance, an indication that RIF was acting via this TCS in potentiating 4,5-linked AG activity. Potentiation was also absent in a RIF-resistant RNA polymerase mutant, consistent with its potentiation of AG activity being dependent on RNA polymerase perturbation. PAR-inducible expression of the AmgRS-dependent genes htpX and yccA was reduced by RIF, suggesting that AG activation of this TCS was compromised by this agent. Still, RIF did not compromise the membrane-protective activity of AmgRS, an indication that it impacted some other function of this TCS. RIF potentiated the activities of 4,5-linked AGs against several AG-resistant clinical isolates, in two cases also potentiating the activity of the 4,6-linked AGs. These cases were, in one instance, explained by an observed AmgRS-dependent expression of the MexXY multidrug efflux system, which accommodates a range of AGs, with RIF targeting of AmgRS undermining mexXY expression and its promotion of resistance to 4,5- and 4,6-linked AGs. Given this link between AmgRS, MexXY expression, and pan-AG resistance in P. aeruginosa, RIF might be a useful adjuvant in the AG treatment of P. aeruginosa infections.

Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions.

Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin-paromomycin, ribostamycin and neamine-each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6'-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6'-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin-ribosome complex, we observe specific contacts between the apical tip of H69 and the 6'-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.

Computational Methods for RNA Structure Validation and Improvement.

With increasing recognition of the roles RNA molecules and RNA/protein complexes play in an unexpected variety of biological processes, understanding of RNA structure-function relationships is of high current importance. To make clean biological interpretations from three-dimensional structures, it is imperative to have high-quality, accurate RNA crystal structures available, and the community has thoroughly embraced that goal. However, due to the many degrees of freedom inherent in RNA structure (especially for the backbone), it is a significant challenge to succeed in building accurate experimental models for RNA structures. This chapter describes the tools and techniques our research group and our collaborators have developed over the years to help RNA structural biologists both evaluate and achieve better accuracy. Expert analysis of large, high-resolution, quality-conscious RNA datasets provides the fundamental information that enables automated methods for robust and efficient error diagnosis in validating RNA structures at all resolutions. The even more crucial goal of correcting the diagnosed outliers has steadily developed toward highly effective, computationally based techniques. Automation enables solving complex issues in large RNA structures, but cannot circumvent the need for thoughtful examination of local details, and so we also provide some guidance for interpreting and acting on the results of current structure validation for RNA.

Epimerization at C-3'' in butirosin biosynthesis by an NAD(+) -dependent dehydrogenase BtrE and an NADPH-dependent reductase BtrF.

Butirosin is an aminoglycoside antibiotic consisting two epimers at C-3'' of ribostamycin/xylostasin with a unique 4-amino-2-hydroxybutyrate moiety at C-1 of the aminocyclitol 2-deoxystreptamine (2DOS). To date, most of the enzymes encoded in the biosynthetic gene cluster for butirosin, from the producing strain Bacillus circulans, have been characterized. A few unknown functional proteins, including nicotinamide adenine dinucleotide cofactor-dependent dehydrogenase/reductase (BtrE and BtrF), are supposed to be involved in the epimerization at C-3'' of butirosin B/ribostamycin but remain to be characterized. Herein, the conversion of ribostamycin to xylsostasin by BtrE and BtrF in the presence of NAD(+) and NADPH was demonstrated. BtrE oxidized the C-3'' of ribostamycin with NAD(+) to yield 3''-oxoribostamycin. BtrF then reduced the generated 3''-oxoribostamycin with NADPH to produce xylostasin. This reaction step was the last piece of butirosin biosynthesis to be described.

Thermodynamic characterization of a thermostable antibiotic resistance enzyme, the aminoglycoside nucleotidyltransferase (4').

The aminoglycoside nucleotidyltransferase (4') (ANT) is an aminoglycoside-modifying enzyme that detoxifies antibiotics by nucleotidylating at the C4'-OH site. Previous crystallographic studies show that the enzyme is a homodimer and each subunit binds one kanamycin and one Mg-AMPCPP, where the transfer of the nucleotidyl group occurs between the substrates bound to different subunits. In this work, sedimentation velocity analysis of ANT by analytical ultracentrifugation showed the enzyme exists as a mixture of a monomer and a dimer in solution and that dimer formation is driven by hydrophobic interactions between the subunits. The binding of aminoglycosides shifts the equilibrium toward dimer formation, while the binding of the cosubstrate, Mg-ATP, has no effect on the monomer-dimer equilibrium. Surprisingly, binding of several divalent cations, including Mg(2+), Mn(2+), and Ca(2+), to the enzyme also shifted the equilibrium in favor of dimer formation. Binding studies, performed by electron paramagnetic resonance spectroscopy, showed that divalent cations bind to the aminoglycoside binding site in the absence of substrates with a stoichiometry of 2:1. Energetic aspects of binding of all aminoglycosides to ANT were determined by isothermal titration calorimetry to be enthalpically favored and entropically disfavored with an overall favorable Gibbs energy. Aminoglycosides in the neomycin class each bind to the enzyme with significantly different enthalpic and entropic contributions, while those of the kanamycin class bind with similar thermodynamic parameters.

Leishmania major protein disulfide isomerase as a drug target: enzymatic and functional characterization.

Leishmaniasis is a major health problem worldwide and tools available for their control are limited. Effective vaccines are still lacking, drugs are toxic and expensive, and parasites develop resistance to chemotherapy. In this context, new antimicrobials are urgently needed to control the disease in both human and animal. Here, we report the enzymatic and functional characterization of a Leishmania virulence factor, Leishmania major Protein disulfide isomerase (LmPDI) that could constitute a potential drug target. LmPDI possesses domain structure organization similar to other PDI family members (a, a', b, b' and c domains), and it displays the three enzymatic and functional activities specific of PDI family members: isomerase, reductase and chaperone. These results suggest that LmPDI plays a key role in assisting Leishmania protein folding via its capacity to catalyze formation, breakage, and rearrangement of disulfide bonds in nascent polypeptides. Moreover, Bacitracin, a reductase activity inhibitor, and Ribostamycin, a chaperone activity inhibitor, were tested in LmPDI enzymatic assays and versus Leishmania promastigote in vitro cultures and Leishmania amastigote multiplication inside infected THP-1-derived macrophages. Bacitracin inhibited both isomerase and reductase activities, while Ribostamycin had no effect on the chaperone activity. Interestingly, Bacitracin blocked in vitro promastigote growth as well as amastigote multiplication inside macrophages with EC(50) values of 39 µM. These results suggest that LmPDI may constitute an interesting target for the development of new anti-Leishmania drugs.

Thermodynamics of nucleic acid "shape readout" by an aminosugar.

Recognition of nucleic acids is important for our understanding of nucleic acid structure as well as for our understanding of nucleic acid-protein interactions. In addition to the direct readout mechanisms of nucleic acids such as H-bonding, shape recognition of nucleic acids is being increasingly recognized as playing an equally important role in DNA recognition. Competition dialysis, UV, flourescent intercalator displacement (FID), computational docking, and calorimetry studies were conducted to study the interaction of neomycin with a variety of nucleic acid conformations (shapes). At pH 5.5, the results suggest the following. (1) Neomycin binds three RNA structures [16S A site rRNA, poly(rA)·poly(rA), and poly(rA)·poly(rU)] with high affinities (K(a) ~ 10(7) M(-1)). (2) The binding of neomycin to A-form GC-rich oligomer d(A(2)G(15)C(15)T(2))(2) has an affinity comparable to those of RNA structures. (3) The binding of neomycin to DNA·RNA hybrids shows a 3-fold variance that can be attributed to their structural differences [for poly(dA)·poly(rU), K(a) = 9.4 × 10(6) M(-1), and for poly(rA)·poly(dT), K(a) = 3.1 × 10(6) M(-1)]. (4) The interaction of neomycin with DNA triplex poly(dA)·2poly(dT) yields a binding affinity (K(a)) of 2.4 × 10(5) M(-1). (5) Poly(dA-dT)(2) shows the lowest association constant for all nucleic acids studied (K(a) < 10(5)). (6) Neomycin binds to G-quadruplexes with K(a) values of ~10(4)-10(5) M(-1). (7) Computational studies show that the decrease in major groove width in the B to A transition correlates with increasing neomycin affinity. Neomycin's affinity for various nucleic acid structures can be ranked as follows: RNAs and GC-rich d(A(2)G(15)C(15)T(2))(2) structures > poly(dA)·poly(rU) > poly(rA)·poly(dT) > T·A-T triplex, G-quadruplex, B-form AT-rich, or GC-rich DNA sequences. The results illustrate the first example of a small molecule-based "shape readout" of different nucleic acid conformations.

The potential of protein disulfide isomerase as a therapeutic drug target.

Protein disulfide isomerase (PDI) is a multifunctional protein of the thioredoxin superfamily. PDI mediates proper protein folding by oxidation or isomerization and disrupts disulfide bonds by reduction; it also has chaperone and antichaperone activities. Although PDI localizes primarily to the endoplasmic reticulum (ER), it is secreted and expressed on the cell surface. In the ER, PDI is primarily involved in protein folding, whereas on the cell surface, it reduces disulfide bonds. The functions of PDI depend on its localization and the redox state of its active site cysteines. The ER-based functions of PDI are linked to cancer invasion and migration. Surface-associated PDI facilitates the entry of viruses, such as HIV-1, and toxins, such as diphtheria and cholera. Thus, based on its involvement in pathological events, PDI is considered a potential drug target. However, a significant challenge in the therapeutic targeting of PDI is discovering function-specific inhibitors for it. To this end, a wide range of therapeutic agents, such as antibiotics, thiol blockers, estrogenic compounds, and arsenical compounds, have been used, although few are bona fide specific inhibitors. In this review, we will describe the potential of PDI as a therapeutic drug target.

Biosynthesis of ribostamycin derivatives by reconstitution and heterologous expression of required gene sets.

Ribostamycin is a 4,5-disubstituted 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics and naturally produced by Streptomyces ribosidificus ATCC 21294. It is also an intermediate in the biosynthesis of butirosin and neomycin. In the biosynthesis of ribostamycin, DOS is glycosylated to generate paromamine which is converted to neamine by successive dehydrogenation followed by amination, and finally ribosylation of neamine gives ribostamycin. Here, we report the biosynthesis of 6'-deamino-6'-hydroxyribostamycin (a ribostamycin derivative or pseudoribostamycin) in Streptomyces venezuelae YJ003 by reconstructing gene cassettes for direct ribosylation of paromamine. A trace amount of pseudoribostamycin was detected with ribostamycin in the isolates of ribostamycin cosmid heterologously expressed in Streptomyces lividans TK24. It has also indicated that the ribosyltransferase can accept both neamine and paromamine. Thus, the present in vivo modification of ribostamycin could be useful for the production of hybrid compounds to defend against bacterial resistance to aminoglycosides.

Aminoglycoside binding to Oxytricha nova telomeric DNA.

Telomeric DNA sequences have been at the center stage of drug design for cancer treatment in recent years. The ability of these DNA structures to form four-stranded nucleic acid structures, called G-quadruplexes, has been perceived as target for inhibiting telomerase activity vital for the longevity of cancer cells. Being highly diverse in structural forms, these G-quadruplexes are subjects of detailed studies of ligand-DNA interactions of different classes, which will pave the way for logical design of more potent ligands in future. The binding of aminoglycosides was investigated with Oxytricha nova quadruplex forming DNA sequence (GGGGTTTTGGGG)(2). Isothermal titration calorimetry (ITC) determined ligand to quadruplex binding ratio shows 1:1 neomycin:quadruplex binding with association constants (K(a)) ∼ 10(5) M(-1) while paromomycin was found to have a 2-fold weaker affinity than neomycin. The CD titration experiments with neomycin resulted in minimal changes in the CD signal. FID assays, performed to determine the minimum concentration required to displace half of the fluorescent probe bound, showed neomycin as the best of the all aminoglycosides studied for quadruplex binding. Initial NMR footprint suggests that ligand-DNA interactions occur in the wide groove of the quadruplex. Computational docking studies also indicate that aminoglycosides bind in the wide groove of the quadruplex.

Role of aromatic rings in the molecular recognition of aminoglycoside antibiotics: implications for drug design.

Aminoglycoside antibiotics participate in a large variety of binding processes involving both RNA and proteins. The description, in recent years, of several clinically relevant aminoglycoside/receptor complexes has greatly stimulated the structural-based design of new bioactive derivatives. Unfortunately, design efforts have frequently met with limited success, reflecting our incomplete understanding of the molecular determinants for the antibiotic recognition. Intriguingly, aromatic rings of the protein/RNA receptors seem to be key actors in this process. Indeed, close inspection of the structural information available reveals that they are frequently involved in CH/pi stacking interactions with sugar/aminocyclitol rings of the antibiotic. While the interaction between neutral carbohydrates and aromatic rings has been studied in detail during past decade, little is known about these contacts when they involve densely charged glycosides. Herein we report a detailed experimental and theoretical analysis of the role played by CH/pi stacking interactions in the molecular recognition of aminoglycosides. Our study aims to determine the influence that the antibiotic polycationic character has on the stability, preferred geometry, and dynamics of these particular contacts. With this purpose, different aminoglycoside/aromatic complexes have been selected as model systems. They varied from simple bimolecular interactions to the more stable intramolecular CH/pi contacts present in designed derivatives. The obtained results highlight the key role played by electrostatic forces and the desolvation of charged groups in the molecular recognition of polycationic glycosides and have clear implications for the design of improved antibiotics.

Heterologous production of ribostamycin derivatives in engineered Escherichia coli.

Aminoglycosides are a class of important antibiotic compounds used for various therapeutic indications. In recent times, their efficacy has been curtailed due to the rapid development of bacterial resistance. There is a need to develop novel derivatives with an improved spectrum of activity and higher sensitivity against pathogenic bacteria. Although efforts have been focused on the development of newer therapeutic agents by chemical synthesis, to our knowledge, there has been no attempt to harness the potential of microorganisms for this purpose. Escherichia coli affords a widely studied cellular system that could be utilized not only for understanding but also for attempting to engineer the biosynthetic pathway of secondary metabolites. The primary metabolic pathway of E. coli can be engineered to divert the precursor pool required for the biosynthesis of secondary metabolites. Utilizing this approach previously, we engineered E. coli host and generated E. coli M1. Here, we produced a ribostamycin derivative in the engineered host by heterologous expression of the recombinants constructed from the genes encoding the biosynthetic pathway in aminoglycoside-producing strains. The products obtained from the transformants were isolated, analyzed and verified to be ribostamycin derivatives. The study further demonstrated the importance of E. coli as surrogate antibiotic producer and also offered future possibility for the production of other aminoglycoside derivatives through genetic engineering and expression in a heterologous background.

Thermodynamics and kinetics of association of antibiotics with the aminoglycoside acetyltransferase (3)-IIIb, a resistance-causing enzyme.

The thermodynamic and kinetic properties of interactions of antibiotics with the aminoglycoside acetyltransferase (3)-IIIb (AAC) are determined with several experimental methods. These data represent the first such characterization of an enzyme that modifies the 2-deoxystreptamine ring common to all aminoglycoside antibiotics. Antibiotic substrates for AAC include kanamycin A, kanamycin B, tobramycin, sisomicin, neomycin B, paromomycin, lividomycin A, and ribostamycin. Kinetic studies show that kanamycin group aminoglycosides have higher k(cat) values than members of the neomycin group. Only small aminoglycosides without intraring constraints show substrate inhibition. Isothermal titration calorimetry (ITC) and fluorescence measurements are consistent with a molecular size-dependent stoichiometry where binding stoichiometries are 1.5-2.0 for small antibiotics and 1.0 for larger. Antibiotic-enzyme interaction occurs with a favorable enthalpy (DeltaH < 0) and a compensating unfavorable entropy (TDeltaS < 0). The presence of coenzyme A significantly increases the affinity of the antibiotic for AAC. However, the thermodynamic properties of its ternary complexes distinguish this enzyme from other aminoglycoside-modifying enzymes (AGMEs). Unlike other AGMEs, the enthalpy of binding becomes more favored by 1.7-10.0-fold in the presence of the cosubstrate CoASH, while the entropy becomes 2.0-22.5-fold less favored. The overall free energy change is still only 1.0-1.9 kcal/mol from binary to ternary for all antibiotics tested, which is similar to those for other aminoglycoside-modifying enzymes. A computationally derived homology model provides structural support for these conclusions and further indicates that AAC is likely a member of the GCN5-related acetyltransferase family of proteins.