Substrate promiscuity of an aminoglycoside antibiotic resistance enzyme via target mimicry

The misuse of antibiotics has selected for bacteria that have evolved mechanisms for evading the effects of these drugs. For aminoglycosides, a group of clinically important bactericidal antibiotics that target the A-site of the 16S ribosomal RNA, the most common mode of resistance is enzyme-catalyzed chemical modification of the drug. While aminoglycosides are structurally diverse, a single enzyme can confer resistance to many of these antibiotics. For example, the aminoglycoside kinase APH(3')-IIIa, produced by pathogenic Gram-positive bacteria such as enterococci and staphylococci, is capable of detoxifying at least 10 distinct aminoglycosides. Here we describe the crystal structures of APH(3')-IIIa in complex with ADP and kanamycin A or neomycin B. These structures reveal that the basis for this enzyme's substrate promiscuity is the presence of two alternative subsites in the antibiotic binding pocket. Furthermore, comparison between the A-site of the bacterial ribosome and APH(3')-IIIa shows that mimicry is the second major factor in dictating the substrate spectrum of APH(3')-IIIa. These results suggest a potential strategy for drug design aimed at circumventing antibiotic resistance.     

Thermodynamics of aminoglycoside binding to aminoglycoside-3'-phosphotransferase IIIa studied by isothermal titration calorimetry

The aminoglycoside-3'-phosphotransferase IIIa [APH(3')-IIIa] phosphorylates aminoglycoside antibiotics and renders them ineffective against bacteria. APH(3')-IIIa is the most promiscuous aminoglycoside phosphotransferase enzyme, and it modifies more than 10 different aminoglycoside antibiotics. A wealth of information exists about the enzyme; however, thermodynamic properties of enzyme-aminoglycoside complexes are still not known. This study describes the determination of the thermodynamic parameters of the binary enzyme-aminoglycoside and the ternary enzyme-metal-ATP-aminoglycoside complexes of structurally related aminoglycosides using isothermal titration calorimetry. Formation of the binary enzyme-aminoglycoside complexes is enthalpically driven and exhibits a strongly disfavored entropic contribution. Formation of the ternary enzyme-metal-ATP-aminoglycoside complexes yields much smaller negative DeltaH values and more favorable entropic contributions. The presence of metal-ATP generally increases the affinity of aminoglycosides to the enzyme. This is consistent with the kinetic mechanism of the enzyme in which ordered binding of substrates occurs. However, the observed DeltaH values neither correlate with kinetic parameters k(cat), K(m), and k(cat)/K(m) nor correlate with the molecular size of the substrates. Comparison of the thermodynamic properties of the complexes formed by structurally similar aminoglycosides indicated that the 2'- and the 6'-amino groups of the substrates are involved in binding to the enzyme. Thermodynamic properties of the complexes formed by aminoglycosides differing only at the 3'-hydroxyl group suggested that the absence of this group does not alter the thermodynamic parameters of the ternary APH(3')-IIIa-metal-ATP-aminoglycoside complex. Our results also indicate that protonation of ligand and protein ionizable groups is coupled to the complex formation between aminoglycosides and APH(3')-IIIa. Comparison of DeltaH values for different aminoglycoside-enzyme complexes indicates that enzyme and substrates undergo significant conformational changes in complex formation.     

Discovery of non-carbohydrate inhibitors of aminoglycoside-modifying enzymes

Chemical modification and inactivation of aminoglycosides by many different enzymes expressed in pathogenic bacteria are the main mechanisms of bacterial resistance to these antibiotics. In this work, we designed inhibitors that contain the 1,3-diamine pharmacophore shared by all aminoglycoside antibiotics that contain the 2-deoxystreptamine ring. A discovery library of molecules was prepared by attaching different side chains to both sides of the 1,3-diamine motif. Several of these diamines showed inhibitory activity toward two or three different representative aminoglycoside-modifying enzymes (AGMEs). These studies yielded the first non-carbohydrate inhibitor N-cyclohexyl-N'-(3-dimethylamino-propyl)-propane-1,3-diamine (Compound G,H) that is competitive with respect to the aminoglycoside binding to the enzyme aminoglycoside-2'-nucleotidyltransferase-Ia (ANT2'). Another diamine molecule N-[2-(3,4-dimethoxyphenyl)-ethyl]-N'-(3-dimethylamino-propyl)-propane-1,3-diamine (Compound H,I) was shown to be a competitive inhibitor of two separate enzymes (aminoglycoside-3'-phosphotransferase-IIIa (APH3') and ANT2') with respect to metal-ATP. Thermodynamic and structural-binding properties of the complexes of APH3' with substrates and inhibitor were shown to be similar to each other, as determined by isothermal titration calorimetry and NMR spectroscopy.     

Aminoglycoside resistance in gramnegative pathogens of nosocomial infections in Russia]

The study of the mechanisms of aminoglycoside resistance in gramnegative pathogens of nosocomial infections in 14 hospitals of Russia showed that the basic mechanism was production of aminoglycoside modifying enzymes, mainly adenylyl transferase ANT(2"), acetyl transferases AAC(3)-V and ACC(6)-I, and phosphotransferases APH(3')-I and APH(3')-VI. In all the hospitals enzymes modifying gentamicin and tobramycin were wide spread while the resistance phenotypes to aminoglycosides were different in separate hospitals. Isepamycin proved to be the most active aminoglycoside. Recommendations for the use of antibiotics in hospital formulas and empiric therapy should be developed on the basis of the local specific features of the resistance in nosocomial pathogens to aminoglycosides.     

Mechanistic and structural analysis of aminoglycoside N-acetyltransferase AAC(6')-Ib and its bifunctional, fluoroquinolone-active AAC(6')-Ib-cr variant

Enzymatic modification of aminoglycoside antibiotics mediated by regioselective aminoglycoside N-acetyltransferases is the predominant cause of bacterial resistance to aminoglycosides. A recently discovered bifunctional aminoglycoside acetyltransferase (AAC(6')-Ib variant, AAC(6')-Ib-cr) has been shown to catalyze the acetylation of fluoroquinolones as well as aminoglycosides. We have expressed and purified AAC(6')-Ib-wt and its bifunctional variant AAC(6')-Ib-cr in Escherichia coli and characterized their kinetic and chemical mechanism. Initial velocity and dead-end inhibition studies support an ordered sequential mechanism for the enzyme(s). The three-dimensional structure of AAC(6')-Ib-wt was determined in various complexes with donor and acceptor ligands to resolutions greater than 2.2 A. Observation of the direct, and optimally positioned, interaction between the 6'-NH 2 and Asp115 suggests that Asp115 acts as a general base to accept a proton in the reaction. The structure of AAC(6')-Ib-wt permits the construction of a molecular model of the interactions of fluoroquinolones with the AAC(6')-Ib-cr variant. The model suggests that a major contribution to the fluoroquinolone acetylation activity comes from the Asp179Tyr mutation, where Tyr179 makes pi-stacking interactions with the quinolone ring facilitating quinolone binding. The model also suggests that fluoroquinolones and aminoglycosides have different binding modes. On the basis of kinetic properties, the pH dependence of the kinetic parameters, and structural information, we propose an acid/base-assisted reaction catalyzed by AAC(6')-Ib-wt and the AAC(6')-Ib-cr variant involving a ternary complex.     

Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase-2��-IVa

Acquired resistance to aminoglycoside antibiotics primarily results from deactivation by three families of aminoglycoside-modifying enzymes. Here, we report the kinetic mechanism and structure of the aminoglycoside phosphotransferase 2″-IVa (APH(2″)-IVa), an enzyme responsible for resistance to aminoglycoside antibiotics in clinical enterococcal and staphylococcal isolates. The enzyme operates via a Bi-Bi sequential mechanism in which the two substrates (ATP or GTP and an aminoglycoside) bind in a random manner. The APH(2″)-IVa enzyme phosphorylates various 4,6-disubstituted aminoglycoside antibiotics with catalytic efficiencies (k_cat/K_m) of 1.5 × 10³ to 1.2 × 10⁶ (M⁻¹ s⁻¹). The enzyme uses both ATP and GTP as the phosphate source, an extremely rare occurrence in the phosphotransferase and protein kinase enzymes. Based on an analysis of the APH(2″)-IVa structure, two overlapping binding templates specifically tuned for hydrogen bonding to either ATP or GTP have been identified and described. A detailed understanding of the structure and mechanism of the GTP-utilizing phosphotransferases is crucial for the development of either novel aminoglycosides or, more importantly, GTP-based enzyme inhibitors which would not be expected to interfere with crucial ATP-dependent enzymes.     

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6′)-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in Gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6′)-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k_cat/K_m = 10⁵-10⁷ m⁻¹ s⁻¹). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.     

The COOH terminus of aminoglycoside phosphotransferase (3')-IIIa is critical for antibiotic recognition and resistance.

The aminoglycoside phosphotransferases (APHs) are widely distributed among pathogenic bacteria and are employed to covalently modify, and thereby detoxify, the clinically relevant aminoglycoside antibiotics. The crystal structure for one of these aminoglycoside kinases, APH(3')-IIIa, has been determined in complex with ADP and analysis of the electrostatic surface potential indicates that there is a large anionic depression present adjacent to the terminal phosphate group of the nucleotide. This region also includes a conserved COOH-terminal alpha-helix that contains the COOH-terminal residue Phe(264). We report here mutagenesis and computer modeling studies aimed at examining the mode of aminoglycoside binding to APH(3')-IIIa. Specifically, seven site mutants were studied, five from the COOH-terminal helix (Asp(261), Glu(262), and Phe(264)), and two additional residues that line the wall of the anionic depression (Tyr(55) and Arg(211)). Using a molecular modeling approach, six ternary complexes of APH(3')-IIIa.ATP with the antibiotics, kanamycin, amikacin, butirosin, and ribostamycin were independently constructed and these agree well with the mutagenesis data. The results obtained show that the COOH-terminal carboxylate of Phe(264) is critical for proper function of the enzyme. Furthermore, these studies demonstrate that there exists multiple binding modes for the aminoglycosides, which provides a molecular basis for the broad substrate- and regiospecificity observed for this enzyme.     

Crystal structures of antibiotic-bound complexes of aminoglycoside 2'-phosphotransferase IVa highlight the diversity in substrate binding modes among aminoglycoside kinases

Aminoglycoside 2'-phosphotransferase IVa [APH(2')-IVa] is a member of a family of bacterial enzymes responsible for medically relevant resistance to antibiotics. APH(2')-IVa confers high-level resistance against several clinically used aminoglycoside antibiotics in various pathogenic Enterococcus species by phosphorylating the drug, thereby preventing it from binding to its ribosomal target and producing a bactericidal effect. We describe here three crystal structures of APH(2')-IVa, one in its apo form and two in complex with a bound antibiotic, tobramycin and kanamycin A. The apo structure was refined to a resolution of 2.05 ?, and the APH(2')-IVa structures with tobramycin and kanamycin A bound were refined to resolutions of 1.80 and 2.15 ?, respectively. Comparison among the structures provides insight concerning the substrate selectivity of this enzyme. In particular, conformational changes upon substrate binding, involving rotational shifts of two distinct segments of the enzyme, are observed. These substrate-induced shifts may also rationalize the altered substrate preference of APH(2')-IVa in comparison to those of other members of the APH(2') subfamily, which are structurally closely related. Finally, analysis of the interactions between the enzyme and aminoglycoside reveals a distinct binding mode as compared to the intended ribosomal target. The differences in the pattern of interactions can be utilized as a structural basis for the development of improved aminoglycosides that are not susceptible to these resistance factors.     

Mechanistic characterization of the bifunctional aminoglycoside-modifying enzyme AAC(3)-Ib/AAC(6')-Ib' from Pseudomonas aeruginosa

A recently discovered bifunctional antibiotic-resistance enzyme named AAC(3)-Ib/AAC(6')-Ib', from Pseudomonas aeruginosa, catalyzes acetylation of aminoglycoside antibiotics. Since both domains are acetyltransferases, each was cloned and purified for mechanistic studies. The AAC(3)-Ib domain appears to be highly specific to fortimicin A and gentamicin as substrates, while the AAC(6')-Ib' domain exhibits a broad substrate spectrum. Initial velocity patterns indicate that both domains follow a sequential kinetic mechanism. The use of dead-end and product inhibition and solvent-isotope effect reveals that both domains catalyze their reactions by a steady-state ordered Bi-Bi kinetic mechanism, in which acetyl-CoA is the first substrate that binds to the active site, followed by binding of the aminoglycoside antibiotic. Subsequent to the transfer of the acetyl group, acetylated aminoglycoside is released prior to coenzyme A. The merger of two genes to create a bifunctional enzyme with expanded substrate profile would appear to be a recent trend in evolution of resistance to aminoglycoside antibiotics, of which four examples have been documented in the past few years.     

Deciphering interactions of the aminoglycoside phosphotransferase(3′)‐IIIa with its ligands

Aminoglycoside phosphotransferase(3′)‐IIIa (APH) is the enzyme with broadest substrate range among the phosphotransferases that cause resistance to aminoglycoside antibiotics. In this study, the thermodynamic characterization of interactions of APH with its ligands are done by determining dissociation constants of enzyme–substrate complexes using electron paramagnetic resonance and fluorescence spectroscopy. Metal binding studies showed that three divalent cations bind to the apo‐enzyme with low affinity. In the presence of AMPPCP, binding of the divalent cations occurs with 7‐to‐37‐fold higher affinity to three additional sites dependent on the presence and absence of different aminoglycosides. Surprisingly, when both ligands, AMPPCP and aminoglycoside, are present, the number of high affinity metal binding sites is reduced to two with a 2‐fold increase in binding affinity. The presence of divalent cations, with or without aminoglycoside present, shows only a small effect (<3‐fold) on binding affinity of the nucleotide to the enzyme. The presence of metal–nucleotide, but not nucleotide alone, increases the binding affinity of aminoglycosides to APH. Replacement of magnesium (II) with manganese (II) lowered the catalytic rates significantly while affecting the substrate selectivity of the enzyme such that the aminoglycosides with 2′‐NH₂ become better substrates (higher V_max) than those with 2′‐OH. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 801–809, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Hydrolysis of ATP by aminoglycoside 3'-phosphotransferases: an unexpected cost to bacteria for harboring an antibiotic resistance enzyme

Aminoglycoside 3'-phosphotransferases (APH(3')s) are common bacterial resistance enzymes to aminoglycoside antibiotics. These enzymes transfer the gamma-phosphoryl group of ATP to the 3'-hydroxyl of the antibiotics, whereby the biological activity of the drugs is lost. Pre-steady-state and steady-state kinetics with two of these enzymes from Gram-negative bacteria, APH(3')-Ia and APH(3')-IIa, were performed. It is demonstrated that these enzymes in both ternary and binary complexes facilitate an ATP hydrolase activity (ATPase), which is competitive with the transfer of phosphate to the antibiotics. Because these enzymes are expressed constitutively in resistant bacteria, the turnover of ATP is continuous during the lifetime of the organism both in the absence and the presence of aminoglycosides. Concentrations of the enzyme in vivo were determined, and it was estimated that in a single generation of bacterial growth there exists the potential that this activity would consume as much as severalfold of the total existing ATP. Studies with bacteria harboring the aph(3')-Ia gene revealed that bacteria are able to absorb the cost of this ATP turnover, as ATP is recycled. However, the cost burden of this adventitious activity manifests a selection pressure against maintenance of the plasmids that harbor the aph(3')-Ia gene, such that approximately 50% of the plasmid is lost in 1500 bacterial generations in the absence of antibiotics. The implication is that, in the absence of selection, bacteria harboring an enzyme that catalyzes the consumption of key metabolites could experience the loss of the plasmid that encodes for the given enzyme.     

Structural analyses of nucleotide binding to an aminoglycoside phosphotransferase

3',5"-Aminoglycoside phosphotransferase type IIIa [APH(3')-IIIa] is a bacterial enzyme that confers resistance to a range of aminoglycoside antibiotics while exhibiting striking homology to eukaryotic protein kinases (ePK). The structures of APH(3')-IIIa in its apoenzyme form and in complex with the nonhydrolyzable ATP analogue AMPPNP were determined to 3.2 and 2.4 A resolution, respectively. Furthermore, refinement of the previously determined ADP complex was completed. The structure of the apoenzyme revealed alternate positioning of a flexible loop (analogous to the P-loop of ePK's), occupying part of the nucleotide-binding pocket of the enzyme. Despite structural similarity to protein kinases, there was no evidence of domain movement associated with nucleotide binding. This rigidity is due to the presence of more extensive interlobe interactions in the APH(3')-IIIa structure than in the ePK's. Differences between the ADP and AMPPNP complexes are confined to the area of the nucleotide-binding pocket. The position of conserved active site residues and magnesium ions remains unchanged, but there are differences in metal coordination between the two nucleotide complexes. Comparison of the di/triphosphate binding site of APH(3')-IIIa with that of ePK's suggests that the reaction mechanism of APH(3")-IIIa and related aminoglycoside kinases will closely resemble that of eukaryotic protein kinases. However, the orientation of the adenine ring in the binding pocket differs between APH(3')-IIIa and the ePK's by a rotation of approximately 40 degrees. This alternate binding mode is likely a conserved feature among aminoglycoside kinases and could be exploited for the structure-based drug design of compounds to combat antibiotic resistance.     

Crystal structures of two aminoglycoside kinases bound with a eukaryotic protein kinase inhibitor

Antibiotic resistance is recognized as a growing healthcare problem. To address this issue, one strategy is to thwart the causal mechanism using an adjuvant in partner with the antibiotic. Aminoglycosides are a class of clinically important antibiotics used for the treatment of serious infections. Their usefulness has been compromised predominantly due to drug inactivation by aminoglycoside-modifying enzymes, such as aminoglycoside phosphotransferases or kinases. These kinases are structurally homologous to eukaryotic Ser/Thr and Tyr protein kinases and it has been shown that some can be inhibited by select protein kinase inhibitors. The aminoglycoside kinase, APH(3')-IIIa, can be inhibited by CKI-7, an ATP-competitive inhibitor for the casein kinase 1. We have determined that CKI-7 is also a moderate inhibitor for the atypical APH(9)-Ia. Here we present the crystal structures of CKI-7-bound APH(3')-IIIa and APH(9)-Ia, the first structures of a eukaryotic protein kinase inhibitor in complex with bacterial kinases. CKI-7 binds to the nucleotide-binding pocket of the enzymes and its binding alters the conformation of the nucleotide-binding loop, the segment homologous to the glycine-rich loop in eukaryotic protein kinases. Comparison of these structures with the CKI-7-bound casein kinase 1 reveals features in the binding pockets that are distinct in the bacterial kinases and could be exploited for the design of a bacterial kinase specific inhibitor. Our results provide evidence that an inhibitor for a subset of APHs can be developed in order to curtail resistance to aminoglycosides.     

Structure of the Antibiotic Resistance Factor Spectinomycin Phosphotransferase from Legionella pneumophila

Aminoglycoside phosphotransferases (APHs) constitute a diverse group of enzymes that are often the underlying cause of aminoglycoside resistance in the clinical setting. Several APHs have been extensively characterized, including the elucidation of the three-dimensional structure of two APH(3′) isozymes and an APH(2″) enzyme. Although many APHs are plasmid-encoded and are capable of inactivating numerous 2-deoxystreptmaine aminoglycosides with multiple regiospecificity, APH(9)-Ia, isolated from Legionella pneumophila, is an unusual enzyme among the APH family for its chromosomal origin and its specificity for a single non-2-deoxystreptamine aminoglycoside substrate, spectinomycin. We describe here the crystal structures of APH(9)-Ia in its apo form, its binary complex with the nucleotide, AMP, and its ternary complex bound with ADP and spectinomycin. The structures reveal that APH(9)-Ia adopts the bilobal protein kinase-fold, analogous to the APH(3′) and APH(2″) enzymes. However, APH(9)-Ia differs significantly from the other two types of APH enzymes in its substrate binding area and that it undergoes a conformation change upon ligand binding. Moreover, kinetic assay experiments indicate that APH(9)-Ia has stringent substrate specificity as it is unable to phosphorylate substrates of choline kinase or methylthioribose kinase despite high structural resemblance. The crystal structures of APH(9)-Ia demonstrate and expand our understanding of the diversity of the APH family, which in turn will facilitate the development of new antibiotics and inhibitors.     

The role of aminoglycoside--modifying enzymes in resistance of Gram-negative rods

The aim of the study was to evaluate the aminoglycoside resistance of Gram-negative bacilli isolated from patients. To the examination 35 strains of Enterobacteriaceae and 18 of non-fermentative bacteria were included. Resistance to aminoglycosides (gentamicin (G), netilmicin (Nt), tobramycin (T), amikacin (A), kanamycin (K), neomycin (N)) was established by disk diffusion method. Interpretation of enzymatic mechanisms was performed by Livermore. The most common enzymes AAC(6')I were found in Enterobacteriaceae group (mostly in E. cloaceae and P. mirabilis) and AAC(3') and in non-fermentative bacteria: AAC(6')I in P. aeruginosa and APH(3')VI and AAC(3')I in A. baumanii. The most frequent phenotype was resistance to six antibiotics (G, Nt, T, A, K, N) Resistance rates were high for gentamicin (>70 %) in both groups and amikacin (88,89 %) in non-fermentatives.     

Studying aminoglycoside modification by the acetyltransferase class of resistance-causing enzymes via microarray

Aminoglycosides are broad-spectrum antibacterials to which some bacteria have acquired resistance. The most common mode of resistance to aminoglycosides is enzymatic modification of the drug by different classes of enzymes including acetyltransferases (AACs). Thus, the modification of aminoglycosides by AAC(2′) from Mycobacterium tuberculosis and AAC(3) from Escherichia coli was studied using aminoglycoside microarrays. Results show that both enzymes modify their substrates displayed on an array surface in a manner that mimics their relative levels of modification in solution. Because aminoglycosides that are modified by resistance-causing enzymes have reduced affinities for binding their therapeutic target, the bacterial rRNA aminoacyl-tRNA site (A-site), arrays were probed for binding to a fluorescently labeled oligonucleotide mimic of the A-site after modification. A decrease in binding was observed when aminoglycosides were modified by AAC(3). In contrast, a decrease in binding of the A-site is not observed when aminoglycosides are modified by AAC(2′). Interestingly, these effects mirror the biological functions of the enzymes: the AAC(3) used in this study is known to confer aminoglycoside resistance, while the AAC(2′) is chromosomally encoded and unlikely to play a role in resistance. These studies lay a direct foundation for studying resistance to aminoglycosides and can also have more broad applications in identifying and studying non-aminoglycoside carbohydrates or proteins as substrates for acetyltransferase enzymes.     

Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis

The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3')-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with (15)N NMR-derived pK(a) and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2'-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3')-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3')-IIIa-induced resistance.     

Aminoglycoside binding and catalysis specificity of aminoglycoside 2″-phosphotransferase IVa: A thermodynamic,structural and kinetic study

BackgroundAminoglycoside O-phosphotransferases make up a large class of bacterial enzymes that is widely distributed among pathogens and confer a high resistance to several clinically used aminoglycoside antibiotics. Aminoglycoside 2″-phosphotransferase IVa, APH(2″)-IVa, is an important member of this class, but there is little information on the thermodynamics of aminoglycoside binding and on the nature of its rate-limiting step.MethodsWe used isothermal titration calorimetry, electrostatic potential calculations, molecular dynamics simulations and X-ray crystallography to study the interactions between the enzyme and different aminoglycosides. We determined the rate-limiting step of the reaction by the means of transient kinetic measurements.ResultsFor the first time, K_d values were determined directly for APH(2″)-IVa and different aminoglycosides. The affinity of the enzyme seems to anti-correlate with the molecular weight of the ligand, suggesting a limited degree of freedom in the binding site. The main interactions are electrostatic bonds between the positively charged amino groups of aminoglycosides and Glu or Asp residues of APH. In spite of the significantly different ratio K_d/K_m, there is no large difference in the transient kinetics obtained with the different aminoglycosides. We show that a product release step is rate-limiting for the overall reaction.ConclusionsAPH(2″)-IVa has a higher affinity for aminoglycosides carrying an amino group in 2′ and 6′, but tighter bindings do not correlate with higher catalytic efficiencies. As with APH(3′)-IIIa, an intermediate containing product is preponderant during the steady state.General significanceThis intermediate may constitute a good target for future drug design.     

Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins

The specific intracellular inhibition of protein activity at the protein level allows the determination of protein function in the cellular context. We demonstrate here the use of designed ankyrin repeat proteins as tailor-made intracellular kinase inhibitors. The target was aminoglycoside phosphotransferase (3')-IIIa (APH), which mediates resistance to aminoglycoside antibiotics in pathogenic bacteria and shares structural homology with eukaryotic protein kinases. Combining a selection and screening approach, we isolated 198 potential APH inhibitors from highly diverse combinatorial libraries of designed ankyrin repeat proteins. A detailed analysis of several inhibitors revealed that they bind APH with high specificity and with affinities down to the subnanomolar range. In vitro, the most potent inhibitors showed complete enzyme inhibition, and in vivo, a phenotype comparable with the gene knockout was observed, fully restoring antibiotic sensitivity in resistant bacteria. These results underline the great potential of designed ankyrin repeat proteins for modulation of intracellular protein function.