The Thermodynamics of Ligand Binding to the Aminoglycoside O-Nucleotidyltransferase(4') and Variants Yields Clues about Thermophilic Properties.

The aminoglycoside nucleotidyltransferase(4') is an enzyme with high substrate promiscuity and catalyzes the transfer of the AMP group from ATP to the 4'-OH site of many structurally diverse aminoglycosides, which results in the elimination of their effectiveness as antibiotics. Two thermostable variants carrying single-site mutations are used to determine the molecular properties associated with thermophilicity. The thermodynamics of enzyme-ligand interactions showed that one variant (T130K) has properties identical to those of the mesophilic wild type (WT) while the other (D80Y) behaved differently. Differences between D80Y and the T130K/WT pair include the change in heat capacity (Δ Cp), which is dependent on temperature for D80Y but not for WT or T130K. The change in Δ Cp with temperature (ΔΔ Cp) with D80Y is dependent on aminoglycoside only in H2O and remains the same with all aminoglycosides in D2O. Furthermore, the offset temperature ( Toff), the temperature difference that yields identical enthalpies in H2O and D2O, becomes larger with an increase in temperature for WT and T130K but remains mostly unchanged for D80Y. Studies in H2O and D2O revealed that solvent reorganization becomes the major contributor to ligand binding with an increase in temperature for WT and T130K, but changes in low-frequency vibrational modes are the main contributors with D80Y. Data presented in this paper suggest that global properties associated with the enzyme-ligand interactions, such as the thermodynamics of ligand binding, may yield clues about thermophilicity and permit us to distinguish those variants that are simply a more thermostable version of the mesophilic protein.

Low-Barrier and Canonical Hydrogen Bonds Modulate Activity and Specificity of a Catalytic Triad.

The position, bonding and dynamics of hydrogen atoms in the catalytic centers of proteins are essential for catalysis. The role of short hydrogen bonds in catalysis has remained highly debated and led to establishment of several distinctive geometrical arrangements of hydrogen atoms vis-à-vis the heavier donor and acceptor counterparts, that is, low-barrier, single-well or short canonical hydrogen bonds. Here we demonstrate how the position of a hydrogen atom in the catalytic triad of an aminoglycoside inactivating enzyme leads to a thirty-fold increase in catalytic turnover. A low-barrier hydrogen bond is present in the enzyme active site for the substrates that are turned over the best, whereas a canonical hydrogen bond is found with the least preferred substrate. This is the first comparison of these hydrogen bonds involving an identical catalytic network, while directly demonstrating how active site electrostatics adapt to the electronic nature of substrates to tune catalysis.

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

BACKGROUND: Aminoglycoside 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. METHODS: We 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. RESULTS: For the first time, Kd 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 Kd/Km, 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. CONCLUSIONS: APH(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 SIGNIFICANCE: This intermediate may constitute a good target for future drug design.

Thermodynamics of an aminoglycoside modifying enzyme with low substrate promiscuity: The aminoglycoside N3 acetyltransferase-VIa.

Kinetic, thermodynamic, and structural properties of the aminoglycoside N3-acetyltransferase-VIa (AAC-VIa) are determined. Among the aminoglycoside N3-acetyltransferases, AAC-VIa has one of the most limited substrate profiles. Kinetic studies showed that only five aminoglycosides are substrates for this enzyme with a range of fourfold difference in kcat values. Larger differences in KM (∼40-fold) resulted in ∼30-fold variation in kcat /KM . Binding of aminoglycosides to AAC-VIa was enthalpically favored and entropically disfavored with a net result of favorable Gibbs energy (ΔG < 0). A net deprotonation of the enzyme, ligand, or both accompanied the formation of binary and ternary complexes. This is opposite of what was observed with several other aminoglycoside N3-acetyltransferases, where ligand binding causes more protonation. The change in heat capacity (ΔCp) was different in H2 O and D2 O for the binary enzyme-sisomicin complex but remained the same in both solvents for the ternary enzyme-CoASH-sisomicin complex. Unlike, most other aminoglycoside-modifying enzymes, the values of ΔCp were within the expected range of protein-carbohydrate interactions. Solution behavior of AAC-VIa was also different from the more promiscuous aminoglycoside N3-acetyltransferases and showed a monomer-dimer equilibrium as detected by analytical ultracentrifugation (AUC). Binding of ligands shifted the enzyme to monomeric state. Data also showed that polar interactions were the most dominant factor in dimer formation. Overall, thermodynamics of ligand-protein interactions and differences in protein behavior in solution provide few clues on the limited substrate profile of this enzyme despite its >55% sequence similarity to the highly promiscuous aminoglycoside N3-acetyltransferase. Proteins 2017; 85:1258-1265. © 2017 Wiley Periodicals, Inc.

Correction to Thermodynamic Characterization of a Thermostable Antibiotic Resistance Enzyme, the Aminoglycoside Nucleotidyltransferase (4').

Biochemistry 2012, 51 (45), 9147−9155. DOI: 10.1021/bi301126g. Page 9148. A corrected version of the Figure 2 legend appears here: Figure 2. Backbone of the ANT D80Y variant in ribbon representation. Two monomer subunits are colored red and green. Bound kanamycin A molecules are colored blue, and Mg-AMPCPP molecules are colored yellow (Protein Data Bank entry 1KNY).14 Page 9148 (last line). The sentence should read, "A thermostable variant of ANT, T130K, was obtained from thermophilic cyanobacterium T. elongatus."

Solvent reorganization plays a temperature-dependent role in antibiotic selection by a thermostable aminoglycoside nucleotidyltransferase-4'.

The aminoglycoside nucleotidyltransferase-4' (ANT) is an enzyme that causes resistance to a large number of aminoglycoside antibiotics by nucleotidylation of the 4'-site on these antibiotics. The effect of solvent reorganization on enzyme-ligand interactions was investigated using a thermophilic variant of the enzyme resulting from a single-site mutation (T130K). Data showed that the binding of aminoglycosides to ANT causes exposure of polar groups to solvent. However, solvent reorganization becomes the major contributor to the enthalpy of the formation of enzyme-aminoglycoside complexes only above 20 °C. The change in heat capacity (ΔCp) shows an aminoglycoside-dependent pattern such that it correlates with the affinity of the ligand for the enzyme. Differences in ΔCp values determined in H2O and D2O also correlated with the ligand affinity. The temperature-dependent increase in the offset temperature (Toff), the temperature difference required to observe equal enthalpies in both solvents, is also dependent on the binding affinity of the ligand, and the steepest increase was observed with the tightest binding aminoglycoside, neomycin. Overall, these data, together with earlier observations with a different enzyme, the aminoglycoside N3-acetyltransferase-IIIb [Norris, A. L., and Serpersu, E. H. (2011) Biochemistry 50, 9309], show that solvent reorganization or changes in soft vibrational modes of the protein are interchangeable with respect to the role of being the major contributor to complex formation depending on temperature. These data suggest that such effects may more generally apply to enzyme-ligand interactions, and studies at a single temperature may provide only a part of the whole picture of thermodynamics of enzyme-ligand interactions.

Protein dynamics are influenced by the order of ligand binding to an antibiotic resistance enzyme.

The aminoglycoside N3 acetyltransferase-IIIb (AAC) is responsible for conferring bacterial resistance to a variety of aminoglycoside antibiotics. Nuclear magnetic resonance spectroscopy and dynamic light scattering analyses revealed a surprising result; the dynamics of the ternary complex between AAC and its two ligands, an antibiotic and coenzyme A, are dependent upon the order in which the ligands are bound. Additionally, two structurally similar aminoglycosides, neomycin and paromomycin, induce strikingly different dynamic properties when they are in their ternary complexes. To the best of our knowledge, this is the first example of a system in which two identically productive pathways of forming a simple ternary complex yield significant differences in dynamic properties. These observations emphasize the importance of the sequence of events in achieving optimal protein-ligand interactions and demonstrate that even a minor difference in molecular structure can have a profound effect on biochemical processes.

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.

Antibiotic selection by the promiscuous aminoglycoside acetyltransferase-(3)-IIIb is thermodynamically achieved through the control of solvent rearrangement.

The results presented here show the first known observation of opposite signs of change in heat capacity (ΔC(p)) of two structurally similar ligands binding to the same protein site. Neomycin and paromomycin are aminoglycoside antibiotics that are substrates for the resistance-conferring enzyme, the aminoglycoside acetyltransferase-(3)-IIIb (AAC). These antibiotics are identical to one another except at the 6' position where neomycin has an amine and paromomycin has a hydroxyl. The opposite trends in ΔC(p) of binding of these two drugs to AAC suggest a differential exposure of nonpolar amino acid side chains. Nuclear magnetic resonance experiments further demonstrate significantly different changes in AAC upon interaction with neomycin and paromomycin. Experiments in H(2)O and D(2)O reveal the first observed temperature dependence of solvent and vibrational contributions to ΔC(p). Coenzyme A significantly influences these effects. Together, the data suggest that AAC exploits solvent properties to facilitate favorable thermodynamic selection of antibiotics.

Coenzyme A binding to the aminoglycoside acetyltransferase (3)-IIIb increases conformational sampling of antibiotic binding site.

NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The (15)N-(1)H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH-enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

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.

Interactions of coenzyme A with the aminoglycoside acetyltransferase (3)-IIIb and thermodynamics of a ternary system.

In this work, the binding of coenzyme A (CoASH) to the aminoglycoside acetyltransferase (3)-IIIb (AAC) is studied by several experimental techniques. These data represent the first thermodynamic and kinetic characterization of interaction of a cofactor with an enzyme that modifies the 2-deoxystreptamine ring (2-DOS) common to all aminoglycoside antibiotics. Acetyl coenzyme A (AcCoA) was the preferred substrate, but propionyl and malonyl CoA were also substrates. CoASH associates with two different sites on AAC as confirmed by ITC, NMR, and fluorescence experiments: one with a high-affinity, catalytic site and a secondary, low-affinity site that overlaps with the antibiotic binding pocket. The binding of CoASH to the high-affinity site occurs with a small, unfavorable enthalpy and a favorable entropy. Binding to the second site is highly exothermic and is accompanied by an unfavorable entropic contribution. The presence of an aminoglycoside alters the binding of CoASH to AAC dramatically such that the binding occurs with a favorable enthalpy (DeltaH < 0) and an unfavorable entropy (TDeltaS < 0). This is irrespective of which aminoglycoside is the cosubstrate and occurs without a significant change in the affinity of CoASH for AAC. Also, antibiotics eliminate binding of CoASH to the second site. These data allowed the enthalpies of all six equilibria present in a ternary system (AAC-antibiotic-coenzyme) to be determined for the first time for an aminoglycoside-modifying enzyme. NMR experiments also shed light on the dynamic nature of AAC as fast, slow, and intermediary exchanges between apoenzyme- and coenzyme-bound forms were observed.

"Catch and Release": A Variation of the Archetypal Nucleotidyl Transfer Reaction.

Nucleotidyl transfer is an archetypal enzyme reaction central to DNA replication and repair. Here we describe a variation of the nucleotidylation reaction termed "catch and release" that is used by an antibiotic modifying enzyme. The aminoglycoside nucleotidyl transferase 4' (ANT4') inactivates antibiotics such as kanamycin and neomycin through nucleotidylation within an active site that shares significant structural, and inferred underlying catalytic similarity, with human DNA polymerase beta. Here we follow the entire nucleotidyl transfer reaction coordinate of ANT4' covalently inactivating neomycin using X-ray crystallography. These studies show that although the underlying reaction mechanism is conserved with polymerases, a short 2.35 A hydrogen bond is initially formed to facilitate tight binding of the aminoglycoside substrate and is subsequently disrupted by the assembly of the catalytically active ternary complex. This enables the release of products post catalysis due to a lower free energy of the product state compared to the starting substrate complex. We propose that this "catch and release" mechanism of antibiotic turnover observed in ANT4' is a variation of nucleotidyl transfer that has been adapted for the inactivation of antibiotics.

NMR detected hydrogen-deuterium exchange reveals differential dynamics of antibiotic- and nucleotide-bound aminoglycoside phosphotransferase 3'-IIIa.

In this work, hydrogen-deuterium exchange detected by NMR spectroscopy is used to determine the dynamic properties of the aminoglycoside phosphotransferase 3'-IIIa (APH), a protein of intense interest due to its involvement in conferring antibiotic resistance to both gram negative and gram positive microorganisms. This represents the first characterization of dynamic properties of an aminoglycoside-modifying enzyme. Herein we describe in vitro dynamics of apo, binary, and ternary complexes of APH with kanamycin A, neomycin B, and metal-nucleotide. Regions of APH in different complexes that are superimposable in crystal structures show remarkably different dynamic behavior. A complete exchange of backbone amides is observed within the first 15 h of exposure to D(2)O in the apo form of this 31 kDa protein. Binding of aminoglycosides to the enzyme induces significant protection against exchange, and approximately 30% of the amides remain unexchanged up to 95 h after exposure to D(2)O. Our data also indicate that neomycin creates greater solvent protection and overall enhanced structural stability to APH than kanamycin. Surprisingly, nucleotide binding to the enzyme-aminoglycoside complex increases solvent accessibility of a number of amides and is responsible for destabilization of a nearby beta-sheet, thus providing a rational explanation for previously observed global thermodynamic parameters. Our data also provide a molecular basis for broad substrate selectivity of APH.

Studies of enzymes that cause resistance to aminoglycosides antibiotics.

Aminoglycoside antibiotics are highly potent, wide-spectrum bactericidals (1, 2). Bacterial resistance to aminoglycosides, however, is a major problem in the clinical use of aminoglycosides. Enzymatic modification of aminoglycosides is the most frequent resistance mode among several resistance mechanisms employed by resistant pathogens (1,3). Three families of aminoglycoside modifying enzymes, O-phosphotransferases, N-acetyltransferases, and N-nucleotidyltransferases, are known to have more than 50 enzymes (1,3,4). In this chapter, determination of enzymatic activity of a single enzyme from each family in the presence and absence of an inhibitor is described.

A low-barrier hydrogen bond mediates antibiotic resistance in a noncanonical catalytic triad.

One group of enzymes that confer resistance to aminoglycoside antibiotics through covalent modification belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. We show how a unique GNAT subfamily member uses a previously unidentified noncanonical catalytic triad, consisting of a glutamic acid, a histidine, and the antibiotic substrate itself, which acts as a nucleophile and attacks the acetyl donor molecule. Neutron diffraction studies allow for unambiguous identification of a low-barrier hydrogen bond, predicted in canonical catalytic triads to increase basicity of the histidine. This work highlights the role of this unique catalytic triad in mediating antibiotic resistance while providing new insights into the design of the next generation of aminoglycosides.

Encoding of Promiscuity in an Aminoglycoside Acetyltransferase.

Aminoglycoside antibiotics are a large family of antibiotics that can be divided into two distinct classes on the basis of the substitution pattern of the central deoxystreptamine ring. Although aminoglycosides are chemically, structurally, and topologically diverse, some aminoglycoside-modifying enzymes (AGMEs) are able to inactivate as many as 15 aminoglycosides from the two main classes, the kanamycin- and neomycin-based antibiotics. Here, we present the crystal structure of a promiscuous AGME, aminoglycoside- N3-acetyltransferase-IIIb (AAC-IIIb), in the apo form, in binary drug (sisomicin, neomycin, and paromomycin) and coenzyme A (CoASH) complexes, and in the ternary neomycin-CoASH complex. These data provide a structural framework for interpretation of the thermodynamics of enzyme-ligand interactions and the role of solvent in the recognition of ligands. In combination with the recent structure of an AGME that does not have broad substrate specificity, these structures allow for the direct determination of how antibiotic promiscuity is encoded in some AGMEs.

Thermophilic Enzyme or Mesophilic Enzyme with Enhanced Thermostability: Can We Draw a Line?

Aminoglycoside nucleotidyltransferase 4' (ANT) is a homodimeric enzyme that modifies the C4'-OH site of aminoglycoside antibiotics by nucleotidylation. A few single- and double-residue mutants of this enzyme (T130K, D80Y, and D80Y/T130K) from Bacillus stearothermophilus show increased thermostability. This article investigates how such residue replacements, which are distant from the active site and monomer-monomer interface, result in various changes of the thermostability of the enzyme. In this work, we show that the thermodynamic properties of enzyme-ligand complexes and protein dynamics may be indicators of a thermophilic behavior. Our data suggests that one of the single-site mutants of ANT, D80Y, may be a thermophilic protein and the other thermostable mutant, T130K, is actually a more heat-stable variant of the mesophilic wild type (WT) with a higher Tm. Our data also suggest that T130K and D80Y adopt different global dynamics strategies to achieve different levels of thermostability enhancement and that the differences between the properties of the species can be described in terms of global dynamics rather than in terms of specific structural features. Thermophilicity of the D80Y comes at the cost of less favorable thermodynamic parameters for ligand binding relative to WT. On the other hand, the T130K species exhibits the same affinity to ligands and the same thermodynamic parameters of complex formation as the WT enzyme. These observations suggest that a quantitative characterization of ligand binding and protein dynamics can be used to differentiate thermophilic proteins from their simply more heat-stable mesophilic counterparts.

Ligand promiscuity through the eyes of the aminoglycoside N3 acetyltransferase IIa.

Aminoglycoside-modifying enzymes (AGMEs) are expressed in many pathogenic bacteria and cause resistance to aminoglycoside (AG) antibiotics. Remarkably, the substrate promiscuity of AGMEs is quite variable. The molecular basis for such ligand promiscuity is largely unknown as there is not an obvious link between amino acid sequence or structure and the antibiotic profiles of AGMEs. To address this issue, this article presents the first kinetic and thermodynamic characterization of one of the least promiscuous AGMEs, the AG N3 acetyltransferase-IIa (AAC-IIa) and its comparison to two highly promiscuous AGMEs, the AG N3-acetyltransferase-IIIb (AAC-IIIb) and the AG phosphotransferase(3')-IIIa (APH). Despite having similar antibiotic selectivities, AAC-IIIb and APH catalyze different reactions and share no homology to one another. AAC-IIa and AAC-IIIb catalyze the same reaction and are very similar in both amino acid sequence and structure. However, they demonstrate strong differences in their substrate profiles and kinetic and thermodynamic properties. AAC-IIa and APH are also polar opposites in terms of ligand promiscuity but share no sequence or apparent structural homology. However, they both are highly dynamic and may even contain disordered segments and both adopt well-defined conformations when AGs are bound. Contrary to this AAC-IIIb maintains a well-defined structure even in apo form. Data presented herein suggest that the antibiotic promiscuity of AGMEs may be determined neither by the flexibility of the protein nor the size of the active site cavity alone but strongly modulated or controlled by the effects of the cosubstrate on the dynamic and thermodynamic properties of the enzyme.

Transient kinetics of aminoglycoside phosphotransferase(3')-IIIa reveals a potential drug target in the antibiotic resistance mechanism.

Aminoglycoside phosphotransferases are bacterial enzymes responsible for the inactivation of aminoglycoside antibiotics by O-phosphorylation. It is important to understand the mechanism of enzymes in order to find efficient drugs. Using rapid-mixing methods, we studied the transient kinetics of aminoglycoside phosphotransferase(3')-IIIa. We show that an ADP-enzyme complex is the main steady state intermediate. This intermediate interacts strongly with kanamycin A to form an abortive complex that traps the enzyme in an inactive state. A good strategy to prevent the inactivation of aminoglycosides would be to develop uncompetitive inhibitors that interact with this key ADP-enzyme complex.