Mutant APH(2″)-IIa enzymes with increased activity against amikacin and isepamicin

Article abstract of DOI:10.1128/AAC.01444-09

Directed evolution by random PCR mutagenesis of the gene for the aminoglycoside 2″-IIa phosphotransferase generated R92H/D268N and N196D/D268N mutant enzymes, resulting in elevated levels of resistance to amikacin and isepamicin but not to other aminoglycoside antibiotics. Increases in the activities of the mutant phosphotransferases for isepamicin are the result of decreases in K_m values, while improved catalytic efficiency for amikacin is the result of both a decrease in K_m values and an increase in turnover of the antibiotic. Enzymes with R92H, D268N, and D268N single amino acid substitutions did not result in elevated MICs for aminoglycosides. Copyright

Full text of DOI:10.1128/AAC.01444-09

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2010, p. 1590–1595
0066-4804/10/$12.00 doi:10.1128/AAC.01444-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 4
Mutant APH(2Љ)-IIa Enzymes with Increased Activity against
Amikacin and Isepamicinᰔ
Marta Toth,1 Hilary Frase,1 Joseph W. Chow,2 Clyde Smith,3 and Sergei B. Vakulenko1*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 465561; Department of
Infectious Diseases Clinical Research, Merck Research Laboratories, North Wales, Pennsylvania 194542; and
Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California 940253
Received 12 October 2009/Returned for modification 22 December 2009/Accepted 28 January 2010
Directed evolution by random PCR mutagenesis of the gene for the aminoglycoside 2؆-IIa phosphotransfer-
ase generated R92H/D268N and N196D/D268N mutant enzymes, resulting in elevated levels of resistance to
amikacin and isepamicin but not to other aminoglycoside antibiotics. Increases in the activities of the mutant
phosphotransferases for isepamicin are the result of decreases in Km values, while improved catalytic efficiency
for amikacin is the result of both a decrease in Km values and an increase in turnover of the antibiotic. Enzymes
with R92H, D268N, and D268N single amino acid substitutions did not result in elevated MICs for
aminoglycosides.
Since the introduction of streptomycin into clinical use in
1944, aminoglycoside antibiotics have been used for treatment
of serious bacterial infections for more than 70 years (8, 14). In
response to their extensive use, bacterial isolates acquired re-
sistance to many aminoglycosides, rendering some of them
obsolete (15, 19). The major mechanism of resistance to ami-
noglycoside antibiotics in clinical bacterial isolates is the pro-
duction of three types of aminoglycoside-modifying enzymes,
aminoglycoside phosphotransferases (APHs), aminoglycoside
acetyltransferases (AACs), and aminoglycoside nucleotidyl-
transferases (ANTs). The APHs and ANTs are the bisubstrate
enzymes that facilitate transfer of the ␥-phosphate and nucle-
otide monophosphate, respectively, from a nucleotide sub-
strate to the hydroxyl groups of aminoglycoside antibiotics,
while AACs acetylate amino groups derived from acetyl coen-
zyme A (acetyl-CoA) (11, 15, 21). Such modifications result in
resistance, as the modified aminoglycosides have decreased
affinity for their target, the bacterial ribosome (12, 20). Ami-
noglycoside 2Љ-phosphotransferases [APH(2Љ)s] phosphorylate
the 2Љ hydroxyl of aminoglycoside antibiotics. Four distantly
related enzymes of this type, APH(2Љ)-Ia, -Ib, -Ic, and -Id,
sharing between 28 and 32% amino acid sequence identity,
have been identified (4–6, 9, 10, 18). Recently, detailed enzy-
mological characterization of these phosphotransferases re-
vealed that contrary to the established dogma, two of these
enzymes utilize GTP and not ATP as the nucleotide substrate.
Moreover, the aminoglycoside substrate profiles of these en-
zymes are not identical. These findings resulted in a revised
nomenclature for the APH(2Љ) enzymes (16), whereby the
APH(2Љ)-Ib, -Ic, and -Id enzymes were renamed APH(2Љ)-IIa,
-IIIa, and -IVa, respectively. The APH(2Љ) enzymes are widely
disseminated among Gram-positive enterococcal and staphy-
lococcal isolates. APH(2Љ)-IIa is the only 2Љ aminoglycoside
phosphotransferase that has been identified also in a Gram-
negative isolate, Escherichia coli (5). While the extensive use of
␤-lactam antibiotics resulted in the emergence of hundreds of
mutant ␤-lactamases with extended-spectrum activity against
␤-lactam antibiotics, no mutant derivatives of aminoglycoside-
modifying enzymes have been reported in clinical isolates de-
spite the long-term use of aminoglycosides. In this paper we
describe PCR-generated mutant derivatives of the APH(2Љ)-
IIa enzyme that produce enhanced levels of resistance to two
clinically important aminoglycoside antibiotics, amikacin and
isepamicin.
MATERIALS AND METHODS
Random PCR mutagenesis. We utilized the gene for APH(2Љ)-IIa phospho-
transferase cloned between the NdeI and HindIII sites of the pHF022 vector as
the template for random PCR mutagenesis. The vector pHF022 was constructed
by combining the pBR origin of replication, an ampicillin resistance gene from
the pUC19 vector, and the strong constitutive expression promoter of the D-
amino acid aminotransferase gene of Geobacillus toebii (13). The pBR origin of
replication was PCR amplified from the pET24a(ϩ) vector using the oligonu-
cleotide primers oHF014 and oHF016 (Table 1). The gene for the ampicillin
antibiotic selection marker was PCR amplified from the pUC19 vector using the
primers oHF021 and oHF022 (Table 1). These two fragments were digested with
EcoRV and PstI and ligated together to generate the plasmid pHF018. A DNA
fragment encoding the constitutive promoter of the D-amino acid aminotrans-
ferase gene, a multicloning site, and the termination signal from the T2 element
of the Escherichia coli rrnB gene was custom synthesized (Celtek Genes). PstI
sites were included at the 5Ј and 3Ј ends of this synthetic DNA fragment.
Following digestion with PstI, the synthetic fragment was cloned into the unique
PstI site of pHF018, resulting in the plasmid pHF022. The sequence of the entire
vector was verified by DNA sequencing. Random PCR mutagenesis of the gene
for the APH(2Љ)-IIa was performed with the GeneMorph II random mutagenesis
kit (Stratagene catalog no. 200550) according to the manufacturer’s recommen-
dations, utilizing conditions that generate a low mutation rate (0 to 4 mutations
per target gene). Briefly, the gene for APH(2Љ)-IIa in the pHF022 vector was
amplified by PCR using Mutazyme II DNA polymerase and two oligonucleotide
primers, IIaPCR-D and IIaPCR-R (Table 1). We performed a total of three
PCRs, using 19, 22, and 25 cycles, respectively. Following this, the PCR products
in the three tubes were combined and fragments generated by PCR were sepa-
rated on a 1% agarose gel and purified with Wizard SV Gel and the PCR
Clean-Up system (Promega). Purified DNA fragments were digested with the
NdeI and HindIII restriction endonucleases, repurified with Wizard SV Gel and
the PCR Clean-Up system, and cloned between the NdeI and HindIII sites of the
pHF022 vector. Purified plasmid DNA of the pHF022 vector containing the
* Corresponding author. Mailing address: Department of Chemistry
and Biochemistry, University of Notre Dame, 417 Nieuwland Science
Hall, Notre Dame, IN 46556. Phone: (574) 631-2935. Fax: (574) 631-
6652. E-mail: svakulen@nd.edu.
ᰔ Published ahead of print on 9 February 2010.
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VOL. 54, 2010
Primer
MUTANT APH(2Љ)-IIa PHOSPHOTRANSFERASES
1591
NaCl gradient (0 to 1 M) in 25 mM HEPES, pH 7.5. Fractions were examined by
measuring enzyme activity and subsequently by SDS-PAGE. Fractions contain-
ing pure enzyme were pooled, concentrated, dialyzed against 25 mM HEPES, pH
7.5, 0.5 mM DTT, and stored at Ϫ80°C. The R92H/D268N mutant enzyme was
purified similarly except that a kanamycin-Affigel 10 affinity column was used for
protein purification with a 0 to 2 M NaCl gradient in 25 mM HEPES, pH 7.5, for
protein elution.
Enzyme kinetics. Phosphorylation of aminoglycosides by APH(2Љ)-IIa and
mutant derivatives was monitored with a continuous spectrophotometric assay in
the presence of ATP or GTP as the second substrate (17). Kinetic assays were
performed in a total volume of 250 ␮l containing 100 mM HEPES, pH 7.5, 10
mM MgCl2, 20 mM KCl, 2 mM phosphoenolpyruvate, 140 ␮M NADH, 15
units/ml pyruvate kinase, and 20 units/ml lactate dehydrogenase. Kinetic param-
eters for aminoglycoside antibiotics were determined using a fixed concentration
of ATP (200 ␮M) and various concentrations of aminoglycosides. Kinetic
parameters for ATP and GTP were determined using kanamycin A at a fixed
concentration (10 ␮M) and ATP or GTP at various concentrations. The reac-
tions were initiated by adding enzyme (5 to 10 nM final concentration). For
determination of kcat, Km, and kcat/Km for aminoglycoside antibiotics, ATP and
GTP data were fit nonlinearly with the Michaelis-Menten equation (equation 1)
using the program Prism 5 (GraphPad Software, Inc.).
TABLE 1. Primers used in this study
Sequencea
oHF014........................GCGGATATCCCCGTAGAAAAGATCAAAGG
oHF016........................GTATCTGCAGAGCGCTGGCATTGACCCTG
oHF021........................GTTTCTGCAGCTAAATACATTCAAATATGTATCC
oHF022........................CTTTTCGATATCGTCTGACGCTCAGTGGAACG
IIaPCR-D....................CTGGAAAAAGGAGATATACCATATG
IIaPCR-R ....................GAGTGCGGCCGCAAGCTT
a Underlined are sites for the following restriction endonucleases: EcoRV
(GATATC), PstI (CTGCAG), NdeI (CATATG), and HindIII (AAGCTT).
mutagenized gene for APH(2Љ)-IIa was used to transform electrocompetent E.
coli NEB 5-alpha cells (NEB catalog no. C2989K). After overnight incubation in
LB broth supplemented with 100 ␮g/ml of ampicillin (selection marker for the
pHF022 vector), the bacterial culture was diluted 500-fold into fresh LB broth
supplemented with amikacin or isepamicin at various concentrations. Bacteria
were incubated overnight at 37°C, and plasmid DNA was isolated from bacterial
cultures that had grown at the highest concentration of each antibiotic. These
DNA samples were used to transform chemically competent E. coli JM83 cells.
Transformed cells were plated on LB agar supplemented with 100 ␮g/ml of
ampicillin, and plates were incubated overnight at 37°C. Resulting individual
colonies were picked for testing susceptibility to amikacin and isepamicin and
subsequent DNA sequencing of the gene for the APH(2Љ)-IIa phosphotransfer-
ase. Since only E. coli double mutants with increased resistance to amikacin and
isepamicin were isolated, E. coli strains containing single mutations in the
aph(2Љ)-IIa gene were constructed by swapping the fragments of the double
mutant and wild-type genes cloned between the NdeI and HindIII sites of the
pHF022 vector. Separation of the individual mutations was possible because of
the existence of the unique BstEII restriction endonuclease site between the
triplets encoding H92 and N268 and D196 and N268 in the double mutant
aph(2Љ)-IIa genes. The R92H single mutant enzyme was constructed by combin-
ing the 649-bp NdeI-BstEII fragment of the gene for the R92H/D268N double
mutant enzyme (this fragment encodes the R92H substitution but not the D268N
substitution) with the 405-bp BstEII-HindIII fragment of the wild-type gene. The
D268N single mutant enzyme was constructed by combining the BstEII-HindIII
fragment of the gene for the R92H/D268N double mutant enzyme (this fragment
encodes only the D268N substitution) with the NdeI-BstEII fragment of the
wild-type gene. Finally, the N196D single mutant enzyme was constructed by
combining the NdeI-BstEII fragment of the gene for the N196D/D268N double
mutant enzyme (this fragment encodes only the N196D substitution) with the
BstEII-HindIII fragment of the wild-type gene.
Vmax ͓A͔
Ka ϩ ͓A͔
v ϭ
(1)
where Vmax represents the maximum velocity, [A] is the concentration of the
variable substrate, and Ka is the Michaelis constant.
RESULTS AND DISCUSSION
Susceptibility profile of E. coli JM83 producing APH(2؆)-IIa
and mutant derivatives. The only E. coli strain isolated that
exhibited increased resistance to amikacin and isepamicin con-
tained double amino acid substitutions in APH(2Љ)-IIa; no
resistant E. coli strain contained single amino acid substitu-
tions. The R92H/D268N and N196D/D268N double mutant
enzymes produce an 8-fold increase in the MICs for both
amikacin and isepamicin but not for any other 4,6-disubsti-
tuted antibiotics (kanamycin A, kanamycin B, gentamicin,
tobramycin, dibekacin, netilmicin, sisomicin, or G418) against the
parental E. coli strain (Table 2). Mutants containing single
amino acid substitutions were constructed to confirm that
these single mutants do not result in higher levels of aminogly-
coside resistance. Antibiotic susceptibility testing demon-
strated that the single R92H, N196D, or D268N substitution in
the APH(2Љ)-IIa enzyme does not significantly change the sus-
ceptibility of E. coli JM83 to any of the aminoglycoside anti-
biotics tested (Table 2).
Antibiotic susceptibility testing. Antibiotic susceptibility profiles of parental E.
coli JM83, E. coli JM83 harboring the pHF022 vector, and E. coli JM83 harboring
the pHF022 vector with cloned genes for the wild-type and mutant APH(2Љ)-IIa
enzymes were determined by the broth microdilution method according to the
recommendations of the Clinical and Laboratory Standards Institute (CLSI) (7).
MIC testing was performed in Mueller-Hinton II broth (Becton Dickinson and
Company) with a bacterial inoculum of 5 ϫ 105 CFU/ml, and results were
recorded after incubation of microtiter plates for 20 h at 35°C. Reported MIC
values are the averages of at least three independent experiments.
Purification of the mutant APH(2؆)-IIa enzymes. For inducible expression of
the wild-type and mutant APH(2Љ) phosphotransferases, their genes were excised
from the pHF022 vector by the restriction endonucleases NdeI and HindIII and
recloned into the NdeI and HindIII sites of the pET22b expression vector.
Ligation mixtures were used to transform E. coli BL21(DE3), and transformants
were selected on the LB agar supplemented with 100 ␮g/ml of ampicillin. For
large-scale protein purification, 5 ml of overnight cultures of E. coli BL21(DE3)
expressing APH(2Љ)-IIa and the R92H/D268N and N196D/D268N mutant de-
rivatives was inoculated into 500 ml of minimal medium supplemented with 100
␮g/ml of ampicillin. Bacteria were incubated at 37°C with agitation until the
optical density at 600 nm (OD600) reached ϳ0.4 and then induced with 0.4 mM
IPTG (isopropyl-␤-D-thiogalactopyranoside) and incubated at 15°C overnight.
Bacterial cells were pelleted by centrifugation (3,000 ϫ g for 20 min at 10°C),
resuspended in 60 ml of buffer A (25 mM HEPES, pH 7.5, 1 mM EDTA, and 0.2
mM dithiothreitol [DTT]), and disrupted by sonication at 4°C. The lysates were
centrifuged (20,000 ϫ g for 30 min at 10°C), and the supernatants were supple-
mented with 1.5% streptomycin sulfate to precipitate nucleic acids. After 0.5 h of
incubation, supernatants were centrifuged (20,000 ϫ g for 30 min at 10°C) and
subsequently dialyzed against 25 mM HEPES, pH 7.5, 200 mM NaCl. Wild-type
APH(2Љ)-IIa and the N196D/D268N mutant derivative were purified by genta-
micin-Affigel 10 (Bio-Rad) affinity chromatography. Enzymes were eluted with a
The level of resistance to aminoglycoside antibiotics is influ-
enced by many factors, including the amount of modifying
enzyme produced (this, in turn, depends on the gene copy
number, promoter strength, and enzyme stability) and the rate
of penetration of the antibiotic into the bacterial cell. The
mutant APH(2Љ)-IIa enzymes described in this study elevate
MIC values of amikacin and isepamicin from 2 to 16 ␮g/ml
when the enzymes are expressed in an E. coli JM83 back-
ground. APH(2Љ)-IIa originated in enterococci, whose faculta-
tive anaerobic metabolism significantly lowers the rate of up-
take of all aminoglycosides, thus producing intrinsic resistance
to these antibiotics at MIC levels ranging from 4 to 256 ␮g/ml.
According to CLSI recommendations, enterococci with genta-
micin MICs of Յ500 ␮g/ml are still considered susceptible, as
gentamicin can successfully be used against such isolates in
combination with cell-wall-active antibiotics such as ␤-lactams
and vancomycin. Acquisition by enterococci of aminoglyco-

1592
TOTH ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Antimicrobial susceptibilities of E. coli JM83 producing APH(2Љ)-IIa and its mutant derivatives
MIC (␮g/ml) of druga:
Enzyme
AMK
ISE
KANA
KANB
GEN
TOB
DBK
NET
SIS
G418
APH(2Љ)-IIa
N196D/D268N
R92H/D268N
R92H
2
16
16
2
2
16
16
4
256
256
256
256
256
256
2
64
64
64
64
64
64
1
16
64
64
64
64
64
32
1
64
64
64
64
64
32
0.5
8
8
8
512
512
512
512
512
512
2
16
4
16
8
8
16
16
8
4
8
N196D
2
2
16
8
D268N
2
2
2
2
16
4
Controlb
0.25
0.25
0.25
a AMK, amikacin; ISE, isepamicin; KANA, kanamycin A; KANB, kanamycin B; GEN, gentamicin; TOB, tobramycin; DBK, dibekacin; NET, netilmicin; SIS,
sisomicin.
b E. coli JM83 harboring the pHF022 vector.
side-modifying enzymes results in a significant increase in re-
sistance to aminoglycoside antibiotics. Thus, the clinical isolate
Enterococcus faecium SF11770, in which the gene encoding the
APH(2Љ)-IIa enzyme was originally identified, produces resis-
tance to amikacin and isepamicin at 512 ␮g/ml while the MICs
of other aminoglycoside antibiotics are higher than 2,000 ␮g/
ml. Thus, one would expect that the appearance of R92H/
D268N or N196D/D268N mutant enzymes in clinical entero-
coccal isolates would produce even higher levels of resistance
to amikacin and isepamicin.
bramycin, and dibekacin with kcat/Km values above 1 ϫ 107
MϪ1 Ϫ1. All three enzymes are characterized by very low
s
(Յ2.1 ␮M) Km values for these aminoglycosides, implying that
the enzyme affinities for kanamycin A, tobramycin, and dibeka-
cin are very high. High binding affinities would allow the en-
zymes to bind and phosphorylate incoming antibiotics when
their concentration in the cells is still very low, below the levels
resulting in irreversible impairment of protein synthesis. The
catalytic efficiencies of APH(2Љ)-IIa for isepamicin and amika-
cin are significantly lower (kcat/Km values of 4.4 ϫ 105 and
Biochemical characterization of the R92H/D268N and N196D/
D268N mutant enzymes. Both double mutant enzymes were
purified to homogeneity by single-step affinity chromatogra-
phy. The kinetic parameters for APH(2Љ)-IIa and the N196D/
D268N and R92H/D268N mutant derivatives are presented in
Table 3. The wild-type enzyme and the double mutant deriv-
atives can utilize both ATP and GTP as phosphate donors,
although ATP is a slightly better substrate than GTP.
APH(2Љ)-IIa turns over ATP 4.6-fold faster than GTP and has
a 4.3-fold lower Km value for ATP than for GTP. The N196D/
D268N mutant enzyme has very similar kcat values for the two
nucleoside triphosphates (NTPs) while the R92H/D268N mu-
tant has almost identical Km values for ATP and GTP but turns
over ATP two times more efficiently than GTP. It is expected
that the wild-type and both double mutant enzymes would
experience saturation with either of these substrates in vivo, as
the concentration of ATP and GTP inside the cell is well above
their Km values (estimated concentrations of ATP and GTP in
E. coli are 3,560 and 1,160 ␮M, respectively) (3). In agreement
with the antibiotic susceptibility profiles, APH(2Љ)-IIa and both
mutant derivatives efficiently phosphorylate kanamycin A, to-
1.3 ϫ 105 MϪ1 Ϫ1, respectively) than for all other 4,6-disub-
s
stituted aminoglycosides tested. These lower catalytic efficien-
cies are the result of both higher Km values (62.9 ␮M for
amikacin and 13.5 ␮M for isepamicin) and lower turnover
numbers for these two antibiotics. The catalytic efficiencies of
the N196D/D268N and R92H/D268N mutant enzymes are in-
creased 7- to 8-fold for amikacin and around 5-fold for isepa-
micin in comparison to the wild-type enzyme. Increase in the
catalytic efficiency of mutant enzymes for amikacin is the result
of a moderate decrease in Km values accompanied by an in-
crease in turnover numbers. The improved catalytic efficiency
of these enzymes for isepamicin results entirely from a signif-
icant decrease in Km values. In contrast to amikacin, the rate of
turnover of isepamicin is slightly decreased for both mutant
phosphotransferases.
Substrate binding in the APH(2؆)-IIa mutant enzymes.
Comparison of the structures of the aminoglycosides used for
this study indicates that unlike other antibiotics, amikacin and
isepamicin contain a bulky functionality at position 1 of the
2-deoxystreptamine ring (Fig. 1). These functionalities [(S)-4-
amino-2-hydroxybutyryl in amikacin and (S)-3-amino-2-hy-
TABLE 3. Kinetic parameters of APH(2Љ)-IIa and its mutant derivatives
APH(2Љ)-IIaa
Km (␮M)
kcat/Km (MϪ1 sϪ1
N196D/D268N mutant
R92H/D268N mutant
Substrateb
kcat (sϪ1
)
)
kcat (sϪ1
)
Km (␮M)
kcat/Km (MϪ1 sϪ1
)
kcat (sϪ1
)
Km (␮M)
kcat/Km (MϪ1 sϪ1
)
KANA
TOB
DBK
ISE
AMK
ATP
GTP
45.1 Ϯ 0.4
26.1 Ϯ 0.5
17.1 Ϯ 0.1
2.1 Ϯ 0.1 (2.2 Ϯ 0.3) ϫ 107 13.8 Ϯ 0.5
1.5 Ϯ 0.2 (1.7 Ϯ 0.4) ϫ 107 16.6 Ϯ 0.6
1.2 Ϯ 0.1 (1.4 Ϯ 0.3) ϫ 107 16.9 Ϯ 0.5
Ͻ1
Ͻ1
Ͻ1
Ͼ1.4 ϫ 107
Ͼ1.7 ϫ 107
Ͼ1.7 ϫ 107
20.5 Ϯ 0.5
26.7 Ϯ 1.5
27.5 Ϯ 1.2
2.8 Ϯ 0.2
Ͻ1
Ͻ1
Ͼ2.0 ϫ 107
Ͼ2.7 ϫ 107
1.2 Ϯ 0.2 (2.3 Ϯ 0.4) ϫ 107
1.2 Ϯ 0.3 (2.3 Ϯ 0.6) ϫ 106
5.7 Ϯ 0.1 13.5 Ϯ 1.1 (4.4 Ϯ 0.6) ϫ 105
4.1 Ϯ 0.1
2.0 Ϯ 0.2 (2.0 Ϯ 0.2) ϫ 106
8.3 Ϯ 0.5 62.9 Ϯ 8.3 (1.3 Ϯ 0.3) ϫ 105 13.8 Ϯ 0.3 13.5 Ϯ 0.8 (1.0 Ϯ 0.5) ϫ 106 22.3 Ϯ 0.4 23.7 Ϯ 1.2 (9.4 Ϯ 0.5) ϫ 105
43.3 Ϯ 1.5 16.3 Ϯ 1.6 (2.7 Ϯ 0.4) ϫ 106 12.1 Ϯ 0.3
5.6 Ϯ 0.5 (2.2 Ϯ 0.2) ϫ 106 21.4 Ϯ 0.5 29.5 Ϯ 2.7 (7.2 Ϯ 0.7) ϫ 105
8.9 Ϯ 0.2 28.2 Ϯ 2.7 (3.2 Ϯ 0.3) ϫ 105
9.4 Ϯ 0.1 70.0 Ϯ 2.0 (1.3 Ϯ 0.1) ϫ 105 10.6 Ϯ 0.3 16.2 Ϯ 1.5 (6.5 Ϯ 0.6) ϫ 105
a Data for the APH(2Љ)-IIa enzyme were reported previously (14, 15).
b KANA, kanamycin A; TOB, tobramycin; DBK, dibekacin; ISE, isepamicin; AMK, amikacin.

VOL. 54, 2010
MUTANT APH(2Љ)-IIa PHOSPHOTRANSFERASES
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the amino acid sequences of the APH(2Љ) enzymes indicates
that R92 is not a conserved amino acid residue in these phos-
photransferases (Fig. 2). In contrast, only two amino acid res-
idues are found in position 196 [residue numbering for
APH(2Љ)-IIa]: APH(2Љ)-Ia and APH(2Љ)-IIa have asparagine at
this position, while the two other enzymes have aspartate (Fig.
2). Thus, the N196D substitution in APH(2Љ)-IIa results in a
negatively charged aspartate residue as found in the APH(2Љ)-
IIIa and -IVa phosphotransferases. Negatively charged resi-
dues [aspartate in APH(2Љ)-IIa or glutamate in APH(2Љ)-Ia
and -IVa] are conserved at position 268. Substitution of aspar-
agine for aspartate at this position produces no significant loss
of resistance to aminoglycoside antibiotics, an indication that a
negatively charged residue at position 268 is not critical for the
ability of the APH(2Љ) enzymes to produce a resistance phe-
notype. As the sizes of aspartate and asparagine residues are
very similar, it remains to be elucidated if size conservation at
this position is important for manifestation of resistance.
Recently, we reported the X-ray structures of the binary
complex of APH(2Љ)-IIa with gentamicin and of the ternary
complex with AMPPCP and the inhibitor streptomycin (22).
These structures allow us to evaluate the precise location of
the mutations within the enzyme’s three-dimensional struc-
ture. Two of the mutations (N196D and D268N) are within the
substrate binding cleft located between the core subdomain
and the helical subdomain (Fig. 3a). In the binary gentamicin
complex, the side chain of N196 accepts a hydrogen bond from
the N1 amine on the central 2-deoxystreptamine ring of the
substrate. D268 is on the opposite side of the binding site on
the long bent helix (␣9) which forms the wall of the cleft. In the
gentamicin complex D268 makes no interactions with the sub-
FIG. 1. Structures of some aminoglycoside antibiotics used in this
study.
droxypropionyl in isepamicin] are likely to reduce affinity of
the APH(2Љ)-IIa for these antibiotics, resulting in high Km
values. This assumption is supported by the 31-fold-elevated
Km of the enzyme for amikacin in comparison to that for
kanamycin A, even though these aminoglycosides are structur-
ally identical except for the functionality at position 1 of the
2-deoxystreptamine ring [an amino group in kanamycin A ver-
sus (S)-4-amino-2-hydroxybutyryl in amikacin]. Alignment of
FIG. 2. Multiple amino acid sequence alignment of the four APH(2Љ) enzymes. The amino acid residues R92, N196, and D268 are highlighted
in gray. Stars indicate fully conserved residues, double dots indicate strongly conserved residues, and single dots indicate weakly conserved residues.

1594
TOTH ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 3. (a) Cartoon representation of the wild-type APH(2Љ)-IIa structure showing the three structural domains in red (N-terminal domain),
green (core subdomain), and blue (helical subdomain). The nucleotide binding site is indicated by the ADP molecule in yellow sticks (between the
N-terminal domain and core subdomain), and the substrate binding site is indicated by the gentamicin in magenta sticks (between the core and
helical subdomains). The location of the three mutations (R92, N196, and D268) in the two double mutants (R92H/D268N and N196D/D268N)
are indicated by cyan ball-and-stick models. (b) Close-up view of the aminoglycoside binding site in wild-type APH(2Љ)-IIa, represented as a
molecular surface with structural domains colored in the same scheme as in panel a. The side chains for R92, N196, and D268 are shown as cyan
sticks, with some of the other important residues shown as green sticks. Gentamicin as observed in wild-type APH(2Љ)-IIa is given as thick magenta
sticks, and the three rings are labeled A, B, and C (the central B ring is the 2-deoxystreptamine ring). Amikacin (thin yellow sticks) and isepamicin
(thin gray sticks) are modeled into the binding site such that the three rings of each aminoglycoside overlie the corresponding rings in the
gentamicin molecule.
strate. The third mutation, R92, is at the C-terminal end of the
linker joining the N-terminal domain and the core subdomain
(Fig. 3a). This linker connects to a loop which passes close to
the substrate binding site before folding into the subdomain.
Modeling of amikacin and isepamicin into the APH(2Љ)-IIa
substrate binding sites (Fig. 3b) allows us to speculate as to the
decreased activity toward these substrates in the wild-type en-
zyme and as to how the point mutations may give rise to
increased activity toward these two aminoglycosides. The 2-de-
oxystreptamine ring (the B ring in Fig. 3b) and the aminogly-
can substituent at position 4 (the A ring in Fig. 3b) of genta-
micin appear to interact with a highly specific binding platform
provided by D228, S230, D232, and D233 from the core sub-
domain and D262 and W265 from helix ␣9 (22). Clearly, were
amikacin and isepamicin to bind to wild-type APH(2Љ)-IIa in
the same manner as that of gentamicin, there would be a
severe steric clash between the N196 side chain and the bulky
hydroxyalkylamide functionalities on the antibiotics (Fig. 3b).
The observation that wild-type APH(2Љ)-IIa is able to turn over
these two substrates indicates that they are able to bind in a
productive manner, which implies that either the hydroxyalky-
lamide functionalities or the N196 side chain needs to move
away. The difference in Km between amikacin (62.9 ␮M) with
an (S)-4-amino-2-hydroxybutyryl group and isepamicin (13.5
␮M) with a shorter (S)-3-amino-2-hydroxypropionyl tail sug-
gests that this steric effect is less significant when the size of the
functionality is decreased by one carbon atom.
to the N1 amide of the 2-deoxystreptamine ring. This region of
the binding site is highly negatively charged, with at least
three other aspartate residues in close proximity (the 2-de-
oxystreptamine ring is surrounded by at least three other as-
partate residues, D210, D232, and D233). Mutation of residue
196 to aspartate would greatly destabilize this position, and if
amikacin or isepamicin were to enter the binding site, this
might result in the side chain and/or the entire peptide flipping
away from its current position, thus allowing for the binding of
the hydroxyalkylamide groups.
The presence of the R92 side chain close to residue 196 is
also intriguing. Although it does not form any specific interac-
tions in the wild-type structure, small changes in the confor-
mation of the arginine side chain would bring the guanidium
group within hydrogen bonding distance of the carbonyl oxy-
gen atoms of residues 195 and 196, which may help stabilize
the protein in the vicinity of residue 196. Upon mutation of this
residue to histidine, which is significantly shorter than arginine,
these putative hydrogen bonding interactions would be lost
and the hydroxyalkylamide tail groups of an incoming amikacin
or isepamicin may be sufficient to force the N196 side chain out
of the way. However, this does not explain the role of residue
268, which is the common point of mutation in both of the
double mutants. It is difficult to understand why loss of the
negative charge at this position, upon going from aspartate to
asparagine, has such a profound effect upon enzyme activity in
conjunction with either the N196D or the R92H mutation. One
intriguing possibility is that amikacin and isepamicin may bind
in a completely different manner, essentially a 180° vertical
rotation relative to their orientation in Fig. 3b, such that the A
In the wild-type APH(2Љ)-IIa structure, the amide nitro-
gen of N196 is approximately 3.6 Å from the side chain of
the catalytic aspartate D192, which is also hydrogen bonded

VOL. 54, 2010
MUTANT APH(2Љ)-IIa PHOSPHOTRANSFERASES
1595
and C aminoglycan rings remain in the same location (with the
2Љ-OH remaining oriented for efficient attack on the ␥-phos-
phate of the ATP). The 2-deoxystreptamine ring and the hy-
droxyalkylamide tail would now point into the pocket occupied
by residue 268. However, given the narrow shape of the bind-
ing cleft and the highly specific A-B ring binding platform, such
a rotation of the substrate is rather unlikely and the specific
role of the D268N mutation must await further structural stud-
ies with these mutants.
zymes should take into account the potential for these enzymes
to evolve.
ACKNOWLEDGMENT
This work was supported by a grant from the National Institutes of
Health (RO1 AI057393).
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