Structure of the full‐length Serratia marcescens acetyltransferase AAC(3)‐Ia in complex with coenzyme A

Acyl‐coenzyme A‐dependent N‐acetyltransferases (AACs) catalyze the modification of aminoglycosides rendering the bacteria carrying such enzymes resistant to this class of antibiotics. Here we present the crystal structure of AAC(3)‐Ia enzyme from Serratia marcescens in complex with coenzyme A determined to 1.8 Å resolution. This enzyme served as an architype for the AAC enzymes targeting the amino group at Position 3 of aminoglycoside main aminocyclitol ring. The structure of this enzyme has been previously determined only in truncated form and was interpreted as distinct from subsequently characterized AACs. The reason for the unusual arrangement of secondary structure elements of AAC(3)‐Ia was not further investigated. By determining the full‐length structure of AAC(3)‐Ia we establish that this enzyme adopts the canonical AAC fold conserved across this family and it does not undergo through significant rearrangement of secondary structure elements upon ligand binding as was proposed previously. In addition, our results suggest that the C‐terminal tail in AAC(3)‐Ia monomer forms intramolecular hydrogen bonds that contributes to formation of stable dimer, representing the predominant oligomeric state for this enzyme.     

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.     

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.     

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     

Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.

BACKGROUND: The predominant mechanism of antibiotic resistance employed by pathogenic bacteria against the clinically used aminoglycosides is chemical modification of the drug. The detoxification reactions are catalyzed by enzymes that promote either the phosphorylation, adenylation or acetylation of aminoglycosides. Structural studies of these aminoglycoside-modifying enzymes may assist in the development of therapeutic agents that could circumvent antibiotic resistance. In addition, such studies may shed light on the development of antibiotic resistance and the evolution of different enzyme classes. RESULTS: The crystal structure of the aminoglycoside-modifying enzyme aminoglycoside 6'-N-acetyltransferase type li (AAC(6')-li) in complex with the cofactor acetyl coenzyme A has been determined at 2.7 A resolution. The structure establishes that this acetyltransferase belongs to the GCN5-related N-acetyltransferase superfamily, which includes such enzymes as the histone acetyltransferases GCN5 and Hat1. CONCLUSIONS: Comparison of the AAC(6')-li structure with the crystal structures of two other members of this superfamily, Serratia marcescens aminoglycoside 3-N-acetyltransferase and yeast histone acetyltransferase Hat1, reveals that of the 84 residues that are structurally similar, only three are conserved and none can be implicated as catalytic residues. Despite the negligible sequence identity, functional studies show that AAC(6')-li possesses protein acetylation activity. Thus, AAC(6')-li is both a structural and functional homolog of the GCN5-related histone acetyltransferases.     

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.     

Effect of solvent and protein dynamics in ligand recognition and inhibition of aminoglycoside adenyltransferase 2″‐Ia

The aminoglycoside modifying enzyme (AME) ANT(2″)‐Ia is a significant target for next generation antibiotic development. Structural studies of a related aminoglycoside‐modifying enzyme, ANT(3″)(9), revealed this enzyme contains dynamic, disordered, and well‐defined segments that modulate thermodynamically before and after antibiotic binding. Characterizing these structural dynamics is critical for in situ screening, design, and development of contemporary antibiotics that can be implemented in a clinical setting to treat potentially lethal, antibiotic resistant, human infections. Here, the first NMR structural ensembles of ANT(2″)‐Ia are presented, and suggest that ATP‐aminoglycoside binding repositions the nucleotidyltransferase (NT) and C‐terminal domains for catalysis to efficiently occur. Residues involved in ligand recognition were assessed by site‐directed mutagenesis. In vitro activity assays indicate a critical role for I129 toward aminoglycoside modification in addition to known catalytic D44, D46, and D48 residues. These observations support previous claims that ANT aminoglycoside sub‐class promiscuity is not solely due to binding cleft size, or inherent partial disorder, but can be controlled by ligand modulation on distinct dynamic and thermodynamic properties of ANTs under cellular conditions. Hydrophobic interactions in the substrate binding cleft, as well as solution dynamics in the C‐terminal tail of ANT(2″)‐Ia, advocate toward design of kanamycin‐derived cationic lipid aminoglycoside analogs, some of which have already shown antimicrobial activity in vivo against kanamycin and gentamicin‐resistant P. aeruginosa. This data will drive additional in silico, next generation antibiotic development for future human use to combat increasingly prevalent antimicrobial resistance.     

Aminoglycoside 2'-N-acetyltransferase from Mycobacterium tuberculosis in complex with coenzyme A and aminoglycoside substrates

AAC(2')-Ic catalyzes the coenzyme A (CoA)-dependent acetylation of the 2' hydroxyl or amino group of a broad spectrum of aminoglycosides. The crystal structure of the AAC(2')-Ic from Mycobacterium tuberculosis has been determined in the apo enzyme form and in ternary complexes with CoA and either tobramycin, kanamycin A or ribostamycin, representing the first structures of an aminoglycoside acetyltransferase bound to a drug. The overall fold of AAC(2')-Ic places it in the GCN5-related N-acetyltransferase (GNAT) superfamily. Although the physiological function of AAC(2')-Ic is uncertain, a structural analysis of these high-affinity aminoglycoside complexes suggests that the enzyme may acetylate a key biosynthetic intermediate of mycothiol, the major reducing agent in mycobacteria, and participate in the regulation of cellular redox potential.     

Kinetic and mutagenic characterization of the chromosomally encoded Salmonella enterica AAC(6')-Iy aminoglycoside N-acetyltransferase

The chromosomally encoded aminoglycoside N-acetyltransferase, AAC(6')-Iy, from Salmonella enterica confers resistance toward a number of aminoglycoside antibiotics. The structural gene was cloned and expressed and the purified enzyme existed in solution as a dimer of ca. 17 000 Da monomers. Acetyl-CoA was the preferred acyl donor, and most therapeutically important aminoglycosides were substrates for acetylation. Exceptions are those aminoglycosides that possess a 6'-hydroxyl substituent (e.g., lividomycin). Thus, the enzyme exhibited regioselective and exclusive acetyltransferase activity to 6'-amine-containing aminoglycosides. The enzyme exhibited Michaelis-Menten kinetics for some aminoglycoside substrates but "substrate activation" with others. Kinetic studies supported a random kinetic mechanism for the enzyme. The enzyme was inactivated by iodoacetamide in a biphasic manner, with half of the activity being lost rapidly and the other half more slowly. Tobramycin, but not acetyl-CoA, protected against inactivation. Each of the three cysteine residues (C70, C109, C145) in the wild-type enzyme were carboxamidomethylated by iodoacetamide. Cysteine 109 in AAC(6')-Iy is conserved in 12 AAC(6') enzyme sequences of the major class I subfamily. Surprisingly, mutation of this residue to alanine neither abolished activity nor altered the biphasic inactivation by iodoacetamide. The maximum velocity and V/K values for a number of aminoglycosides were elevated in this single mutant, and the kinetic behavior of substrates exhibiting linear vs nonlinear kinetics was reversed. Cysteine 70 in AAC(6')-Iy is either a cysteine or a threonine residue in all 12 AAC(6') enzymes of the major class I subfamily. The double mutant, C109A/C70A, was not inactivated by iodoacetamide. The double mutant exhibited large increases in the K(m) values for both acetyl-CoA and aminoglycoside substrates, and all aminoglycoside substrates exhibited Michaelis-Menten kinetics. Solvent kinetic isotope effects on V/K were normal for the WT enzyme and inverse for the double mutant. We discuss a chemical mechanism and the likely rate-limiting steps for both the wild-type and mutant forms of the enzyme.     

An aminoglycoside sensing riboswitch controls the expression of aminoglycoside resistance acetyltransferase and adenyltransferases

The emergence of antibiotic resistance in human pathogens is an increasing threat to public health. The fundamental mechanisms that control the high levels of expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are one of the earliest classes of antibiotics that were introduced in the 1940s. In the clinic aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug although resistance through enzymatic modification of the target rRNA through methylation or the overexpression of efflux pumps is also appearing. An aminoglycoside sensing riboswitch has been identified that controls expression of the aminoglycoside resistance genes that encode the aminoglycoside acetyltransferase (AAC) and aminoglycoside nucleotidyltransferase (ANT) (adenyltransferase (AAD)) enzymes. AAC and ANT cause resistance to aminoglycoside antibiotics through modification of the drugs. Expression of the AAC and ANT resistance genes is regulated by aminoglycoside binding to the 5′ leader RNA of the aac/aad genes. The aminoglycoside sensing RNA is also associated with the integron cassette system that captures antibiotic resistance genes. Specific aminoglycoside binding to the leader RNA induces a structural transition in the leader RNA, and consequently induction of resistance protein expression. Reporter gene expression, direct measurements of drug RNA binding, chemical probing and UV cross-linking combined with mutational analysis demonstrated that the leader RNA functioned as an aminoglycoside sensing riboswitch in which drug binding to the leader RNA leads to the induction of aminoglycoside antibiotic resistance. This article is part of a Special Issue entitled: Riboswitches.     

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.     

Silver potentiates aminoglycoside toxicity by enhancing their uptake

The predicted shortage in new antibiotics has prompted research for chemicals that could act as adjuvant and enhance efficacy of available antibiotics. In this study, we tested the effects of combining metals with aminoglycosides on Escherichia coli survival. The best synergizing combination resulted from mixing aminoglycosides with silver. Using genetic and aminoglycoside uptake assays, we showed that silver potentiates aminoglycoside action in by‐passing the PMF‐dependent step, but depended upon protein translation. We showed that oxidative stress or Fe–S cluster destabilization were not mandatory factors for silver potentiating action. Last, we showed that silver allows aminoglycosides to kill an E. coli gentamicin resistant mutant as well as the highly recalcitrant anaerobic pathogen Clostridium difficile. Overall this study delineates the molecular basis of silver's potentiating action on aminoglycoside toxicity and shows that use of metals might offer solutions for battling against increased bacterial resistance to antibiotics.     

Thermodynamics of aminoglycoside and acyl-coenzyme A binding to the Salmonella enterica AAC(6')-Iy aminoglycoside N-acetyltransferase

Kinetic and mechanistic studies on the chromosomally encoded aminoglycoside 6'-N-acetyltransferase, AAC(6')-Iy, of Salmonella enterica that confers resistance toward aminoglycosides have been previously reported [Magnet et al. (2001) Biochemistry 40, 3700-3709]. In the present study, equilibrium binding and the thermodynamic parameters of binding of aminoglycosides and acyl-coenzyme A derivatives to AAC(6')-Iy and of two mutants, C109A and the C109A/C70A double mutant, have been studied using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Association constants for different aminoglycosides varied greatly (4 x 10(4)-150 x 10(4)) while the association constants of several acyl-coenzyme A derivatives were similar (3.2 x 10(4)-4.5 x 10(4)). The association constants and van't Hoff enthalpy changes derived from intrinsic protein fluorescence changes were in agreement with independently measured values from isothermal titration calorimetry studies. Binding of both aminoglycosides and acyl-coenzyme A derivatives is strongly enthalpically driven and revealed opposing negative entropy changes, resulting in enthalpy-entropy compensation. The acetyltransferase exhibited a temperature-dependent binding of tobramycin with a negative heat capacity value of 410 cal mol(-1) K(-1). Isothermal titration studies of acetyl-coenzyme A and tobramycin binding to mutant forms of the enzyme indicated that completely conserved C109 does not play any direct role in the binding of either of the substrates, while C70 is directly involved in aminoglycoside binding. These results are discussed and compared with previous steady-state kinetic studies of the enzyme.     

Overexpression and mechanistic analysis of chromosomally encoded aminoglycoside 2'-N-acetyltransferase (AAC(2')-Ic) from Mycobacterium tuberculosis

The chromosomally encoded aminoglycoside N-acetyltransferase, AAC(2')-Ic, of Mycobacterium tuberculosis has a yet unidentified physiological function. The aac(2')-Ic gene was cloned and expressed in Escherichia coli, and AAC(2')-Ic was purified. Recombinant AAC(2')-Ic was a soluble protein of 20,000 Da and acetylated all aminoglycosides substrates tested in vitro, including therapeutically important antibiotics. Acetyl-CoA was the preferred acyl donor. The enzyme, in addition to acetylating aminoglycosides containing 2'-amino substituents, also acetylated kanamycin A and amikacin that contain a 2'-hydroxyl substituent, although with lower activity, indicating the capacity of the enzyme to perform both N-acetyl and O-acetyl transfer. The enzyme exhibited "substrate activation" with many aminoglycoside substrates while exhibiting Michaelis-Menten kinetics with others. Kinetic studies supported a random kinetic mechanism for AAC(2')-Ic. Comparison of the kinetic parameters of different aminoglycosides suggested that their hexopyranosyl residues and, to a lesser extent, the central aminocyclitol residue carry the major determinants of substrate affinity.     

Domain-domain interactions in the aminoglycoside antibiotic resistance enzyme AAC(6')-APH(2')

The most common determinant of aminoglycoside antibiotic resistance in Gram positive bacterial pathogens, such as Staphylococcus aureus, is a modifying enzyme, AAC(6')-APH(2' '), capable of acetylating and phosphorylating a wide range of antibiotics. This enzyme is unique in that it is composed of two separable modification domains, and although a number of studies have been conducted on the acetyltransferase and phosphotransferase activities in isolation, little is known about the role and impact of domain interactions on antibiotic resistance. Kinetic analysis and in vivo assessment of a number of N- and C-terminal truncated proteins have demonstrated that the two domains operate independently and do not accentuate one another's resistance activity. However, the two domains are structurally integrated, and mutational analysis has demonstrated that a predicted connecting alpha-helix is especially critical for maintaining proper structure and function of both activities. AAC(6')-APH(2' ') detoxifies a staggering array of aminoglycosides, where one or both activities make important contributions depending on the antibiotic. Thus, to overcome antibiotic resistance associated with AAC(6')-APH(2' '), aminoglycosides resistant to modification and/or inhibitors against both activities must be employed. Domain-domain interactions in AAC(6')-APH(2' ') offer a unique target for inhibitor strategies, as we show that their disruption simultaneously inhibits both activities >90%.     

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.     

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.     

The molecular basis of the expansive substrate specificity of the antibiotic resistance enzyme aminoglycoside acetyltransferase-6'-aminoglycoside phosphotransferase-2". The role of ASP-99 as an active site base important for acetyl transfer

The most frequent determinant of aminoglycoside antibiotic resistance in Gram-positive bacterial pathogens is a bifunctional enzyme, aminoglycoside acetyltransferase-6'-aminoglycoside phosphotransferase-2" (AAC(6')- aminoglycoside phosphotransferase-2", capable of modifying a wide selection of clinically relevant antibiotics through its acetyltransferase and kinase activities. The aminoglycoside acetyltransferase domain of the enzyme, AAC(6')-Ie, is the only member of the large AAC(6') subclass known to modify fortimicin A and catalyze O-acetylation. We have demonstrated through solvent isotope, pH, and site-directed mutagenesis effects that Asp-99 is responsible for the distinct abilities of AAC(6')-Ie. Moreover, we have demonstrated that small planar molecules such as 1-(bromomethyl)phenanthrene can inactivate the enzyme through covalent modification of this residue. Thus, Asp-99 acts as an active site base in the molecular mechanism of AAC(6')-Ie. The prominent role of this residue in aminoglycoside modification can be exploited as an anchoring site for the development of compounds capable of reversing antibiotic resistance in vivo.     

X-ray structure of the AAC(6')-Ii antibiotic resistance enzyme at 1.8 A resolution; examination of oligomeric arrangements in GNAT superfamily members

The rise of antibiotic resistance as a public health concern has led to increased interest in studying the ways in which bacteria avoid the effects of antibiotics. Enzymatic inactivation by several families of enzymes has been observed to be the predominant mechanism of resistance to aminoglycoside antibiotics such as kanamycin and gentamicin. Despite the importance of acetyltransferases in bacterial resistance to aminoglycoside antibiotics, relatively little is known about their structure and mechanism. Here we report the three-dimensional atomic structure of the aminoglycoside acetyltransferase AAC(6')-Ii in complex with coenzyme A (CoA). This structure unambiguously identifies the physiologically relevant AAC(6')-Ii dimer species, and reveals that the enzyme structure is similar in the AcCoA and CoA bound forms. AAC(6')-Ii is a member of the GCN5-related N-acetyltransferase (GNAT) superfamily of acetyltransferases, a diverse group of enzymes that possess a conserved structural motif, despite low sequence homology. AAC(6')-Ii is also a member of a subset of enzymes in the GNAT superfamily that form multimeric complexes. The dimer arrangements within the multimeric GNAT superfamily members are compared, revealing that AAC(6')-Ii forms a dimer assembly that is different from that observed in the other multimeric GNAT superfamily members. This different assembly may provide insight into the evolutionary processes governing dimer formation.     

Substrate specificities and structure-activity relationships for acylation of antibiotics catalyzed by kanamycin acetyltransferase

Antibiotic resistance caused by the presence of the plasmid pMH67 is mediated by the aminoglycoside acetyltransferase AAC(6')-4, also known as kanamycin acetyltransferase. Bacteria harboring the plasmid are resistant to the kanomycins plus a broad range of other deoxystreptamine-containing aminoglycosides but not to the gentamicins XK62-2 and C1 which are substituted at the 6'-position. Substrate specificity studies on the purified enzyme, however, now show that the enzyme acetylates an even broader range of aminoglycosides, including the gentamicins XK62-2 and C1. The enzyme also accepts several acyl-CoA esters, which differ in nucleotide as well as in acyl chain length. Application of the method of analysis of structure-activity data developed earlier for gentamicin acetyltransferase [Williams, J. W., & Northrop, D. B. (1978) J. Biol. Chem. 253, 5908-5914] to the kinetic data obtained for AAC(6')-4 shows that the turnover of the acylation reaction is limited by catalysis and not by the rate of release of either the acetylated antibiotic or CoA. Most structural changes in aminoglycosides cause changes in rates of release, and only drastic changes, near the 6'-amino group, affect catalysis. The structural requirements on aminoglycosides for enzymatic activity run parallel to the structural requirements for antibacterial activity.