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*

David D. Boehr, Stephen I. Jenkins, and Gerard D. WrightDagger

From the Antimicrobial Research Centre, Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada

Received for publication, November 15, 2002, and in revised form, January 31, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Clinical usage of aminoglycoside-aminocylitol antibiotics is blocked by the presence of aminoglycoside modifying enzymes (AMEs)1 in resistant organisms (for review see Refs. 1 and 2). Bacteria become protected from aminoglycosides, because the modified antibiotics can no longer bind with high affinity to their target, the A-site of the small ribosomal subunit, because of unfavorable steric and/or electrostatic constraints (3). The AMEs are a diverse set of proteins composed of three families: aminoglycoside nucleotidyltransferases, aminoglycoside acetyltransferases (AACs), and aminoglycoside phosphotransferases (APHs).

The most clinically important AME in Gram-positive bacterial pathogens such as Staphylococcus aureus is AAC(6')-APH(2") (4). This enzyme is unique in that it is bifunctional, comprising activities from two classes of AMEs: an N-terminal AAC (AAC(6')-Ie) and a C-terminal APH (APH(2")-Ia) (Fig. 1) (5, 6). AAC(6')-APH(2") has an extraordinary ability to detoxify a wide selection of aminoglycosides (7, 8) not only as a result of its bifunctional nature but also because of the special characteristics of each activity. For example, APH(2")-Ia has a tremendous ability to accommodate a vast range of antibiotics and binding conformations as evidenced by the remarkably broad regiospecificity of phosphoryl transfer that enables modification to occur on hydroxyl groups from four different aminoglycoside ring systems (see Fig. 1) (8). Conversely, AAC(6')-Ie, has a very stringent regiospecificity of acetyl transfer, because it is restricted to acetylation of the 6'-position of aminoglycosides only (see Fig. 1) (8). AAC(6')-Ie is the sole member of the very large AAC(6') subclass known to acetylate the antibiotic fortimicin A (9), and moreover, in addition to N-acetylation activity, this enzyme has been shown to have O-acetylation capabilities (8). The unusual activities of AAC(6')-Ie may be related to residues that can bind fortimicin A and 6'-OH aminoglycosides, or to unique residues that are required to catalyze the acetylation of these antibiotics.

The APH class of enzyme is relatively well characterized (10), mostly owing to structural and functional studies on APH(3')-IIIa (Refs. 11 and 12, and references therein). Our mechanistic studies on APH(2")-Ia (13)2 are consistent with results obtained with APH(3')-IIIa. On the other hand, the molecular mechanisms of AACs are comparatively less understood, despite the fact that crystal structures for AAC(3')-Ia (14), AAC(2')-Ic (15), and AAC(6')-Ii (16) are known. The clinical importance of AAC(6')-APH(2") and the substrate specificity differences between AAC(6')-Ie and other aminoglycoside acetyltransferases warrant more thorough study of the acetyltransferase domain of AAC(6')-APH(2") to understand the distinct properties of this enzyme.

Structural studies on AACs have demonstrated that the aminoglycoside acetyltransferases belong to the GNAT (GCN-5 related N-acetyltransferases) superfamily (for review, see Ref. 17). The kinetic mechanisms of characterized superfamily members (18-22), including AAC(6')-Ie (23), are sequential Bi Bi suggesting that acetyl transfer occurs directly from acetyl-CoA to acceptor substrate via the formation of a ternary enzyme·acetyl-CoA·aminoglycoside complex. Crystal structures of Tetrahymena GCN5 (tGCN5) histone acetyltransferase bound with acetyl-CoA and histone H3 (24) and serotonin N-acetyltransferase (also known as arylalkylamine N-acetyltransferase or AANAT) bound with a bisubstrate analog (25-28), further argue for a direct acetyl transfer mechanism. For such a transfer to occur, the amine of the acceptor substrate must react with the carbonyl carbon of acetyl-CoA. However, at physiological pH, amines tend to be fully protonated and, hence, are not in a chemically competent state to accept the acetyl group. Consequently, an active site base has been proposed to deprotonate the amine to generate a more potent nucleophile capable of being acetylated.

Structural and functional analyses of AANAT (25, 26, 29) and tGCN5 (24, 30, 31) have implicated His-122 and Glu-173, respectively, as the catalytic base in these enzymes, and mutation of Glu-173 to Gln results in an ~1000-fold decrease in GCN5 acetyltransferase activity (30, 31). However, studies with AANAT have been less enlightening, as mutation of His-122 to Gln only results in a 6-fold decrease in activity (29). It has been suggested that substrate deprotonation does not occur directly, but through a series of well-ordered water molecules, or "proton wire," connecting the substrate amine and the catalytic base (reviewed in Refs. 17). In such a scenario, other residues in the active site that are capable of accepting hydrogen from these water molecules may compensate in part for the loss of the normal proton acceptor. Indeed, His-120 in AANAT appears to play this role and only when both His-122 and His-120 are mutated to Gln is there a much more substantial effect consistent with base catalysis (29).

In contrast to either tGCN5 or AANAT, glucosamine 6-phosphate N-acetyltransferase (GNA1) appears to lack an active site base since mutational analysis of Glu-98, which is at the same geometrical position as His-120 in AANAT, is inconsistent with base catalysis (32). In this case, an active site base may not be required considering that the pKa of its substrate (~7.75) is sufficiently low that a significant proportion will be in the chemically competent form at physiological pH (33). A very complex picture of catalysis emerges from these findings where the molecular details of acetyl transfer in the GNAT family depend on the specific enzyme and/or substrate. This is not surprising, taking into account the limited sequence similarity shared between these proteins where the conserved GNAT core is mostly involved in providing a binding site for acetyl-CoA and not in providing common scaffolding for catalysis.

Here we have addressed the role of an active base in the catalytic mechanism of AAC(6')-Ie. We demonstrate through solvent isotope effects, pH studies, and mutational analysis that Asp-99 fulfills the role of an active site base and is responsible for the ability of AAC(6')-Ie to catalyze O-acetyl transfer. Although Asp-99 provides the enzyme with an enhanced detoxification profile, we further demonstrate that compounds such as 1-(bromomethyl)phenanthrene can inactivate AAC(6')-Ie by covalently modifying this residue. These compounds have the potential to be further developed as potent inhibitors of AAC(6')-APH(2") to overcome aminoglycoside resistance in vivo.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Reagents-- Kanamycin A was from Bioshop (Burlington, Ontario, Canada). 4,4'-Dithiodipyridine was from Amersham Biosciences (Baie d'Urfe, Quebec, Canada). All other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise noted. All oligonucleotide primers were synthesized at the Central Facility of the Institute for Molecular Biology and Biotechnology, McMaster University. The purification of N-terminal hexahistidine-tagged AAC(6')-APH(2") has been previously described (13), and mutant enzymes were purified similarly.

AAC(6')-Ie Kinetic Assays-- Aminoglycoside acetylation by AAC(6')-Ie was normally monitored by the in situ titration of free coenzyme A product with 4,4'-dithiodipyridine as previously described (8). However, assays were scaled down from 1-ml to 250-µl volumes, so they could be conducted in 96-well microtiter plates using a Molecular Devices SpectraMax Plus microtiter plate reader. For more sensitive assays, acetyltransferase activity was determined using [1-14C]acetyl-CoA and a phosphocellulose binding assay described previously (16). Reaction mixtures typically contained 25 mM HEPES-NaOH, pH 7.5, 0.1 µCi of [1-14C]AcCoA (200 µM final concentration), 1 mM aminoglycoside substrate, and 0.3 nmol of pure AAC(6')-Ie, and were allowed to proceed for 10 min at 37 °C.

Initial rates were fit to the Michaelis-Menten equation (Equation 1) using Grafit 4.0 (34),
v=(k<SUB><UP>cat</UP></SUB>/E<SUB><UP>t</UP></SUB>)[<UP>S</UP>]<UP>/</UP>(K<SUB>m</SUB>+[<UP>S</UP>]) (Eq. 1)
where v is the initial velocity, Et is the total amount of enzyme in the assay and [S] is the concentration of substrate. The concentrations of acetyl-CoA and kanamycin A were held at 300 µM when measuring the steady-state kinetic parameters for aminoglycoside substrate and acetyl-CoA, respectively.

Solvent Viscosity, Solvent Isotope, and pH Effects for AAC(6')-Ie-- The solvent viscosity, solvent isotope, and pH effects for AAC(6')-Ie were determined by varying the buffer conditions of the standard assay. For the solvent viscosity effect experiments, steady-state kinetic parameters were determined with varying concentrations of the microviscosogen glycerol (0, 15, 22.5, and 30% (w/v)). The viscosity of the solutions was determined using an Ostwald viscometer in triplicate, and the slope of a plot of relative viscosity versus rateo/rateviscogen reveals the solvent viscosity effect (SVE). Steady-state kinetic parameters were also performed in the macroviscosogen polyethylene glycol 8000 (6.7% w/v) and were not found to have a significant effect on the rate constants.

Solvent isotope effects were measured by conducting kinetic analyses in D2O (99.9% from Isotec), where the final amount of H2O was not more than 5%. pD values were determined by measuring pH and adding 0.4 unit (pD = pH + 0.4). The proton inventory study was conducted by adding ratios of buffer in H2O and D2O and making appropriate corrections for the addition of enzyme and substrates to calculate the final amount of D2O in the enzyme assay solution.

Buffers with overlapping pH ranges were used to investigate the effect of pH on enzyme activity. Buffers used were: 25 mM MOPS-NaOH (pH 6.0-6.5), 25 mM MES-NaOH (pH 6.5-7.5), 25 mM HEPES-NaOH (pH 7.0-8.0), and 25 mM glycylglycine (pH 8.0-9.5). None of the buffers gave any significant nonspecific effects. The data were analyzed using Grafit 4 (34). For AAC(6')-Ie WT, which displays two ionizations, the data can be fit to the following equation through nonlinear regression,
<UP>log </UP>v=<UP>log</UP><FENCE>C/<FENCE>1+<FR><NU>H</NU><DE>K<SUB>a</SUB></DE></FR>+<FR><NU>K<SUB>b</SUB></NU><DE>H</DE></FR></FENCE></FENCE> (Eq. 2)

For single ionization analysis with AAC(6')-Ie Asp-99 right-arrow Ala, the data can be fit using nonlinear regression analysis of the following equation,
<UP>log </UP>v=<UP>log</UP><FENCE>C/<FENCE>1+<FR><NU>K<SUB>b</SUB></NU><DE>H</DE></FR></FENCE></FENCE> (Eq. 3)
where v is the first-order (kcat) or second-order (kcat/Km) rate constant, Ka and Kb are the acid and base equilibrium constants, respectively, C is the pH-independent value, and H is the proton concentration.

Site-directed Mutagenesis-- Site-directed mutagenesis was performed using the QuikChange method (Stratagene, La Jolla, CA). The appropriate mutagenic primers (Tyr-96 right-arrow Phe, GAGATAGTCTTTGGTATGGATCAATTTATAGGAGAGCC; Asp-99 right-arrow Ala, GAGATAGTCTATGGTATGGCTCAATTTATAGGAGAGCC; Asp-99 right-arrow Asn, GAGATAGTCTATGGTATGGAACAATTTATAGGAGAGCC; and Asp-99 right-arrow Glu, GAGATAGTCTATGGTATGAATCAATTTATAGGAGAGCC) and their reverse complements were used in combination with 20 ng of template DNA (pET15AACAPH (13)) in Pfu DNA polymerase (Stratagene, La Jolla, CA)-catalyzed PCR reactions. After parental DNA was digested with DpnI, mutant plasmid DNA was transformed into CaCl2-competent Escherichia coli XL-1 Blue. Positive clones were sequenced in their entirety and then used to transform into E. coli BL21(DE3) for subsequent protein purification.

Minimum Inhibitory Concentration Determinations with Fortimicin A-- Minimum inhibitory concentration (MIC) determinations were performed as described in Ref. 35, where the MICs for E. coli BL21(DE3) carrying control plasmid pET15b(+) were compared with E. coli BL21(DE3) carrying pET15AACAPH and appropriate plasmids with site mutants in the aac(6')-aph(2") gene.

Synthesis of 1-(Bromomethyl)phenanthrene, 1-(Bromomethyl)naphthalene, and 9-(Chloromethyl)phenanthrene-- The preparation of 1-(bromomethyl)phenanthrene and 9-(chloromethyl)phenanthrene using N-bromo- and N-chlorosuccinimide, respectively, has been previously reported (36). 1-(Bromomethyl)naphthalene was prepared in an analogous manner using N-bromosuccinimide: thin-layer chromatography, Rf (SiO2, 2% (v/v) EtOAc in hexanes), 0.42; 1H NMR (200 MHz, CDCl3): delta  8.21 (d, J = 8.57 Hz, 1H, CH), 7.90 (t, J = 9.18 Hz, 2H, CH), 7.43-7.67 (m, 4 H, CH), 4.99 (s, 2H, CH2Br); 13C NMR (50 MHz, CDCl3): delta  133.9, 133.2, 131.0, 129.7, 128.8, 127.7, 126.5, 126.1, 125.3, 123.6, and 31.7.

Inactivation of AAC(6')-Ie by (Halomethyl)phenanthrenes and (Halomethyl)naphthalenes-- Inactivation experiments were carried out by incubating enzyme (0.2-2 µM) with compound (0-200 µM) dissolved in Me2SO (0.04% v/v total) in 240 µl of 25 mM HEPES-NaOH, pH 7.5, and 2 mM 4,4'-dithiodipyridine at 37 °C for 10-40 min, prior to the addition of kanamycin A and acetyl-CoA (300 µM final concentrations) to measure acetyltransferase activity. For the substrate protection experiments, kanamycin A (500 µM) and/or desulfo-CoA (100 µM) was incubated with the inactivation mixture for 10 min before the addition of acetyl-CoA (500 µM final). The concentration of desulfo-CoA used in these experiments (100 µM) was in the range of the IC50 for desulfo-CoA using 300 µM kanamycin A and 300 µM acetyl-CoA.2 The effect of pH on inactivation was determined by conducting inactivation experiments in the following buffers: 25 mM MOPS-NaOH (pH 6.5), 25 mM HEPES-NaOH (pH 7.0-8.0), and 25 mM glycylglycine (pH 8.5).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

AAC(6')-Ie Can Acetylate Aminoglycoside 6'-OH and 6'- NH2-- AAC(6')-Ie is unique in that it is the only known member of the AAC(6') subclass that can acetylate fortimicin A, and it also has the ability to O-acetylate aminoglycosides at the 6' position as represented by paromomycin (8) (Fig. 1 and Table I). The pKa of the 6'-NH<UP><SUB>3</SUB><SUP>+</SUP></UP> of aminoglycosides related to neamine (37), and kanamycin A (38) has previously been determined to be 8.6. As such, a significant proportion of the aminoglycoside will be in the fully protonated state at physiological pH, and a catalytic base would be predicted to increase the efficiency of the reaction, although there is no absolute requirement for an active site base. An active site base would be predicted to be more critical with 6'-OH aminoglycosides, because the pKa of the hydroxyl will be in the range of 14-16.


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Fig. 1.   Regiospecificity of acetyl and phosphoryl modification of aminoglycosides catalyzed by AAC(6')-APH(2"). Arrows indicate sites of modification.


                              
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Table I
Steady-state kinetic parameters of wild type and mutant AAC(6')-Ie

Solvent Viscosity and Solvent Isotope Effects for AAC(6')-Ie-- AAC(6')-Ie has previously been shown to follow a random order Bi Bi kinetic mechanism (23). To further define the kinetic mechanism, we performed solvent viscosity effect (SVE) experiments with glycerol as the microviscosogen to identify the rate-determining step(s) in the catalytic cycle. A significant SVE indicates that one or more rate-determining steps are diffusion-controlled, with either substrate coming to the enzyme, product leaving the enzyme, or a diffusion-controlled conformational change, whereas the lack of a significant SVE effect indicates a diffusion-independent rate-determining step, such as a viscosity-independent conformational change or chemistry at the active site.

There was a minor solvent viscosity effect for both kcat and kcat/Km with acetyl-CoA as the variable substrate, but there was not a significant SVE for either kcat or kcat/Km with kanamycin A as the variable substrate (Table II). The lack of a SVE is consistent with the chemical step(s) being rate-limiting.


                              
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Table II
Solvent viscosity and solvent isotope effects for AAC(6')-Ie

The importance of proton extraction in the chemical step(s) was investigated by determining solvent isotope effects (SIEs) (Hkcat/Dkcat). The only significant SIE determined for AAC(6')-Ie was for the second order rate constant (kcat/Km) with respect to kanamycin A (Table II). As solvent isotope effects may be related to one or multiple exchangeable hydrogens, we further defined the solvent isotope effect for kanamycin A by performing a proton inventory study, where the steady-state kinetic parameters are determined in varying ratios of H2O to D2O (Fig. 2). The linearity of the curve is consistent with the SIE being due to mobilization of only one hydrogen (39), likely a hydrogen extracted from the 6'-NH<UP><SUB>3</SUB><SUP>+</SUP></UP> position of kanamycin A. Thus, these results are consistent with deprotonation of kanamycin A being important in productive aminoglycoside capture.


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Fig. 2.   Proton inventory study of AAC(6')-Ie action using kanamycin A as the variable substrate. The steady-state parameters for kanamycin A were determined in varying ratios of D2O:H2O in the presence of 300 µM acetyl-CoA.

pH Effects for AAC(6')-Ie-- The effects of pH on steady-state kinetic parameters can give insight into the required ionization state of important enzyme active site residues. The pH effects for AAC(6')-Ie showed two important ionizations whether acetyl-CoA or neamine was the variable substrate (Fig. 3, A and B, and Table III). These pKa values may be related to the required ionization states of enzymatically important residues. Thus, for example, the acidic limb of the pH curve (pKa = 7.2-7.3) could be associated with a residue that needs to be deprotonated for maximum enzyme activity. The putative active site base required to deprotonate the antibiotic would normally need to be deprotonated to accept a hydrogen from the aminoglycoside substrate. Thus, the pH profiles are consistent with a chemical mechanism employing an active site base and an active site acid, and we can tentatively assign the first pKa to the active site base.


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Fig. 3.   pH dependence of AAC(6')-Ie WT with variable substrate acetyl-CoA (A) or neamine (B) and AAC(6')-Ie Asp-99 right-arrow Ala activity with the variable substrate acetyl-CoA (C). The other substrate was held at 300 µM. Steady-state kinetic parameters were determined at various pH levels with the following buffers: 25 mM MES-NaOH (pH 6.0), 25 mM MOPS-NaOH (pH 6.5), 25 mM HEPES-NaOH (pH 7.0-8.0), and 25 mM glycylglycine (pH 8.5-9.5).


                              
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Table III
Summary of the dependence of AAC(6')-Ie WT and Asp-99 right-arrow Ala steady-state kinetics on pH

Mutational Analyses of AAC(6')-Ie Tyr-96 and Asp-99-- These results suggest that AAC(6')-Ie employs an active site base that acts to deprotonate the aminoglycoside substrate, thereby generating a more potent nucleophile to facilitate acetyl transfer from acetyl-CoA. In members of the GNAT superfamily, there are two amino acid positions that have been suggested to act as the active site base. In the histone acetyltransferase tGCN5, Glu-173 has been suggested to fulfill this role (30, 31), whereas in AANAT, the active site base appears to be His-122 (29). The three-dimensional structure of AAC(6')-APH(2") has not been determined, so we used available sequence alignments (16, 40) to generate a partial alignment of GNAT family members that could aid in identifying the putative active site base in AAC(6')-Ie (Fig. 4).


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Fig. 4.   Alignment of beta -strand 4 segments of motif A from selected GNAT family members. Shaded text indicates conserved hydrophobic regions, and asterisks indicate positions of residues suggested to play the role of an active site base in different family members. Glu-173 has been suggested to be the active site base in tGCN5 (30, 31), and His-122 has been suggested to be the active site base in AANAT (29). The alignment is based on previous structure-based alignments (16, 40).

The residues in AAC(6')-Ie that align with known or predicted active site bases in GNAT family members are Tyr-96 and Asp-99 (Fig. 4). To test the possibility that these residues could act as the active site base, we generated the following mutant proteins: AAC(6')-Ie Tyr-96 right-arrow Phe, AAC(6')-Ie Asp-99 right-arrow Ala, AAC(6')-Ie Asp-99 right-arrow Asn, and AAC(6')-Ie Asp-99 right-arrow Glu. The mutation of Asp-99 to Ala is a more drastic change, whereas mutation to Asn conserves R group length but not the proton extracting carboxylate moiety and mutation to Glu conserves the carboxylate moiety but extends R group length by one methylene unit.

There were only minor effects on the steady state kinetic parameters when Tyr-96 was mutated to Phe (Table I). The largest effects were seen for paromomycin (e.g. change in kcat/Km compared with WT was 5.5-fold). These results are not consistent with Tyr-96 acting as an active site base, although it may have a minor effect on substrate binding with some aminoglycosides.

The changes in steady-state parameters for AAC(6')-Ie Asp-99 right-arrow Ala and AAC(6')-Ie Asp-99 right-arrow Asn were similar (Table I). For kanamycin A, there were significant decreases in both kcat (45- to 52-fold) and kcat/Km (131- to 215-fold) parameters. The changes for neamine were smaller but still significant for both kcat (15- to 16-fold) and kcat/Km (85- to 101-fold). In contrast, there were more drastic changes for kcat with fortimicin A (350- to 1400-fold) and paromomycin (>2500).

The decreases in activity determined with AAC(6')-Ie Asp-99 right-arrow Glu were generally less than for either Ala or Asn mutations (Table I). This result suggests that the carboxylate moiety of Asp-99 is most critical for WT enzyme activity. This would be expected if Asp-99 acts as the active site base, because it would require the ability to ionize and accept a hydrogen from the aminoglycoside substrate. A Glu at this position would have a similar capacity, although it would not likely be optimally positioned for such a task, whereas Asn or Ala could not fulfill this role. Moreover, the fact that AAC(6')-Ie Asp-99 right-arrow Ala and AAC(6')-Ie Asp-99 right-arrow Asn become increasingly impaired in their ability to catalyze acetyl transfer with paromomycin is consistent with Asp-99 acting as the active site base, because it will become exceedingly important to have an active site base to deprotonate the aminoglycoside when the pKa of the acceptor group increases and a greater proportion of the substrate will be in a noncompetent form for efficient acetyl group transfer at physiological pH.

To correlate in vitro effects to biological impact, we performed minimum inhibitory concentration (MIC) determinations for E. coli expressing AAC(6')-APH(2") WT or Asp-99 mutant enzymes (Table IV). Because APH(2")-Ia cannot phosphorylate fortimicin A, protection of bacteria from this particular antibiotic is solely the responsibility of the AAC(6')-Ie activity in the bifunctional enzyme. The MIC determinations were consistent with the kinetic data, where E. coli carrying pET15AACAPH Asp-99 right-arrow Ala and pET15AACAPH Asp-99 right-arrow Asn provided no protection against fortimicin A (Table IV). This finding further underscores the importance of Asp-99 in the acetylation and detoxification of fortimicin A. 


                              
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Table IV
MIC determinations with fortimicin A for E. coli BL21(DE3) expressing HisAAC(6')-APH(2") wild type and Asp-99 mutants

pH Effects for AAC(6')-Ie Asp-99 right-arrow Ala-- We have previously assigned the first ionization in the pH profile of AAC(6')-Ie to the putative active site base. To further test this hypothesis and more fully develop the role of Asp-99 in the catalytic mechanism of AAC(6')-Ie, we determined the effects of pH on kcat and kcat/Km for AAC(6')-Ie Asp-99 right-arrow Ala (Fig. 3C and Table III). The results do not show the double ionization curves as seen in AAC(6')-Ie WT. The pH curves for kcat with either neamine or acetyl-CoA as the variable substrate and for kcat/Km with acetyl-CoA as the variable substrate show only a single ionization associated with the second pKa in AAC(6')-Ie (Table III). Thus, these results are consistent with Asp-99 serving as the active site base, and we can assign the first pKa in the pH profile of AAC(6')-Ie WT to Asp-99.

When neamine is the variable substrate, there is not a distinct ionization (Table III). This is likely related to the form of neamine that preferentially binds to AAC(6')-Ie WT compared with AAC(6')-Ie Asp-99 right-arrow Ala. With AAC(6')-Ie WT, neamine is bound as the fully protonated form, however, as AAC(6')-Ie Asp-99 right-arrow Ala lacks an active site base to deprotonate the incoming aminoglycoside, neamine likely binds in the unprotonated form in order to be chemically active. This effect complicates analysis and essentially conceals the basic limb of the pH profile.

Inhibition of AAC(6')-Ie by (Halomethyl)naphthalene and (Halomethyl)phenanthrene Derivatives-- A screen of a library of small planar molecules against AAC(6')-Ie identified 1-(bromomethyl)phenanthrene and 9-(chloromethyl)phenanthrene as inhibitors of AAC(6')-Ie, where 1-(bromomethyl)phenanthrene gave much more significant inhibition than 9-(chloromethyl)phenanthrene (Fig. 5). This difference may be related to either the position or nature of the halogen group. We also assayed 1-(bromomethyl)naphthalene and 1-(chloromethyl)naphthalene to assess the importance of the halo-group and the additional benzene ring to inhibition by 1-(bromomethyl)phenanthrene. The bromomethyl- derivative demonstrated more significant inhibition of AAC(6')-Ie activity than did the chloromethyl- derivative, however, 1-(bromomethyl)naphthalene was a much poorer inhibitor than was 1-(bromomethyl)phenanthrene (Fig. 5), highlighting the importance of both the bromo- group and the additional benzene ring in inhibition.


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Fig. 5.   Inhibition of AAC(6')-Ie by 1-(bromomethyl)naphthalene, 1-(chloromethyl)naphthalene, 1-(bromomethyl)phenanthrene, and 9-(chloromethyl)phenanthrene (left to right). Enzyme (0.2 µM) was incubated with 25 µM compound at 37 °C for 20 min before assaying for acetyltransferase activity with the addition of acetyl-CoA and kanamycin A to final concentrations of 300 µM.

1-(Bromomethyl)phenanthrene Is a Potent Irreversible Inhibitor of AAC(6')-Ie-- The increased potency of the bromo- derivatives compared with the equivalent chloro- compounds suggested that these molecules can act as covalent modifiers of AAC(6')-Ie. One hallmark of an irreversible inhibitor is time-dependent inhibition of enzyme activity, where there is typically an initial enzyme·inhibitor complex described by the dissociation constant KI before covalent modification of an active site residue with a first order rate constant kmax, according to the following reaction,


E+I <LIM><OP><ARROW>⇌</ARROW></OP><UL>K<SUB><UP>I</UP></SUB></UL></LIM> EI <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB><UP>max</UP></SUB></UL></LIM> EI*

<UP>R<SC>eaction</SC> 1</UP>
Both 1-(bromomethyl)phenanthrene (Fig. 6A) and 1-(bromomethyl)naphthalene (not shown) showed time-dependent inhibition of AAC(6')-Ie. Unfortunately, due to the reactivities and relative insolubilities of these compounds, a complete kinetic profile of the inactivation reactions could not be achieved, and so, KI and kmax values could not be determined. However, a linear plot of the first order rate constants versus time yields a slope that estimates kmax/KI. For 1-(bromomethyl)phenanthrene (Fig. 6B) and 1-(bromomethyl)naphthalene (not shown), this gives approximate kmax/KI values of 11.7 ± 1.0 M-1s-1 and 1.82 ± 0.17 M-1s-1, respectively. Because the leaving group is identical in both compounds, the difference in reactivities is likely related to the initial binding event. Thus, 1-(bromomethyl)phenanthrene binds to the enzyme 6.4-fold more efficiently than 1-(bromomethyl)naphthalene, indicating the importance of the third benzene ring in ligand recognition.


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Fig. 6.   Time- and concentration-dependent inhibition of AAC(6')-Ie by 1-(bromomethyl)phenanthrene. To determine the kinetics of inactivation for 1-(bromomethyl)phenanthrene (A), enzyme (0.2 µM) was incubated with 0 (open circle ), 2 (), 5 (), 10 (black-square), 15 (triangle ), or 20 (black-triangle) µM compound prior to assaying acetyltransferase activity with the addition of acetyl-CoA and kanamycin A to final concentrations of 300 µM. The first order rate constants determined from A were used to construct the curves in B.

Neither extensive dialysis nor gel filtration could return enzyme activity following inactivation by 1-(bromomethyl)phenanthrene, again consistent with 1-(bromomethyl)phenanthrene acting as a covalent modifier of AAC(6')-Ie. Moreover, matrix-assisted laser desorption ionization time-of-flight mass spectral analysis was consistent with covalent modification of the enzyme by 1-(bromomethyl)phenanthrene (data not shown). These results suggest that 1-(bromomethyl)phenanthrene inactivates AAC(6')-Ie by covalently modifying an important residue in the enzyme.

1-(Bromomethyl)phenanthrene Inactivates AAC(6')-Ie by Covalently Modifying Asp-99-- To gain insight into which residue or residues are modified by 1-(bromomethyl)phenanthrene, we performed substrate/inhibitor protection experiments, where the enzyme was incubated with 1-(bromomethyl)phenanthrene and kanamycin A and/or desulfo-CoA prior to measurement of acetyl transfer activity with the addition of acetyl-CoA. Kanamycin A and desulfo-CoA both weakly protected AAC(6')-Ie from inactivation by 1-(bromomethyl)phenanthrene (Fig. 7A). Moreover, when the enzyme was incubated with both kanamycin A and desulfo-CoA, protection was much more substantial and was additive with respect to the single compound protection experiments (Fig. 7A). This result is consistent with 1-(bromomethyl)phenanthrene having a binding site that overlaps that of both the kanamycin A and desulfo-CoA/acetyl-CoA binding sites.


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Fig. 7.   Characterization 1-(bromomethyl)phenanthrene inactivation of AAC(6')-Ie. A, protection of AAC(6')-Ie from inactivation by 1-(bromomethyl)phenanthrene. Enzyme (0.2 µM) was incubated in the presence of 20 µM 1-(bromomethyl)phenanthrene (1-bmp) in the presence or absence of kanamycin A (500 µM) and desulfo-CoA (100 µM) for 10 min at 37 °C before the addition of acetyl-CoA (500 µM) to measure the acetyltransferase activity. Activities were compared with incubations without 1-(bromomethyl)phenanthrene. B, susceptibility of AAC(6')-Ie and mutants to inactivation by 1-(bromomethyl)phenanthrene. Enzyme (0.2-2 µM) was incubated with 40 µM 1-(bromomethyl)phenanthrene for 10 min at 37 °C before the addition of acetyl-CoA and kanamycin A to final concentrations of 300 µM to assay for acetyltransferase activity.

Taking into account the results of the protection experiments, only a few select residues in AAC(6')-Ie could be potential candidates to serve as the active site nucleophile in the reaction. Because our attempts to identify the residue through a series of experiments involving large-scale enzyme inactivation, proteolysis of the complex, and mass spectral analysis of the proteolytic fragments failed due to relative insolubility of 1-(bromomethyl)phenanthrene and nonspecific protein losses during work-up, we opted to use an alternative approach. We previously determined that, although 1-(bromomethyl)phenanthrene can inactivate AAC(6')-Ie, it cannot inactivate AAC(6')-Ii.3 Its inability to inactivate AAC(6')-Ii could be because 1-(bromomethyl)phenanthrene can not efficiently bind to AAC(6')-Ii or because AAC(6')-Ii lacks an appropriate active site residue to serve as the nucleophile in the reaction.

Close inspection of the active site of AAC(6')-Ii, together with the alignment between AAC(6')-Ii and AAC(6')-Ie (Fig. 4), suggested that Asp-99 in AAC(6')-Ie could be responsible for the different reactivities of the enzymes. The equivalent residue in AAC(6')-Ii (His-74) would not likely act as a nucleophile in the reaction with 1-(bromomethyl)phenanthrene, and there does not appear to be any other candidate residues in the vicinity. To test this hypothesis, we determined the sensitivities of Tyr-96 and Asp-99 mutant proteins to 1-(bromomethyl)phenanthrene. AAC(6')-Ie Tyr-96 right-arrow Phe was just as sensitive as wild type enzyme to inactivation by 1-(bromomethyl)phenanthrene, indicating that the hydroxyl group of Tyr-96 is not required for inactivation (Fig. 7B). However, AAC(6')-Ie Asp-99 right-arrow Ala and AAC(6')-Ie Asp-99 right-arrow Asn were completely resistant to inactivation by 1-(bromomethyl)phenanthrene (Fig. 7B). This suggested that either Asp-99 is the active site residue that is modified by 1-(bromomethyl)phenanthrene or Asp-99 is important in the initial binding event between compound and enzyme. Consistent with the former hypothesis, AAC(6')-Ie Asp-99 right-arrow Glu is sensitive to inactivation, albeit less sensitive than AAC(6')-Ie WT (Fig. 7B). This result suggests that the carboxylate moiety provided by Asp-99 (or Asp-99 right-arrow Glu) is most critical to the inactivation reaction and implicates Asp-99 as the active site residue that is covalently modified by 1-(bromomethyl)phenanthrene.

To further validate this conclusion, we determined the effect that pH has on the inactivation of AAC(6')-Ie by 1-(bromomethyl)phenanthrene. AAC(6')-Ie was most sensitive to inactivation at lower pH, whereas it became more resistant as the assay pH increased (Fig. 8). This result yielded a pKa for the inactivation reaction of 7.2. We have previously assigned a pKa of 7.2 to Asp-99, thus, together these results are consistent with Asp-99 serving as the active site nucleophile that is covalently modified by 1-(bromomethyl)phenanthrene. Therefore, although the presence of Asp-99 in AAC(6')-Ie leads to a wider resistance profile by enabling AAC(6')-APH(2") to acetylate fortimicin A and paromomycin, it also makes the enzyme sensitive to inactivation by 1-(bromomethyl)phenanthrene.


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Fig. 8.   pH dependence of the inactivation of AAC(6')-Ie WT by 1-(bromomethyl)phenanthrene. Enzyme (0.2 µM) was incubated with 20 µM 1-(bromomethyl)phenanthrene for 10 min at 37 °C before the addition of acetyl-CoA and kanamycin A to final concentrations of 300 µM to assay for acetyltransferase activity. These inactivation reactions were conducted at different pH levels using the following buffers: 25 mM MOPS-NaOH (pH 6.5), 25 mM HEPES-NaOH (pH 7.0-8.0), and 25 mM glycylglycine (pH 8.5).

Molecular Mechanism of AAC(6')-Ie and Comparison to Other GNAT Family Members-- Because there is no available crystal structure for AAC(6')-APH(2"), it is not known if Asp-99 acts directly upon aminoglycoside substrate or if base catalysis is mediated through a chain of water molecules similar to that proposed for both tGCN5 histone acetyltransferase and AANAT (17). However, recently the structure of AAC(2')-Ic from Mycobacterium tuberculosis, an aminoglycoside acetyltransferase also known to catalyze O-acetylation, has been solved (15). As with other GNAT superfamily members, there is not a direct interaction with the substrate 2'-NH2 and a potential active site base, but rather, a series of water molecules that connects the amine to Glu-82, the residue that aligns with Asp-99 in AAC(6')-Ie. This suggests that with a sufficiently potent active site base, O-acetylation may be catalyzed through a "proton-wire" of appropriately positioned water molecules.

These studies identify Asp-99 as an active site base required for O-acetylation of aminoglycoside substrates and N-acetylation of fortimicin A and provides the molecular basis for these unique properties in an AAC(6'). It also identifies this residue as sensitive to modification by small molecule inhibitors. This provides a unique anchoring site for the development of specific inhibitors of AAC(6')-Ie that could find use as leads in the development of anti-resistance molecules.

    FOOTNOTES

* This work was supported by the Canadian Institutes of Health Research Grant MT-13536 and by a Canada Research Chair in Antibiotic Biochemistry (to G. D. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 905-525-9140 (ext. 22454); Fax: 905-525-9033; E-mail: wrightge@mcmaster.ca.

Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M211680200

2 D. D. Boehr and G. D. Wright, unpublished.

3 K.-a. Draker and G. D. Wright, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: AME, aminoglycoside modifying enzyme; AAC, aminoglycoside acetyltransferase; APH, aminoglycoside phosphotransferase; AANAT, arylalkylamine N-acetyltransferase; SVE, solvent viscosity effect; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; MIC, minimum inhibitory concentration; SIE, solvent isotope effect; WT, wild type; GNAT, superfamily of GCN-5 related N-acetyltransferases.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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