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.     

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.     

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.     

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%.     

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.     

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.     

Nucleotide sequence analysis of the gene specifying the bifunctional 6''-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities.

The gene specifying the bifunctional 6'-aminoglycoside acetyltransferase [AAC(6')] 2"-aminoglycoside phosphotransferase [APH(2")] enzyme from the Streptococcus faecalis plasmid pIP800 was cloned in Escherichia coli. A single protein with an apparent molecular weight of 56,000 was specified by this cloned determinant as detected in minicell experiments. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 479 amino acids and with a molecular weight of 56,850. The deduced amino acid sequence of the bifunctional AAC(6')-APH(2") gene product possessed two regions of homology with other sequenced resistance proteins. The N-terminal region contained a sequence that was homologous to the chloramphenicol acetyltransferase of Bacillus pumilus, and the C-terminal region contained a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. Subcloning experiments were performed with the AAC(6')-APH(2") resistance determinant, and it was possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities. These data suggest that the gene specifying the AAC(6')-APH(2") resistance enzyme arose as a result of a gene fusion.     

Primary structure of an aminoglycoside 6''-N-acetyltransferase AAC(6'')-4, fused in vivo with the signal peptide of the Tn3-encoded beta-lactamase.

The gene aacA4 encoding an aminoglycoside 6'-N-acetyltransferase, AAC(6')-4, was cloned from a natural multiresistance plasmid, and its nucleotide sequence was determined. The gene was 600 base pairs (bp) long, and the AAC(6')-4 had a calculated molecular size of 22.4 kilodaltons and an isoelectric point of 5.35. The sequence of the 17 N-terminal amino acids was determined from the purified enzyme. The AAC(6')-4 gene was part of a resistance gene cluster, and its expression was under the control of the regulatory sequences of the beta-lactamase encoded by Tn3. The five N-terminal amino acids were identical to those of the signal peptide of the Tn3-encoded beta-lactamase, and the entire 5' region of aacA4, as far as it was sequenced (354 bp, including the promoter and the ribosome-binding site sequences), was identical to that of the beta-lactamase gene. This led us to presume an in vivo fusion between the beta-lactamase and the acetyltransferase genes. The latter was followed, in a polycistronic arrangement, by an aminoglycoside 3",9-adenylyltransferase gene, aadA, with an intergenic region of 68 bp. At a distance of ca. 1.3 kilobases in the 3' direction, we found remnants of a second Tn3-like element specifying an active beta-lactamase. At their 5' extremities, the two incomplete copies of Tn3, which were in tandem orientation, were interrupted within the resolvase gene. We speculate that Tn3-related sequences have played a role in the process of selection and dissemination of the AAC(6')-4 gene, which specifies resistance to amikacin and related aminoglycosides.     

Substrate specificities and structure-activity relationships for the nucleotidylation of antibiotics catalyzed by aminoglycoside nucleotidyltransferase 2'-I

Aminoglycoside nucleotidyltransferase 2'-I (formerly gentamicin adenylyltransferase) conveys antibiotic resistance to Gram-negative bacteria by transfer of AMP to the 2'-hydroxyl group of 4,6-substituted deoxystreptamine-containing aminoglycosides. The kinetics constants of thirteen aminoglycoside antibiotics and the magnesium chelates of eight nucleotide triphosphates were determined with purified enzyme. Eleven of the antibiotics exhibit substrate inhibition attributed to secondary binding of the aminoglycoside to an enzyme-AMP-aminoglycoside complex. Maximal velocities vary by only 4-fold, versus variation of values of Vmax/Km for the aminoglycosides of nearly 4000-fold, consistent with a Theorell-Chance kinetic mechanism as proposed for this enzyme [Gates, C. A., & Northrop, D. B. (1988) Biochemistry (second of three papers in this issue)] with the added specification that the binding of aminoglycosides is in rapid equilibrium. Under these conditions, Vmax/Km becomes kcat/Kd, where kcat is the net rate constant for catalysis (but not turnover) and Kd is the dissociation constant of aminoglycosides from a complex with enzyme and nucleotide. Values of kcat fall closely together into three distinct sets, with the 3',4'-dideoxygentamicins greater than gentamicins greater than kanamycins. These sets reflect unusual structure-activity correlations which are specific for catalysis but have nothing to do with the maximal velocity of this enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative study of apramycin-resistant microorganisms isolated from man and animals

Apramycin-modifying strains isolated from pigs with coli bacteriosis, from humans and hospital environment were studied comparatively. Production of enzymes modifying the aminoglycoside was estimated with the radioactive cofactor procedure. E. coli isolates from the animals were phenotypically resistant to apramycin and a number of other aminoglycosides. They produced acetyltransferase AAC(3)IV, phosphotransferase APH(3')(5"), APH(3") and other enzymes. Resistance of the strains to gentamicin was also conditioned by AAC(3)IV since these strains did not produce AAD(2") and AAC(6'). In the resistant strains of E. coli and their transconjugates there were detected plasmids with a relative molecular weight of 60-80 MD. Some of the belonged to the compatibility group I1, the others belonged to the compatibility group H1. Strains of S. marcescens, K. pneumoniae. K. oxytoca and S. aureus isolated from humans and hospital environment were sensitive to apramycin. Only isolates of P. aeruginosa were resistant to this antibiotic. However, all the isolates produced AAC(3)IV. Some of them additionally produced AAC(6'), an enzyme modifying amikacin, kanamycin and other antibiotics and not acetylating apramycin. Almost all the strains produced kanamycin- and streptomycin phosphotransferases. Possible coselection of strains resistant to apramycin and gentamicin using one of these aminoglycosides is discussed.     

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.     

Aminoglycoside resistance patterns in clinical isolates of E. coli and Klebsiella sp. from Czechoslovakia and the United States

Resistance of gram-negative bacilli to aminoglycoside antibiotics differs by region and country. Previous studies have demonstrated predominance of the nucleotidyltransferase ANL(2") as the mechanism of enzymatic resistance to gentamicin in the United States and many European countries (Federal Republic of Germany, Switzerland, Greece, Turkey) whereas the acetylating enzymes AAC(6') and AAC(3) were the principal causes of resistance to aminoglycosides in Japan and Chile. In the present comparison of 18 drug resistant isolates of E. coli and Klebsiella sp. from Czechoslovakia and the United States, with aminoglycoside-inactivating enzymes, ANT(2") characterized the most strains from both countries. In a higher number of isolates from Czechoslovakia however, the aminoglycoside resistance was mediated by AAC(3). In the majority of strains a simultaneous occurrence of two gentamicin-inactivating enzymes i.e. ANT(2"), plus AAC (2'), or AAC(6') or AAC(3) was observed. In amikacin resistant E. coli strains the mechanism of resistance was represented by production of AAC(6') or AAC*--an acetyltransferase with uncommon substrate profile. In all E. coli and K. pneumoniae strains from the United States apart from ANT(2") also AAC(2') was detected. This represents a broadening of the host range of aac(2') gene, the occurrence of which has been limited only to Providencia and Proteus strains.     

Enzyme structural plasticity and the emergence of broad-spectrum antibiotic resistance

The emergence of multi-resistant pathogenic bacteria is a worldwide health issue. Recently, clinical variants of a single antibiotic-modifying acetyltransferase, AAC(6')-Ib-a variant of aminoglycoside 6'-N-acetyltransferase-have been identified that confer extended resistance to most aminoglycosides and, more surprisingly, to structurally unrelated fluoroquinolones. The corresponding gene is carried by mobile genetic elements and is present in most multi-resistant pathogenic strains, hence making it a serious threat to current therapies. Here, we report the crystal structures of both narrow- and broad-spectrum resistance variants of this enzyme, which reveal the structural basis for the emergence of extended resistance. The active site shows an important plasticity and has adapted to new substrates by a large-scale gaping process. We have also obtained co-crystals with both substrates, and with a simple transition state analogue, which provides new clues for the design of inhibitors of this resistance mechanism.     

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.     

Kinetic mechanism of the GCN5-related chromosomal aminoglycoside acetyltransferase AAC(6')-Ii from Enterococcus faecium: evidence of dimer subunit cooperativity

The aminoglycoside 6'-N-acetyltransferase AAC(6')-Ii from Enterococcus faecium is an important microbial resistance determinant and a member of the GCN5-related N-acetyltransferase (GNAT) superfamily. We report here the further characterization of this enzyme in terms of the kinetic mechanism of acetyl transfer and identification of rate-contributing step(s) in catalysis, as well as investigations into the binding of both acetyl-CoA and aminoglycoside substrates to the AAC(6')-Ii dimer. Product and dead-end inhibition studies revealed that AAC(6')-Ii follows an ordered bi-bi ternary complex mechanism with acetyl-CoA binding first followed by antibiotic. Solvent viscosity studies demonstrated that aminoglycoside binding and product release govern the rate of acetyl transfer, as evidenced by changes in both the k(cat)/K(b) for aminoglycoside and k(cat), respectively, with increasing solvent viscosity. Solvent isotope effects were consistent with our viscosity studies that diffusion-controlled processes and not the chemical step were rate-limiting in drug modification. The patterns of partial and mixed inhibition observed during our mechanistic studies were followed up by investigating the possibility of subunit cooperativity in the AAC(6')-Ii dimer. Through the use of AAC-Trp(164) --> Ala, an active mutant which exists as a monomer in solution, the partial nature of the competitive inhibition observed in wild-type dead-end inhibition studies was alleviated. Isothermal titration calorimetry studies also indicated two nonequivalent antibiotic binding sites for the AAC(6')-Ii dimer but only one binding site for the Trp(164) --> Ala mutant. Taken together, these results demonstrate subunit cooperativity in the AAC(6')-Ii dimer, with possible relevance to other oligomeric members of the GNAT superfamily.     

Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001

Resistance to the aminoglycosides gentamicin, tobramycin and kanamycin (GmTmKmR) in Australian clinical strains of Staphylococcus aureus is commonly carried on the composite transposon Tn4001. The resistance gene aacA-aphD of Tn4001, which encodes a bifunctional AAC(6')-APH(2") modifying enzyme, is flanked by two 1324-bp inverted repeats, IS256L and IS256R, that are identical in sequence. Analysis of the IS256 sequence revealed structural features characteristic of IS elements including 26-bp imperfect terminal inverted repeats and a single open reading frame with coding capacity for a 45.6 kDa protein. The nucleotide sequence of IS256 described here, together with the sequence of the aacA-aphD gene reported previously [Rouch et al., J. Gen. Microbiol. 133 (1987) 3039-3052], completes the entire sequence of Tn4001, which totals 4566 bp.     

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.     

The use of aminoglycoside derivatives to study the mechanism of aminoglycoside 6'-N-acetyltransferase and the role of 6'-NH2 in antibacterial activity

Aminoglycoside antibiotics act by binding to 16S rRNA. Resistance to these antibiotics occurs via drug modifications by enzymes such as aminoglycoside 6'-N-acetyltransferases (AAC(6')s). We report here the regioselective and efficient synthesis of N-6'-acylated aminoglycosides and their use as probes to study AAC(6')-Ii and aminoglycoside-RNA complexes. Our results emphasize the central role of N-6' nucleophilicity for transformation by AAC(6')-Ii and the importance of hydrogen bonding between 6'-NH(2) and 16S rRNA for antibacterial activity.     

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 k_cat values. Larger differences in K_M (～40‐fold) resulted in ～30‐fold variation in k_cat/K_M. 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 H₂O and D₂O 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.     

Resistance to apramycin in Escherichia coli isolated from animals: detection of a novel aminoglycoside-modifying enzyme

The mechanisms of resistance to apramycin of five isolates of Escherichia coli from animals were investigated. Three isolates, which were resistant to all the aminoglycosides tested, did not transfer their resistance and did not produce aminoglycoside-modifying enzymes. The fourth isolate, which was resistant to apramycin, tobramycin, gentamicin, kanamycin and neomycin but not to amikacin, owed its resistance to production of the acetyltransferase AAC(3)IV. The gene specifying this enzyme was carried on a transposon, Tn800, on a plasmid designated R1535. The fifth isolate was resistant to apramycin, neomycin and kanamycin but not to gentamicin, tobramycin or amikacin. It produced an acetyltransferase that readily acetylated only apramycin, neomycin and paromomycin, a compound that is closely related to neomycin. Synthesis of this enzyme was specified by a chromosomal gene located near pyrD at about 20 min on the map of the E. coli K12 chromosome.