Azucena and Mobashery
Aminoglycoside-modifying
enzymes: mechanisms of
catalytic processes and
inhibition
Eduardo Azucena, Shahriar Mobashery
Institute for Drug Design, Departments of Chemistry, Pharmacology and
Biochemistry and Molecular Biology,Wayne State University, Detroit, Michigan,
USA
Abstract The most prevalent mechanism for resistance to
aminoglycoside antibiotics is mediated through their enzymatic
modification in resistant organisms. Dozens of aminoglycoside-
modifying enzymes are known at the gene sequence level, but
few have been characterized in the details of their mechanism.
This review summarizes the state of knowledge of the best
studied of these enzymes, focusing on their catalytic
mechanisms and inhibition. © 2001 Harcourt Publishers Ltd
INTRODUCTION
A
minoglycosides constitute a large family of water-
soluble, polycationic amino sugars of considerable
structural diversity (Fig. 1).The structural aspects of
these antibiotics has been reviewed recently and will not be
discussed here extensively.1,2 They are often broad-spectrum
antibacterial agents that are products of bacterial or fungal
metabolism. The structures of several of these natural prod-
ucts have been altered by chemists to generate semi-
synthetic aminoglycosides with expanded activities, which
usually show aversion to modification by known important
resistance enzymes.
Shortly after the isolation of the first aminoglycoside,
streptomycin, from Streptomyces griseus in the 1940s,3 the
clinical importance of these mostly bactericidal agents was
realized in the treatment of tuberculosis.4,5 Over the decades
following this discovery, a series of aminoglycosides were
identified and characterized for clinical use in the treatment
of bacterial infections caused by gram-positive and gram-
negative organisms. Some aminoglycosides such as paro-
momycin have also been used in treatment of amoeboid or
protozoan infections. In recent years, enthusiasm for the
therapeutic use of aminoglycosides for purposes has been
waning because of toxicity issues, and there has been a trend
towards the use of less toxic alternative drugs such as
β-lactams. Nevertheless, aminoglycosides remain important
weapons in the modern antimicrobial armamentarium, as a
vast majority of gram-negative aerobes, including some mul-
tiresistant strains, remain vulnerable to some of these agents.
Bacterial resistance to aminoglycosides manifests itself
by one of the following mechanisms: (a) the presence of
aminoglycoside-modifying enzymes that inactivate them;
(b) decreased cell membrane permeability towards amino-
glycoside uptake into the cell; (c) structural alteration in
106 Drug Resistance Updates (2001) 4, 106–117  2001 Harcourt Publishers Ltd
doi: 10.1054/drup.2001.0197, available online at http://www.idealibrary.com on
the ribosomal target of the drug; or (d) extrusion of the
aminoglycosides from the cell by efflux pumps. Of these
resistance mechanisms, inactivation by aminoglycoside-
modifying enzymes is the most important both in terms of
level and frequency of resistance conferred to the bac-
terium.
There are three types of aminoglycoside-modifying en-
zymes,each of which transfers a functional group to the amino-
glycoside structure thereby inactivating the antibiotic: (a)
aminoglycoside nucleotidyltransferases (ANTs) transfer a
nucleotide triphophates; (b) aminoglycoside acetyltransferases
(AACs) transfer the acetyl group from acetyl-CoA; and (c)
aminoglycoside phosphotransferases (APHs) transfer the phos-
phoryl group from ATP.6,7 Each family of enzymes consists of
different isozymic forms that differ in substrate regiospecificity
for their reactions. At least 50 different genes for aminoglyco-
side-modifying enzymes are known in bacteria.7 A number of
bacteria also harbor a unique bifunctional resistance enzyme
that catalyzes both the AAC and APH reactions.6,8,9
Bacterial action of aminoglycosides in susceptible strains
involves an energy-requiring membrane transport into the
cell10–12 as well as interaction of the drug with the ribo-
some.13–15 The rate-limiting step in the drug action is the slow
process of initial transport of the aminoglycoside across the
cytoplasmic membrane.11 Once inside the cell, aminoglyco-
side binds with the 16S rRNA of the 30S ribosomal frag-
ment.16–18 This aminoglycoside-ribosome interaction causes
misreading of mRNA, leading to biosynthesis of non-
functional, misfolded proteins.14,15 Some of the resulting non-
active proteins then interact with the cell membrane and
cause the creation of aberrant membrane structure and a
subsequent enhancement of cellular penetration by the
aminoglycoside. This event augments the drug’s pleiotropic
effects, which in turn collectively contribute to bacterial
death.14,15 In resistant strains that possess aminoglycoside-
inactivating enzymes, a different scenario occurs: drugs that
manage to enter the cell are modified by the resistance
enzyme, the modified drug is unable to interact with the
ribosome effectively, and the enhanced rate of aminoglyco-
side accumulation observed in susceptible strains does not
occur.19,20 Because drug uptake remains suppressed at low
rates, the cell survives and remains viable for as long as the
rate of drug modification matches or surpasses the rate of
drug uptake.19 This requirement is often met readily: resis-
tance enzymes are often expressed in high concentrations
and some of these enzymes have been shown to operate at
the diffusion limit.6
Structure-activity relationship studies on aminoglycosides
have established that their antibiotic activity depends little
on their hydroxyl groups. On the other hand, the number
and positions of amino groups play a significant role in
antibiotic activity in the order 2′, 6′-diamino > 6′-amino > 2′
amino > absence of amine.21 Thus, according to this study, an
aminoglycoside with unsubstituted amino groups at posi-
tions 2′ and 6′ is generally found to possess greater antibac-
terial activity than one with an unsubstituted amino group at
position 2′. Since the discovery of the antibiotic-inactivating
enzymes, there have been efforts to chemically modify the
existing antibiotics to produce new drugs that are immune
to inactivation, but at the same time retain their antibacterial
 Aminoglycoside-modifying enzymes

R4O 6" R3 R4
4" R2 4" CH2OH
5" O 4' "O'
HO 2' 3' HO
H 3N 3" 2"
1" R1O OH HO
1"
OH
6' 3" 2" O
+ HO 1' O NH3 O HO
O 6 4 5' NH3+ IV +
+ 2'
3' 4'
III 5 O H3N OH
R3NH NH3+ O 1' O
1
2
3
I HO 6 4 5' 6'
R1
5
II III HO
R2NH 2 NH3+ I
1 3
aminoglycoside R1 R2 R3 R4 II

kanamycin A OH OH H H aminoglycoside R1 R2 R3 R4
kanamycin B NH3 + OH H H
neomycin NH3+ H H CH2NH3+
amikacin OH OH L-AHBA H
butirosin NH3+ L-AHBA (no ring IV)
tobramycin NH3+ OH R3 CONH2
lividomysin OH H H CH2NH3+
dibekacin NH3+ H H H
paromomycin OH H CH2NH3 + H
r`ibostamycin NH3 + H (no ring IV)

OH
4" 5" 2'
R2
R7 O 3' 4'
1"
R1 6' R3
H3N 2" O
+ 3" HO 1' 5'
OH O 6 5 4
O R5
III R4
R6NH 2 NH3+
1 3

II I

aminoglycoside R1 R2 R3 R4 R5 R6 R7

gentamicin A NH3+ OH OH H OH H H
gentamicin B OH OH OH H NH3+ H CH3
gentamicin B1 OH OH OH CH3 NH3+ H CH3
gentamicin C1 NH3 + H H CH3 NH2CH3 H CH3
gentamicin C1a NH3+ H H H NH3+ H CH3
gentamicin C2 NH3 + H H CH3 NH3 + H CH3
sisomicin NH3 + H 4' H NH3 + H CH3
netilmicin NH3+ H 4' H NH3+ CH2CH3 CH3

AHBA = 4-amino-1-hydroxybutyryl.
Fig. 1 Structure of representative aminoglycosides.

activity. This strategy has led to considerable success in the been forthcoming. First, resistance to aminoglycosides has
fight against penicillin resistance. For instance, the use of emerged gradually and not explosively, as documented for
methicillin, a derivative of the early penicillins, overcame the case of β-lactam antibiotics.22 This is in part due to the
enzyme-mediated inactivation in most penicillin-resistant, fact that β-lactams have been used very extensively clinically,
gram-positive organisms.22 Such strategy has been relatively in contrast to aminoglycosides. On the other hand, the
modest for aminoglycosides, since many of the structural enzymes of resistance to aminoglycosides have not been as
requirements for their antimicrobial activity run parallel to numerous as β-lactamases. They have also been difficult to
those for their enzymatic inactivation.23,24 Nonetheless, semi- study and few laboratories have been willing to undertake
synthetic aminoglycosides have enjoyed clinical success in such efforts. The cornerstone of success in development of
the past several years. Combinations of inhibitors of modify- inhibitors for these enzymes hinges on our ability to under-
ing enzymes and enzyme-susceptible aminoglycosides have stand the basic properties of these resistance enzymes.
not entered clinical use. This contrasts to β-lactam antibi- This report presents a discussion of the best studied of
otics, where clinical use of combinations of β-lactamase these enzymes and presents the results for inhibitors that
inhibitors and penicillins trace back to the mid-1980s.22 have been reported for members of each class of amino-
There are a number of reasons that such a strategies have not glycoside-modifying enzymes. Most of these inhibitors were

 2001 Harcourt Publishers Ltd Drug Resistance Updates (2001) 4, 106–117 107
 Azucena and Mobashery
designed based on the information obtained from kinetics
and mechanisms of the particular groups of enzymes. Hence,
only enzymes on which mechanistic studies were per-
formed, and against which inhibitors have been designed, are
discussed here.
AMINOGLYCOSIDE NUCLEOTIDYL TRANSFERASES
Mechanism
Most of the mechanistic studies on aminoglycoside
nucleotidyltransferases have been carried out on two
enzymes: ANT(4′) and ANT(2′′)-I. The crystal structure of
ANT(4′) isolated from Staphylococcus aureus has been elu-
cidated.25,26 Several members of the kanamycin and
neomycin family of aminoglycosides, as well as five
nucleotide triphosphates (ATP, GTP, CTP, TTP, and UTP) are
substrates for this enzyme.27 Kanamycins and neomycins are
similar in structure for rings I and II, but differ in ring III.
While kanamycins have a six-membered ring III, neomycins
have a five-membered ring III (some members have a six-
membered ring IV) (Fig. 1).The X-ray crystal structure of the
enzyme reveals that several active-site residues interact via
hydrogen bonds with rings I and II of aminoglycosides, but
relatively fewer residues interact with the rest of the antibi-
otic structure (Fig. 2).The lack of interaction between active-
site residues and ring III (and IV) accounts for both the
broad substrate specificity of the enzyme and the compara-
ble rates of turnover for kanamycins and neomycins.
Similarly, the small differences in V and V/Km values among
the five nucleotide substrates are due to the relatively few
interactions between active-site residues and nucleotide
base. A catalytic mechanism proposed by Chen-Goodspeed
et al. for ANT(4′) based on the crystal structure and on pH
dependence of the kinetic parameters postulates that Glu-
145 (Fig. 2) acts as a general base for the activation of the 4′-
hydroxyl of the aminoglycoside, and in so doing, prepares
the antibiotic for its subsequent nucleophilic attack at the
α-phosphoryl group of the nucleotide triphosphate.27
Moreover, the close proximity between the positively
charged Lys-149 and the nucleotide α-phosphoryl group
Glu-141
Ser-94
O
O H
O−
NH3+ NH3+
II
O Ser-39
O O
HO O NH3+
III OH
I O
HO OH OH
HO
NH3+ OH
−O
O O
O−
NH3+ −O
O O
Glu-67
Lys-74
Glu-76
Fig. 2 Representation of the interactions between active site residues of ANT(4′) with the substrates,ATP and kanamycin A.27
108 Drug Resistance Updates (2001) 4, 106–117  2001 Harcourt Publishers Ltd
could enhance the electrophilicity of the latter and thus
would facilitate the nucleophilic attack by the antibiotic.
These authors also proposed the possibility of rotation for
ring II of kanamycins such that the 4′′-hydroxyl of ring III
could also be adenylated by ATP.This, however, is not possi-
ble for the neomycins, as they lack the internal symmetry in
their structure that exist within kanamycins.
The order of binding of the substrates to ANT(4′)
was determined by kinetic studies using spectinomycin, a
kanamycin A analog.28 Inhibition by spectinomycin was found
to be competitive versus kanamycin and noncompetitive
versus ATP, indicating that spectinomycin binds to the
free enzyme and not to the E.ATP complex. On the other
hand, the nonhydrolyzable ATP analogs AMP and AMPCP
(β,γ-methyleneadenosine 5′-triphosphate) are competitive
against ATP but uncompetitive against kanamycin A.Therefore,
these inhibitors bind not to the free enzyme but rather to the
E. kanamycin A complex. Substrate binding to the active
site is an ordered process, with antibiotic binding taking
place prior to nucleotide binding. Determination of the
order of product release, which adds further support for
the proposed order of substrate binding, was provided by
product-inhibition experiments using the turnover product
AMP-kanamycin A. AMP-kanamycin was found to be a com-
petitive inhibitor of kanamycin A and noncompetitive
inhibitor of ATP.This finding implicates the nucleotidylated-
aminoglycoside (not pyrophosphate) as the last product
released from the enzyme, since it supposes that the amino-
glycoside portion of the product inhibitor binds only to the
free enzyme.The kinetic mechanism that is most consistent
with these inhibition data is an ordered Bi-Bi mechanism. In
this mechanism, aminoglycoside binds first, followed by the
nucleotide.After bond-making and bond-breaking, pyrophos-
phate leaves the enzyme first, before the nucleotidylated
aminoglycoside does (Fig. 3A). Substrate inhibition is ob-
served with all aminoglycoside substrates tested for ANT(4′).
This observation predicates that aminoglycoside, the sub-
strate that binds first to the enzyme, may bind to the antibi-
otic-binding site of the E-AMP-aminoglycoside (E.Q) before
the product is completely released, thereby forming a
Thr-187 Lys-149
O
H H
Glu-145 NH2
+H3N
O H N N
O O O
−O P O P O P O N N
Glu-145
−O O− O
Mg2+
HO OH

Fig. 3 (A) Scheme for the ordered Bi-Bi kinetic mechanism of
ANT(4′).The horizontal line represents the reaction
proceeding from left to right. E = enzyme;
A = aminoglycoside; B = nucleotide triphosphate;
P = pyrophosphate; Q = nucleotidylated aminoglycoside.
(B) Scheme for the Theorell-Chance kinetic mechanism of
ANT(2′′)-I.The horizontal line represents the reaction
proceeding from left to right. E = enzyme;A = nucleotide
triphosphate; B = aminoglycoside; P = pyrophosphate;
Q = nucleotidylated aminoglycoside.
dead-end complex, E.Q.A, that prevents the full release of the
product. In this case, the aminoglycoside portion of Q is
released first from its binding site, while the AMP portion
remains enzyme-bound.
Another well-studied nucleotidyltransferase is ANT(2′′)-I.
This enzyme can turn over several aminoglycosides from
both the gentamicin and the kanamycin families of antibi-
otics (Fig. 1).29 The significant differences in the kcat values
for turnover of gentamicins and kanamycins might be attrib-
utable to structural differences in ring III, the site of
nucleotidylation.Although a variety of aminoglycosides with
considerable diversity of structures can be modified by
ANT(2′′)-I, these aminoglycosides have an equatorial 5-
hydroxyl group in common—presumably a requisite for
optimal binding to the enzyme.Apart from this, there seems
to be no other requirement for a hydroxyl group at any other
site except for the site of nucleotidylation. It was suggested
that the relative positions of rings I and III may switch in
binding to the active site, reminiscent of the situation for
ANT(4′).29 Thus, the site occupied by the 6′-amino group of
kanamycins can be occupied by the 3′′-amino group of the
gentamicins.
Inhibition kinetics of ANT(2′′)-I supports a Theorell-
Chance kinetic mechanism,30 with nucleotide binding to the
enzyme occurring prior to aminoglycoside binding, and the
release of pyrophosphate preceding that of the nucleotidy-
lated aminoglycoside (Fig. 3B). Release of the adenylated
product is the rate-limiting step. Substrate inhibition is
observed with nearly all aminoglycosides at concentrations
only slightly above their Km values. Substrate inhibition
by aminoglycosides of ATP binding was shown to be
partial uncompetitive and it arises as a result of binding of
aminoglycoside (B) to the E.Q complex (enzyme.
Aminoglycoside-modifying enzymes
NMP-aminoglycoside) in a dead-end fashion (Fig. 3B).
Analogous to the mechanism of ANT(4′), binding of B to E.Q
involves the release of the aminoglycoside portion of NMP-
aminoglycoside, while the nucleotide portion remains bound
to the enzyme.The release of the aminoglycoside moiety of
the product is believed to be driven by the exothermic
hydrolysis of a phosphate anhydride bond of NTP, together
with the stereochemical inversion of the α-phosphate, which
is consistent with a mechanism of direct attack by the amino-
glycoside.31 This initial step also exposes the specific aminogly-
coside binding site in the E.Q complex and allows a second
aminoglycoside molecule to bind to the enzyme, a process
that reaches rapid equilibrium, as confirmed by pH- and vis-
cosity-effects data.32 A step in which only a portion of the
product is detached from the enzyme occurs. This event is
subsequent to a slow conformational change, which delays
the release of the product from the enzyme, thereby allow-
ing the E.Q.B to accumulate and the inhibition to occur at
aminoglycoside levels only slightly above the Km values.
Secondary binding of a substrate inhibitor to the E.Q
complex can be tested by the addition of Q to reaction mix-
tures to increase the steady-state levels of E.Q, and thus
enhance substrate inhibition.33,34 However, increased Q con-
centration does not increase, but rather decreases, substrate
inhibition for the ANT(2′′)-I enzyme.This is due to the unique
feature of the Theorell-Chance mechanism, wherein virtually
all of the enzyme already exists as E.Q during steady-state
turnover. Therefore, the observed decrease in substrate in-
hibition with increase in concentration of Q indicates the
formation of an E.Q.Q complex. Product inhibition by AMP-
tobramycin (product Q, Fig. 3) was found to be noncompeti-
tive against ATP.30 Normally, for enzymes that follow the
Theorell-Chance mechanism, product inhibition should be
competitive with ATP since Q and ATP both compete for free
enzyme.Therefore, for the Theorell-Chance mechanism, it is
necessary to presuppose that the tobramycin portion of Q
binds to the exposed aminoglycoside-binding site of the E.Q
and forms E.Q.Q rather than to the free enzyme. The
nucleotidylated tobramycin resembles what may be termed a
bisubstrate analog inhibitor that normally would have a dis-
sociation constant much less than those of either tobramycin
or ATP. However,the dissociation constant for AMP-tobramycin
was instead found to be an order of magnitude greater than
that for tobramycin. An explanation for this comes from pH-
and viscosity-dependence kinetic studies32 that suggest that
the aminoglycoside portion of AMP-aminoglycoside is released
prior to the release of pyrophosphate and that pyrophosphate
blocks binding of AMP-aminoglycoside to the enzyme.
The ‘natural’ aminoglycoside substrates of ANT(2′′)-I are
turned over to products with such high-affinity enzyme bind-
ing properties that they practically behave as suicide
inhibitors.30 Hence, the mechanism by which these enzymes
confer resistance to bacteria during the initial encounter with
the aminoglycoside is an open question. One potential expla-
nation to this paradox might be that antibiotic resistance is
the result of the fast rate of capture of aminoglycosides by
the enzyme upon their entry into the cell, rather than their
subsequent rate of inactivation by catalytic turnover.30
For this strategy to be practical, either the uptake of the
antibiotic into bacteria must be relatively slow, or the levels of
 2001 Harcourt Publishers Ltd Drug Resistance Updates (2001) 4, 106–117 109
 Azucena and Mobashery
expression of the enzymes must be high. Unfortunately, the
literature does not shed light on these issues. O
OR1
R4 7 2
1
Inhibition 3

As discussed above for the mechanism of ANT(2′′), there 6 R2

5
appears to be a requirement for an equatorial hydroxyl group at 4

the 5 position of the 2-deoxystreptamine ring for effective bind- R3

ing of the aminoglycoside to this enzyme. Since the 5-hydroxyl
group is not critical for the antibiotic activity, this position is a
logical candidate for modification as a strategy for development aminoglycoside R1 R2 R3 R4
of effective antimicrobial aminoglycoside.21,35 Thus, semisyn- tropolone H H H H
thetic drugs such as 5-epi-sisomicin36,37,38 and 5-epi-gentamicin39 7-hydroxytropolone (7-HT) H H H OH
(Fig. 4), both of which have the 5-hydroxyl group at the axial 3,5,7-Tri(hydroxymethyl)- H CH2OH CH2OH CH2OH
position, are found to be effective against a broad spectrum of tropolone
7-hydroxytropolone COMe H H COMe
bacteria that harbor resistance enzymes, including ANT(2′′), diacetate
AAC(3),AAC(2′).ANT(2′′) was shown to have about 9- and 55-
fold higher Km for 5-epi-sisomicin and 5-epi-gentamicin B, Fig. 5 Structures of tropolone and tropolone derivatives.
respectively, compared to the corresponding parent com-
pounds, sisomicin and gentamicin.39
The results of these in vitro inhibition studies were found
7-Hydroxytropolone (7-HT, Fig. 5), a fermentation product
to parallel those of in vivo experiments, demonstrating
of certain Pseudomonas and Streptomyces species, and some
potentiation of tobramycin activity by 7-HT against
of its derivatives were found to be potent inhibitors specific for
tobramycin-resistant strains. For example, growth of
ANT(2′′) and ANT(4′).40,41,42 Kinetic studies with 7-HT showed
Escherichia coli strains that harbor ANT(2′′) was not affected
that in combination with aminoglycosides tobramycin, gen-
by tobramycin, gentamicin, amikacin, or dibekacin alone.
tamicin, kanamycin A or dibekacin, this compound can inhibit
However, each of these antibiotics was shown to inhibit
ANT(2′′).40 Certain derivatives of 7-HT, such as 7-hydrox-
growth of the same E. coli strains when combined with 7-HT.
ymethyltropolone, 3,5,7-tri(hydroxymethyl)-tropolone, and 7-
The synergistic effect between 7-HT and various aminogly-
HT diacetate can cause complete inhibition of adenylation of
cosides was not observed in E. coli strains that harbor APHs
certain aminoglycosides at inhibitor concentrations of 50
or AACs, which is an indication of the specificity of the inhi-
µg/mL.42 Replacement of the hydroxyl group at the 7-position
bition towards aminoglycoside nucleotidylation. It is interest-
with bromo, nitro, carboxyl, or amino group gave inactive
ing to note, however, that 7-HT did not inhibit the APH
derivatives,40 emphasizing the importance of the 7-hydroxyl
enzymes, despite the requirement by these enzymes for
group for the inhibitory activity. Inhibition due to 7-HT is com-
ATP.40
petitive with respect to ATP (Ki = 10 µM) and not competitive
The effects of a combination of tobramycin plus 7-HT
with respect to tobramycin. Whether inhibition against
and of tobramycin alone on [3H]dihydrostreptomycin
tobramycin was uncompetitive or noncompetitive has not
([3H]DHS) uptake was examined in an E. coli strain that har-
been clearly determined.40
bors tobramycin-adenylating and streptomycin-phosphory-
lating enzymes and in a susceptible E. coli strain.40 In the
susceptible strain, accumulation of [3H]DHS occurs after a
short lag period in the absence of tobramycin. However, the
uptake of [3H]DHS occurred at an increased rate and with-
OH
out a lag when susceptible cells were pre-exposed to
+
H3C O H 3N tobramycin, indicating that preincubation with tobramycin
H3CHN O
facilitates the further entry of aminoglycosides. When the
OH 5 susceptible cells were preincubated with 7-HT or 7-HT plus
O O
tobramycin prior to incubation with [3H]DHS, the cellular
H 3N NH3+ NH3+
+ OH uptake of [3H]DHS was unaffected. In contrast, uptake of
[3H]DHS was inadequate in the ANT/APH-bearing strain
5-epi-sisomicin
without pre-exposure to tobramycin and was only slightly
OH enhanced with pre-exposure to tobramycin. Preincubation
HO
with a combination of tobramycin and 7-HT, however,
O OH resulted in a dramatic increase in [3H]DHS uptake. These
H3 C HO
H3CHN O results may be due to the observed reduced antibiotic
NH3+
OH 5 uptake that accompanies aminoglycoside modification in
O O
H3N NH3+ resistant strains.10,19,20 The effectiveness of the combination
+ OH of 7-HT and aminoglycosides in inhibiting bacterial cell
5-epi-gentamicin B growth is therefore credited to the aminoglycoside-ribo-
some interaction, which results in increased aminoglyco-
Fig. 4 Structures of 5-epi-sisomicin and 5-epi-gentamicin B. side uptake.

110 Drug Resistance Updates (2001) 4, 106–117  2001 Harcourt Publishers Ltd

AMINOGLYCOSIDE ACETYLTRANSFERASES
Mechanism
The first aminoglycoside-modifying enzyme ever reported in
bacteria (second only to penicillinase as an antibiotic-
modifying enzyme) was aminoglycoside 6′-N-acetyltrans-
ferase type-IV [AAC(6′)-IV, also known as AAC(6′)-Ib].43
Radika and Northrop established the kinetic mechanism for
this enzyme as rapid equilibrium sequential random Bi-Bi
(Fig. 6A)44,45 using a kinetic diagnostic that they developed
for distinguishing the ping-pong (Fig. 6B) from the sequential
mechanisms, as well as for distinguishing among sequential
mechanisms.46 In this diagnostic analysis, concentration of
substrate A is varied against a fixed saturating concentration
of the alternative substrates B, B′, B′′, etc., and B is similarly
varied against a series of the alternative substrates A. This
method was amenable to the study of AAC(6′)-IV, since it can
transfer a variety of acyl groups (acetyl, n-propionyl, n-
butyryl, n-valeryl, isovaleryl, and isohexyl).45 The initial-
velocity kinetics for discriminating the ping-pong
mechanism from the sequential mechanisms requires the
use of substrate concentrations at the vicinity of the value for
Km.This may prove difficult when the Km value is very low, as
is the case for substrates of AAC(6′)-IV (Km for acetyl-CoA =
0.8 µM).45 In addition, initial-velocity patterns show differ-
ences in intercepts and slopes for the disparate kinetic
mechanisms of only a factor of 2 or less, which in turn may
produce imprecise results.The Radika-Northrop method cir-
cumvents the requirement for near-Km substrate concentra-
tions and shows greater variance in V/Km among kinetic
mechanisms.
The data obtained for AAC(6′)-IV using this method indi-
cated that V/Km for the second substrate was dependent on
the identity of the antibiotic, and the V/Km for an aminoglyco-
side was dependent on the nature of the nucleotide.44 This
mutual dependence of V/Km for one substrate on the identity
of the other substrate is consistent only with a random
sequential mechanism. In the ping-pong mechanism, the V/Km
for a substrate should not exhibit dependence on the identity
Fig. 6 Kinetic scheme for (A) random Bi-Bi and (B) ping-pong
mechanisms.A and B = substrates; P and Q = products;
E = enzyme; F = modified enzyme.
Aminoglycoside-modifying enzymes
of the other substrate, while in an ordered sequential mecha-
nism only one set of data should show this dependence.
Additional data from kinetics studies established a rapid equi-
librium feature for substrate binding in this mechanism.45
Another aminoglycoside acetyltransferase enzyme,
AAC(3)-I, has been subjected to detailed kinetic analysis.47,48
Sixteen aminoglycosides and derivatives were found to be
substrates for this enzyme, including members of the gen-
tamicin and kanamycin families. Since the site of aminogly-
coside modification is the 2-deoxystreptamine ring,
derivatives containing only two rings, one of which contains
the site of modification, are among the substrates for this
enzyme.These include neamine, sisomine, gentamine Cla, and
tobramine—two-ring derivatives of neomycin, sisomicin,
gentamicin Cla, and tobramycin, respectively.47 Initial-velocity
kinetic experiments with tobramycin, sisomicin, and gentam-
icin Cla revealed that AAC(3)-I exhibits a sequential mecha-
nism.48 Gentamicin Cla was found to be a substrate inhibitor
of the enzyme, and this inhibition is partially uncompetitive
with respect to acetyl-CoA.This is evidence for a sequential
mechanism, arising from binding of the second gentamicin
Cla to the enzyme after catalysis, forming E.CoA.gentamcin
Cla complex and preventing the release of the CoA.This par-
tially uncompetitive substrate inhibition suggests an incom-
plete trapping of CoA by gentamicin Cla. It was therefore
postulated that a degree of randomness in substrate binding
exists that is coupled to partial rate limitation of turnover by
the rate of product release. Based on these results, a random
Bi-Bi mechanism (Fig. 6A) was proposed for this enzyme.This
proposed mechanism was further validated by product
inhibition experiments in which CoA was shown to be
competitive against acetyl-CoA and noncompetitive against
tobramycin, while acetyl-tobramycin was competitive and
noncompetitive with reference to tobramycin and acetyl-
CoA, respectively.48 An important characteristic of both prod-
uct and substrate inhibitions by gentamicin Cla is the
synergism that exists between CoA binding and gentamicin
Cla binding, i.e. the presence of CoA on the enzyme enhances
binding of gentamicin Cla and vice versa.Additional support
for the proposed mechanism came from dead-end inhibition
and multiple inhibition patterns.48
The initial-velocity pattern for sisomicin showed an
upward curve at low concentrations. This indicates a non-
rapid equilibrium for enzyme-substrate binding in which the
reaction Vmax is greater than the rate constant for the release
of the substrate from the enzyme.49 In fact, the rate constant
for binding of sisomicin to enzyme during catalytic turnovers
is found to approach the normal diffusion-controlled limits,
making the product release rate limiting. On the other hand,
the initial-velocity pattern for tobramycin is linear, with its
binding rate constant two orders of magnitude less than that
for sisomicin.48 In this case, substrate binding takes place as a
rapid equilibrium, and catalytic steps of bond-making and
bond-breaking become primarily rate limiting. Thus, an
antibiotic-dependent shift from rapid to nonrapid equili-
brium random mechanism occurs as one shifts from to-
bramycin (poor substrate) to sisomicin (good substrate).48
Crystal structures of two aminoglycoside acetyltrans-
ferases have recently been determined. These are AAC(3)-Ia
from Serratia marcescens50 and AAC(6′)-li from Enterococcus
 2001 Harcourt Publishers Ltd Drug Resistance Updates (2001) 4, 106–117 111
 Azucena and Mobashery
faecium.51 AAC(3)-Ia was observed to be similar in folding to
the yeast histone acetyltransferase HATI and N-myristoyltrans-
ferase—both protein-acylating enzymes and members of the
GCN5-related N-acetyltransferase superfamily.
Comparison of the crystal structure of AAC(6′)-Ii in com-
plex with acetyl-CoA with S. marcescens AAC(3)-la and Hat1
revealed that it belongs to this superfamily of enzymes as
well. Although the sequence identity among these enzymes
was negligible, functional studies show that this aminoglyco-
side-modifying enzyme possesses protein-acetylation activity.
Inhibition
A tight-binding, bisubstrate analog has been synthesized as
an inhibitor of AAC(3)-I.52 The design of this inhibitor was
based on the proposed kinetic mechanism and aminoglyco-
side structure-function studies for this enzyme, as described
above.48 (1) Substrate binding is sequential, that is both
antibiotic and CoA must be present on the enzyme before
any product is released and no covalent bonds would be
formed between the enzyme and either of the two sub-
strates; (2) substrate binding is synergistic; (3) interactions of
gentamicin C1a with active site residues occur primarily on
the purpurosamine and garosamine rings (rings I and III),
with few interactions with 2-deoxystraptamine; (4) acyl
transfer by the enzyme accommodates acetyl-CoA and propi-
onyl-CoA, but not butyryl-CoA or malonyl-CoA.The inhibitor
(Fig. 7) was enzymatically prepared using chloroacetyl-CoA
in place of acetyl-CoA. During the enzymatic reaction with
gentamicin C1a, the chloroacetyl moiety was transferred to
the 3 position of gentamicin C1a, and the sulfur of the CoA
product displaced chlorine on the transferred acyl group via
nucleophilic attack.
The bisubstrate inhibitor gave a Ki of 0.5 to 20 nM against
AAC(3)-I.53 When tested against a resistant E. coli strain that
harbored AAC(3)-I, the inhibitor did not show any effect on
the antibiotic activity of gentamicin presumably due to its
inability to cross the cell membrane. In addition to this
inhibitor, several aminoglycosides that were tested as non-
substrates for AAC(3)-I exhibited competitive inhibition
against substrate aminoglycosides, neomycin B being the
best inhibitor having a submicromolar iK.47 Also, two semi-
synthetic kanamycin derivatives, 6′-N-methylkanamycin A
and 3′, 4′-dideoxy-6-′-N-methylkanamycin B have been shown
to possess strong antibacterial activity against strains having
6′-N-acetylating enzymes, as they are resistant to modifica-
tion at the 6′-position.54
Detailed structure-function relationships for acylation of
several aminoglycyosides with AAC(6′)-IV and AAC(3)-I have
been reported.45,47 These data provide insights for strategies
OH
+ supporting the suggestion that phosphoryl transfer occurs
H3C O H3N
H3N HO
NH2 + NH3+
OH O
H3N
ON
N N + O
−OO−O O O O
N N O P P S
O O O N N
OH H H hydroxyl group, acts as the catalytic base that abstracts the
HO P O−
−O
O
Fig. 7 Structure of acetylgentamicin-CoA inhibitor.
112 Drug Resistance Updates (2001) 4, 106–117  2001 Harcourt Publishers Ltd
in modifying the structures of existing aminoglycosides to
produce drugs that are refractory to enzyme inhibition with-
out impairing their antibacterial activity. Unfortunately, from
the correlation of the antimicrobial activity with the amino-
glycoside structures, many of the structural requirements of
aminoglycosides for enzymatic activity run parallel to those
for antimicrobial activity.Thus, there are very few chemically
modified aminoglycoside derivatives that have been found to
retain activity against bacterial strains harboring these
enzymes. Derivatives that possess antimicrobial properties
include 5-epi-sisomicin36,38 and netilmicin55,56 which are
shown to be resistant to modification by AAC(3)-I.
AMINOGLYCOSIDE PHOSPHOTRANSFERASES
Mechanism
Among the known APHs, the best studied is APH(3′)-IIIa.This
enzyme confers resistance to a broad spectrum of aminogly-
cosides, including members of the kanamycin and neomycin
groups of aminoglycosides, most of which were also found to
be relatively poor substrate inhibitors for this enzyme.57 The
structural basis for substrate inhibition is not well elucidated.
One possible explanation is that the drug may bind improp-
erly to the active site of the enzyme.The substrate specificity
of this enzyme indicates that there may be little stringency for
4,6-disubstituted aminoglycosides (e.g. kanamycins) and 4,5-
disubstituted aminoglycoside (e.g. neomycins), and that the
aminocyclitol ring is significant for binding. Interestingly,
lividomycin A, which lacks the 3′-hydroxyl group, was found
to be a substrate for APH(3′)-IIIA.57 The site of modification of
this aminoglycoside was found to be the 5′′-hydroxyl group.58
Butirosin and neomycin, both of which possess 3′ and 5′′
hydroxyl groups, were found to be phosphorylated at either
sites.59 NMR analysis of butirosin bound to a ternary
Cr.ATP.APH(3′)-IIIa complex shows that both 3′-and 5′′-
hydroxyl groups are in close proximity to the γ-phosphoryl
group of ATP.60 It is speculated that the first phosphorylation
occurs at random at either position, followed by a conforma-
tional change that precedes the second phosphorylation.This
conformational change draws the negatively charged phos-
phoryl group on the modified aminoglycoside away from the
incoming second ATP molecule.
It was proposed that the chemical mechanism of the
phosphoryl transfer reaction between the aminoglycoside
and ATP among APH(3′)s involves a conserved histidine
residue that would relay the phosphoryl group from the γ-
position of ATP to the aminoglycoside 3′-hydroxyl group.61
This notion was disproved, at least in the case of APH(3′)-IIIa,
when conversion of the equivalent His residue (His-188) in
APH(3′)-IIIa failed to abolish the enzyme activity, thereby
O through direct attack of the 3′-hydroxyl group of the amino-
glycoside on the γ-phosphoryl group of ATP.62 The crystal
structure of this enzyme predicted that residue Asp-190,
which is in close proximity to the aminoglycoside 3′-
3′-hydroxyl proton to facilitate attack at ATP.63 This predic-
tion was validated by site-directed mutagenesis, which
demonstrated that replacement of this Asp-190 with alanine

resulted in at least a 650-fold decrease in kcat.63 Site-directed
mutagenesis also established the importance of interaction
of Lys-44 with the α-and β-phosphates of ATP, as this residue
may impart greater electrophilicity to the γ-phosphoryl
group for enhanced nucleophilic attack by the aminogly-
coside by its electrostatic interaction.63 Further structural
analyses of APH(3′)-IIIa-bound aminoglycosides by NMR
demonstrated that amikacin and butirosin can assume sev-
eral different arrangements at the active site of the
enzyme.64,65 It is presumed that not all of the bound confor-
mations lead to productive phosphoryl transfer. This may
provide explanation for the observed low kcat/Km for these
aminoglycosides.57
APH(3′)-IIIa was subjected to kinetic studies to determine
its catalytic mechanism.66 Initial velocity patterns ruled out
the ping-pong mechanism and supports a sequential mecha-
nism.ATP would bind first, followed by the aminoglycoside,
as established by dead-end inhibition patterns.Tobramycin is
not a substrate for this enzyme, but was found to be a strong
competitive inhibitor with reference to kanamycin A, but
uncompetitive inhibitor relative to ATP. Both AMP and AMP-
PNP (a nonhydrolizable ATP isostere) exhibited competitive
inhibition versus ATP, and noncompetitive versus amikacin.
The uncompetitive substrate inhibition observed with
kanamycin against ATP is indicative of dead-end binding of
kanamycin to the E.ADP complex, suggesting a partial rate-
limiting release of ADP. Noncompetitive product inhibition
with kanamycin phosphate versus ATP can be circumvented
by increasing kanamycin A concentration. This establishes
the order of product release as follows: rapid release of
kanamycin phosphate, followed by a slow release of ADP.
These results collectively argue for a Theorell-Chance mecha-
nism for this enzyme in which ATP binds first, followed by
aminoglycoside, and the rapid release of kanamycin phos-
phate taking place before the slow release of ADP. These
results are corroborated further by solvent-isotope effect
measurements and thio-and viscosity-effect experiments.67 A
small solvent isotope effect measured for reactions carried
out in deuterium oxide ruled out the possibility that the
enzyme-mediated deprotonation of the aminogycoside 3′-
hydroxyl group is the rate-limiting step. Likewise, the phos-
phate-transfer step (from ATP to the aminoglycoside) was
ruled out as the predominant contributor to the overall rate
of reaction, since it produced only a small thio-effect when
ATP was replaced by ATPγS.Alteration of the viscosity of the
assay solution did not affect the V/Km for aminoglycosides,
but had a slope effect on the V/Km for ATP.This identified ATP
binding and ADP release as diffusion-controlled processes.
However, since under physiologic conditions, aminoglyco-
side (not ATP) is limiting, ADP release was identified as the
primary contributor to the enzymatic reaction rate.This pro-
posal is consistent with the Theorell-Chance mechanism (see
Figure 3B).
Two additional APHs,APH(3′)-Ia and APH(3′)-IIa, have also
been studied.APH(3′)-I is the most common APH is gram-neg-
ative bacteria. Kinetic analyses by the alternative-substrate
method46 (using amikacin, kanamycin A, and neomycin as
aminoglycoside substrates, and ATP, GTP, and UTP as
nucleotide substrates), together with product inhibition
(with kanamycin A 3′-phosphate and ADP) determined a
Aminoglycoside-modifying enzymes
rapid equilibrium random mechanism for this enzyme.68
Thus, similarly to the case of APH(3′)-IIIa, this enzyme
exhibits a direct displacement mechanism for the γ-phospho-
ryl of ATP to the aminoglycoside. In the absence of aminogly-
cosides, this enzyme was also shown to hydrolyze ATP with a
Km.ATP value in the same range as that for the aminoglycoside
phosphotransferase activity of the enzyme (Km = 49 ± 1 µM).
This indicates that aminoglycoside binding does not influ-
ence the enzyme affinity for ATP.68 Insofar as the physiologic
ATP concentration in bacteria is well above this Km value, it
would appear that this enzyme is constantly hydrolyzing ATP
in vivo. However, the kcat for the ATP hydrolysis is signifi-
cantly lower than that for the aminoglycoside phosphoryla-
tion, such that the ATPase activity does not compete with
phosphorylation of aminoglycosides when these drugs are
present.
APH(3′)-IIa differs from APH(3′)-Ia in that it modifies both
butirosin and lividomycin.This enzyme also possesses ATPase
activity in the absence of aminoglycosides (Km.ATP = 52 ± 19
µM), similarly to what is observed for the type Ia counter-
part.69 It is believed that electrostatic interactions (ion pair-
ing and hydrogen binding) are significant for both substrate
binding and catalysis among aminoglycoside-modifying en-
zymes.To assess the significance of these interactions, seven
distinct deaminated analogs of neamine and kanamycin A
were synthesized by Roestamadji et al., and were tested with
APH(3′) types Ia, IIa, and IIIa.70,71 All seven molecules were
shown to be exceedingly poor substrates for both APH(3′)-Ia
and APH(3′)-IIa, arguing for the critical role of electrostatic
interactions between the aminoglycoside and the active sites
of these enzymes.Although the Km values for these deamino
derivatives increased only modestly relative to those for the
parent aminoglycosides, the kcat values were dramatically
decreased by several orders of magnitutde.70,71 Surprisingly,
APH(3′)-IIIa seemed to interact with the deaminated amino-
glycoside derivatives in quite a different fashion than
APH(3′)-Ia and -IIa, as it showed no clear kinetic pattern
among most of these deaminated aminoglycosides and the
parent compounds.
Inhibition
The deaminated analogs of neamine and kanamycin A,
described above, were shown to possess biological activity
against strains that harbor aminoglycoside 3′-phoaphotrans-
ferases types Ia and IIa, indicating that these compounds
were not modified by these enzymes in vivo.70 Thus, the syn-
thesis of such antibiotic derivatives that have reduced elec-
trostatic interactions with their corresponding enzymes may
prove to be valuable in the circumvention of resistance to
antibiotics. Roestamadji et al. also synthesized mechanism-
based inactivators (Fig. 8) whose design was based on mech-
anistic studies with APH(3′)s implicating an active-site
catalytic base in activation of the 3′-hydroxyl proton of
the substrate aminoglycoside.72 These compounds are 2′-
deamino-2′-nitro derivatives of neamine and kanamycin B.
Upon phosphorylation of 2′-deamino-2′-nitro-neamine at its
3′ position, the phosphoryl group is spontaneously elimi-
nated.As a result, an electrophilic inactivating species is gen-
erated, which then captures an active-site nucleophilic
residue.A covalent bond is thus formed between the enzyme
 2001 Harcourt Publishers Ltd Drug Resistance Updates (2001) 4, 106–117 113
 Azucena and Mobashery

HO
2' 4'
3'
O2N OH 6" OH
HO 1' O 6' 4" HO
5' NH3+ 5" O 2' 4'
6 4 3'
5 O HO O2N OH
HO 1" 6'
NH3+ +H3N 3" 2" HO 1' O
2 4 NH3+
+H3N 1 3 OH
6 5 5'
O O
+H3N 2 NH3+
1 3

Fig. 8 Structures of mechanism-based inactivators for APH(3′)s.The structure on the left is 2′-deamino-2′-nitro-neamine; the
structure on the right is 2′-deamino-2′-nitro-kanamycin B.

HO 4'
2'
H2N
3'
OH 2' HO 4'
3'
1' O H2N OH
HO 6'
4 5' NH2 1' O
6
O HO 6'
HO 5
6 4
5' NH2
5 O
NH2 HO
HN 2
1 3 NH
H2N 1
2
3
Br Br
O O

O
Br HO 4' 2' HO 4'
2' 3' 3'
OH O
N OH H2N
H O HO 1' O 6' Br
HO 5'
6'
4
5' NH
6 4 NH2 6
5 O
5 O HO
HO
NH2 H2N 2 NH2
H2N 2 1 3
1 3

Fig. 9 Structures of bromoacretylated analogs of neamine.

and the inactivator and an irreversible inactivation of the γ-CH2ATP and/or kanamycin A, the observed rate of inactiva-
enzyme ensues.This mechanism is validated by the detection tion was attenuated. Therefore, inactivation is directed
of the released inorganic phosphate during the inactivation towards the active site.To see whether the inactivators react
process, which is rapid and time-dependent, among other with any of the five thiols of cysteine residues69—potential
experiments.72 sites of preferential modification by the inactivators—the
The 2′-deamino-2′-nitro derivative of kanamycin B was cyanylated enzyme, which has all its cysteine thiols pro-
also found to inactivate APH(3′) via a mechanism similar to tected with the nitrile group, was prepared. The activity of
that proposed for 2′-deamino-2′-nitro-neamine, except that the enzyme upon cyanylation was found to be unaffected,
the enzyme inactivated by 2′-deamino-2′-nitro-kanamycin B and the kinetic parameters for the enzyme inactivation with
regained activity over several hours.This observed difference the cyanylated and uncyanylated enzymes were virtually the
in the inactivation profile could be attributed to interaction same.
of the two compounds with different active-site residues. The close similarity in the chemical mechanism and overall
This is the only account of a mechanism-based inhibition for three dimensional structure between APH(3′)-IIIa and protein
aminoglycoside resistance enzymes.72 kinases led to the investigation of possible sensitivity of APHs
Four bromoacetylated analogs of neamine were synthe- to known inhibitors of protein kinases. Of the three classes of
sized by Roestamadji et al. (Fig. 9) as affinity inactivators for inhibitors tested, the isoquinolinesulfonamides were found to
APH(3′)-IIa.73 In the presence of ATP, the enzyme could phos- be good inhibitors of APH(3′)-IIIa and the bifunctional
phorylate these compounds, but only at higher Km values, AAC(6′)-APH(2′′).74 Unfortunately, these compounds did not
ranging from 3-to 86-fold greater than that for the unmodi- reverse antibiotic resistance in enterococcal strains that har-
fied neamine. Since Km may approximate Ks here, the effect bor either the aph(3′)-IIIa gene or aac(6′)-aph(2′′) gene.
of structural modification of neamine in these compounds is In a recent publication, Wong et al. Synthesized various
largely on binding, and not turnover (i.e. kcat). On the other dimers of neamine linked via either amides or 1,2-hydrox-
hand, in the absence of ATP, all four compounds inactivated yamine moieties.75 These dimers were designed to identify
APH(3′)-IIa in a time-dependent and saturable fashion. The bivalent aminoglycosides that would interact with biotiny-
inactivated protein did not recover activity despite extensive lated E. coli 16S rRNA A-site—a model for the target site of
dialysis to remove the inactivator molecules, suggestive of aminoglycosides in bacteria.Three of these compounds were
covalent bond formation between the enzyme and the in- shown to be potent inhibitors of the APH(2′′) activity of the
activators. When the enzyme was preincubated with β, bifunctional enzyme AAC(6′)-APH(2′′), with a Kis as low as 0.1

114 Drug Resistance Updates (2001) 4, 106–117  2001 Harcourt Publishers Ltd

µM. Furthermore, these dimers show antimicrobial activity,
with the most active dimer exhibiting MIC of 6.25 µM
against E. coli, and a Kd of 40 nM for the biotinylated 16s
rRNA A-site.
CONCLUDING REMARKS
In this review we have attempted to provide biochemical
information for each of the three classes of bacterial resis-
tance enzymes for aminoglycosides.The emphasis was placed
on surveying the published body of information on the
respective mechanisms of these enzymes, along with infor-
mation on their inhibition.There are scores of such inactivat-
ing enzymes for these antibiotics, yet mechanistic knowledge
exists only for a handful. To date, there are no examples of
combination drugs of aminoglycoside-modifying enzyme
inhibitors and aminoglycosides. However, in order to counter
the resistance produced by these enzymes, detailed mecha-
nistic understanding of them is a critical first step.
Received 23 February 2001;Accepted 6 April 2001
Correspondence to: Shahriar Mobashery, Institute for Drug Design,
Departments of Chemistry, Pharmacology and Biochemistry and Molecular
Biology, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, USA.
Tel.: +313 577 3924; Fax: +1 313 577 8822;
E-mail: som@gordon.chem.wayne.edu
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