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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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),
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(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,
|
(Eq. 2)
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For single ionization analysis with AAC(6')-Ie Asp-99
Ala, the data can be fit using nonlinear regression analysis of
the following equation,
|
(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
Phe,
GAGATAGTCTTTGGTATGGATCAATTTATAGGAGAGCC; Asp-99
Ala,
GAGATAGTCTATGGTATGGCTCAATTTATAGGAGAGCC; Asp-99
Asn,
GAGATAGTCTATGGTATGGAACAATTTATAGGAGAGCC; and Asp-99
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):
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):
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 |
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
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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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.
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
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.
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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 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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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 -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).
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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
Phe, AAC(6')-Ie Asp-99
Ala, AAC(6')-Ie Asp-99
Asn, and
AAC(6')-Ie Asp-99
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
Ala
and AAC(6')-Ie Asp-99
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
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
Ala
and AAC(6')-Ie Asp-99
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
Ala and pET15AACAPH Asp-99
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
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pH Effects for AAC(6')-Ie Asp-99
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
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
Ala. With AAC(6')-Ie WT, neamine is bound as the fully
protonated form, however, as AAC(6')-Ie Asp-99
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.
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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,
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 ( ), 2 ( ), 5 ( ), 10 ( ), 15 ( ), or 20 ( )
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.
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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.
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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
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
Ala and AAC(6')-Ie Asp-99
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
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
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).
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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.