(Received for publication, April 17, 1995; and in revised form, May 24, 1995)
From the
TEM-35 (inhibitor resistant TEM (IRT)-4) and TEM-36 (IRT-7)
clavulanic acid-resistant
The production of To overcome this constant evolution of TEM-1,
suicide inhibitors of the enzyme such as clavulanic acid and sulbactam
are used clinically in combination with penicillins. Unfortunately,
mutations occur leading to TEM-1-derived
The M182T
substitution does not affect the level of inhibitor resistance
(Blasquez et al., 1993). The R165W mutation results in a small
increase of resistance (Lenfant et al., 1993), whereas the
major impact of the substitutions at positions 69 and 244 were
described by Delaire et al.(1992) and Imtiaz et
al.(1993). The role of the N276D substitution encountered in
clavulanic acid-resistant TEM-35 and TEM-36 was not clear because this
substitution has never been isolated. In order to assess this point,
the N276D variant TEM-1
Bacterial cells were grown at 30 °C on Luria broth
supplemented with 0.5% MgSO This extract was then concentrated and desalted by ultrafiltration
using Amicon system 8200 (Amicon GmbH, Witten, Germany) with a PM-10
membrane. The concentrated volume was approximately 5 ml. This
suspension was then purified by preparative electrofocusing using a
Multiphor II system (Pharmacia, Uppsala, Sweden) on a 4-6.5 pH
gradient. The gel portion containing the
For evaluation of potential fields, we used
the DelPhi package (Nicholls and Honig, 1991; Sharp and Nicholls,
1989), which solves the linearized Poisson-Boltzmann equation by a
finite difference method. The TEM-1 structure has a length of about 57
Å, which, together with the original DelPhi grid (65
The antibiograms of the exponential cultures of these transformed
strains (Table 3) show that all the variant proteins are less
active than the wild type TEM-1
The antibiotic disc assays of the N276D
mutant show that this variant is as active as the wild type
As opposed to the behavior with substrates, the
effect of the N276D substitution on the inhibitory capacity of
clavulanic acid is drastic (Table 5). The main feature is the
very poor affinity of the enzyme for this inhibitor, with a 23-fold
increased K
Figure 1:
Electrospray mass spectra of the
reaction of clavulanic acid (inhibitor-to-enzyme ratio of 150) at 37
°C for 3 h with TEM-1 (A) and the N276D mutant (B). The spectra were obtained by applying the MaxEnt
deconvolution procedure to raw data. Measurements from at least 6
independent experiments were used to compute mass averages of the major
inhibited enzyme complex (Complex). The mass complex is 29,028
± 8 Da with TEM-1 and 29,025 ± 13 Da with the N276D
mutant.
The IRT-4 (TEM-35) The antibiotic
disc assays show that, except for the glycine and serine variants,
which remain active, all the substitutions obtained by suppression of
the amber-mutated codon lead to enzymes with very poor activity, even
against penicillins. On the contrary, the decreased activity due to the
substitution of the asparagine by an aspartic acid is not significant,
because the growth inhibition diameters with all the Kinetic and ESMS analyses show that the N276D
mutant's main property is its resistance to clavulanic acid
inhibition. Imtiaz et al.(1993) suggested that irreversible
inactivation involves the capture of the The resistance in the N276D mutant is probably related to the
considerable loss of affinity for the inhibitor (23-fold increase in K
Figure 2:
Stereo view of the environment of residues
244 and 276 in the wild type (thin lines) and the N276D mutant
enzymes (thick lines). The distance between the Arg
These electrostatic
effects in the substrate binding cavity may be responsible for the
increased K The decrease of
the binding constant of clavulanic acid, assumed to be positioned as a
penicillin substrate in the catalytic site (Imtiaz et al.,
1993), may also result from the weakened electrostatic interaction
between the carboxylate at C-3 of the clavulanic acid molecule and the
arginine 244 guanidium group in the acyl-enzyme complex. As a
consequence, deacylation could be sufficiently facile to compete with
the inactivation reaction and thereby explain the regeneration of
N276D. A faster release of the penicilloic acid reaction product would
also be in line with the increased k In conclusion, even though the N276D
substitution in TEM
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-lactamases have evolved from TEM-1
-lactamase by two substitutions: a methionine to a leucine or a
valine at position 69 and an asparagine to an aspartic acid at position
276. The substitutions at position 69 have previously been shown to be
responsible for the resistance to clavulanic acid, and they are the
only mutations encountered in TEM-33 (IRT-5) and TEM-34 (IRT-6).
However, the N276D substitution has never been found alone in
inhibitor-resistant
-lactamases, and its role in resistance to
clavulanic acid was thus unclear. The N276D mutant was constructed,
purified, and kinetically characterized. It was shown that the
substitution has a direct effect on substrate affinities and leads to
slightly decreased catalytic efficiencies and that clavulanic acid
becomes a poor substrate of the enzyme. Electrospray mass spectrometry
demonstrated the simultaneous presence of free and inhibited enzymes
after incubation with clavulanic acid and showed that a cleaved moiety
of clavulanic acid leads to the formation of the major inactive
complex. The kinetic properties of the N276D mutant could be linked to
a salt-bridge interaction of aspartic acid 276 with arginine 244 that
alters the electrostatic properties in the substrate binding area.
-lactamases (EC 3.5.2.6) is the major
mechanism used by Gram-negative bacteria to develop resistance to
-lactam antibiotics, and TEM-1 is the most currently encountered
plasmid-mediated
-lactamase within this group. This enzyme, which
efficiently hydrolyzes penicillins and first and second generation
cephalosporins, is not active against third generation cephalosporins.
However, it is able to evolve and rapidly confer bacterial resistance
to the newer
-lactam drugs, such as cefotaxime and ceftazidime
(Paul et al., 1988; Sougakoff et al., 1989). This new
hydrolysis ability results from one to four substitutions in the
vicinity of the active site of TEM-1
-lactamase (Jacoby and
Medeiros, 1991).
-lactamases that are
resistant to these inhibitors, and several inhibitor-resistant TEM
-lactamases (or IRT (
)
-lactamases) have been
characterized in hospital strains since 1992 (Thomson and Amyes, 1992).
Few amino acid mutations are involved in inhibitor resistance, but they
differ from those encountered in extended spectrum
-lactamases (Table 1): a leucine, an isoleucine, or a valine instead of
methionine at position 69; a serine or a cysteine instead of arginine
244; an arginine instead of tryptophane 165; a threonine for a
methionine at position 182; and, more recently, an asparagine for an
aspartic acid at position 276 (N276D) or an arginine for a leucine at
position 275 (Henquell et al., 1995).
-lactamase was constructed by
site-directed mutagenesis and studied for its ability to hydrolyze
penicillins and cephalosporins and for the inhibitory effect of
clavulanic acid.
Site-directed Mutagenesis
The following
oligonucleotides were synthesized on an Applied Biosystem 38A
synthesizer. They were phosphorylated as described by Normanly et
al.(1986) (N276Am, 5`-GATCTGTCTCTATCGTTCATC-3`; N276D,
5`-GATCTGTCTATCTCGTTCATC-3`). The mutations were introduced by
Kunkel's method(1985) on the bla gene, which encodes
-lactamase TEM-1, in the plasmid pCT-1 (Lenfant et al.,
1991). The single-stranded wild type pCT-1 DNA was isolated from strain
BW313, after infection by the helper phage M13K07 (Pharmacia). The
N276am mutant colonies were selected for their loss of resistance to
ampicillin. Missense revertant N276D was obtained in the same manner
from the amber-mutated gene, and the mutant colonies were then selected
for their resistance to ampicillin. The mutations were confirmed by
sequencing by the dideoxy method (Sanger et al., 1977) with
the Pharmacia T7 polymerase sequencing kit.
S-dCTP was
provided by Amersham Corp.
Informational Suppression
This method and the Escherichia coli K12 strains and plasmids have already been
described by Lenfant et al.(1990). It allows us to obtain 15
variants of -lactamase TEM-1 by introducing an amber mutation at
position 276 of the
-lactamase gene by site-directed mutagenesis
and transforming a set of suppressor strains. The variants obtained by
suppression were assayed for growth in the presence of various
antibiotics at 30 °C. The disc agar diffusion method was used
(Lenfant et al., 1990).
The
genes coding for wild type -Lactamase Expression and Purification
-lactamase or the N276D mutant were
cloned into plasmid pCT3, a pCT1 derivative with a stronger promoter
(Lenfant et al., 1990). All enzymes for genetic engineering
were obtained from Pharmacia LKB Biotechnology Inc. E. coli strain XAC-1 (Normanly et al., 1986) was used to produce
TEM-1
-lactamase and the N276D mutant. This strain was
electrotransformed (Dower et al., 1988) with the pCT-3
plasmids.
, 0.2% glycerol, and 100
µg/ml ampicillin (Sigma). Cells were harvested by centrifugation
during the exponential phase (A
= 2)
at 5,000
g for 10 min and washed with 200 ml of 200
mM Tris-HCl, pH 8. The cell pellet was resuspended to 60 A
with 200 mM Tris-HCl, pH 8. One
volume of 1 M sucrose in 200 mM Tris-HCl, pH 8, 0.1
volume of 100 mM EDTA, pH 7.6, and 30 mg of lysozyme were
added. The suspension was incubated for 20 min at 4 °C, and 0.2
volume of 0.5 M MgCl
was added. The periplasmic
fluid was collected by centrifugation at 18,000 g for 20 min and
dialyzed against 200 mM Tris-HCl, pH 8, to eliminate sucrose.
-lactamase was eluted
with 20 mM Tris-HCl, pH 7.5, and extensively dialyzed against
the same buffer. The protein extract was chromatographed on a Pharmacia
Mono Q HR5/5 anion exchanger column. The proteins were eluted with a
0-0.2 M linear NaCl gradient in 20 mM Tris-HCl,
pH 7.5, at a flow rate of 0.5 ml/min. Active fractions detected with
nitrocephin (Oxoid, Batingstoke, Hampshire, UK) were found to be
homogeneous as judged by analytical SDS Phastgel (Pharmacia) and silver
staining. Protein concentration was determined according to the
Bradford method (Bradford, 1976).
Determination of Kinetic Parameters of Substrate
Hydrolysis
The Michaelis-Menten constants k and K
were determined at 37 °C
in 50 mM sodium phosphate buffer, pH 7. The kinetic
measurements were carried out spectrophotometrically. The cleavage of
the
-lactam ring of antibiotics was monitored with an UVIKON 930
(Kontron). The wavelengths for each antibiotic and the corresponding
values of
calculated according to Samuni(1975) are presented
in Table 2. The catalytic efficiency is defined as the ratio k
/K
.
Determination of Inhibition Kinetic
Parameters
These parameters were determined
spectrophotometrically at 37 °C in 50 mM sodium phosphate
buffer, pH 7. K was determined from
competition procedures with a good substrate, amoxicillin. The rate
constant of irreversible inactivation by clavulanic acid, k
, was determined by incubating the inhibitor
in saturating concentration with the enzyme for various times. A large
excess of amoxicillin was then added, and the remaining activity was
measured. The half-time (t
) is the time
necessary to inhibit 50% of the enzymatic activity; it allows
calculation of the k
value: k
=ln2/t
(Kitz and Wilson, 1962). To
measure the turnover (t
) of the enzyme
for clavulanic acid, the inhibitor and the enzyme were incubated at
different molar ratios for 30 min at 37 °C, and the residual
activity was measured. The turnover value was deduced from the
extrapolated value for 100% inactivation from the plot of the residual
activity versus the inhibitor-to-enzyme ratio. The inhibition
efficiency is defined as k
/K
(for details, see Delaire et al., 1992).
Visualization of Free and Complexed Enzymes by
Electrospray Mass Spectrometry
Analyses were performed using a
TRIO 2000 quadrupole mass spectrometer (VG Biotech, Altrincham, UK)
equipped with an electrospray source. Inhibition reactions were carried
out at 37 °C by mixing 10 µl of the enzyme solution (1 mg/ml in
10 mM ammonium bicarbonate, pH 7) with 3 µl of the
clavulanic acid solution (18 mM in water). After 3 h of
incubation, reaction mixtures were directly introduced into the
electrospray source via a 10-µl injection loop at 15 µl/min
using a 50:48:2 (v/v/v) mixture of acetonitrile:water:formic acid
delivered by a syringe pump (140 A Solvent Delivery System, Applied
Biosystems, Foster City, CA). Mass spectra were acquired in
multichannel analyzer mode at a scan rate of 15 s/scan.Enzyme Coordinates and Electrostatic
Calculations
The atomic coordinates of TEM-1 were from the
1.8-Å resolution refined x-ray structure (Jelsch et al.,
1993). The structure includes 199 water molecules and 1 sulfate ion
provided by the crystallization conditions. This ion has no biological
significance and was discarded from the calculations as well as 33
water molecules that do not exchange hydrogen bonds to protein atoms.
All other water molecules were considered as part of the protein and
have an average temperature factor of 14.6 Å. The
N276D mutant was modelled from the structure of the wild type enzyme
with the program O (Jones et al., 1991). Polar hydrogen
positions were built and energy was minimized using the program X-PLOR
(Brnger, 1992). The acyl-enzyme model was
constructed according to the description given by Strynadka et
al.(1992). In this model, five more water molecules were removed
from the active site.
65
65 grid points), gives a resolution of about one grid
point/Å. This was considered insufficient, and the grid was
extended to 129
129
129 points. Two dummy atoms were
introduced to control the position of the molecule within the grid. At
a ``perfil'' of 100%, these dummy atoms are located at
opposite ends of the diagonal of the cubic grid in such a way that the
longest dimension of the protein molecule, including its
solvent-accessible surface (computed with a probe radius of 1.4
Å), falls exactly within the grid. The atoms were assigned
Connolly radii provided with the DelPhi package. A 2.0-Å-thick
ion exclusion layer was added, and the ionic strength of the bulk was
set to 145 mM. The protein moiety was assigned a relative
dielectric constant of 3.0, whereas that of the bulk was set to 80.
Atomic charges were taken from the ``toph19.pro'' file in
X-PLOR (Brnger, 1992; Brooks et al.,
1983) and distributed over the grid points according to an algorithm
described by Gilson et al.(1987). This method gives more
accurate potentials at close distances than a traditional distribution
over the eight closest grid points. All residues were kept in their
normal protonation state at pH 7.8. Independence on the chosen grid
origin was verified by repeating the calculations with the geometric
center of the protein plus dummy atoms placed at seven different grid
points (the grid center itself and its six closest neighbors). A
three-step focusing protocol (15, 60, and 100% perfils) was used with
the Debye-Hckel potential of the equivalent dipole
to the molecular charge distribution as a boundary condition in the
initial step (Sharp and Nicholls, 1989). The grid size was 0.52 Å
(1.92 grid points/Å) in the last step.
Hardware
The program O was run on an Evans and
Sutherland ESV/30-33. DelPhi and X-PLOR were run on a Digital DEC
3000/400 Alpha workstation (96 Mb RAM).
Informational Suppression and Antibiotic Disc
Assays
The N276am and N276D mutant bla genes were
obtained by site-directed mutagenesis. The N276am pCT-1 plasmid was
transformed in the 15 different available strains containing the amber
suppressor genes, which introduce 15 different amino acids at position
276. Thus, with only one mutagenesis step, 15 variants of the TEM-1
-lactamase were generated. This plasmid was also transformed in
the XAC-1 strain, which did not express any suppressor gene as a
control. The N276D pCT-3 plasmid was transformed in the XAC-1 strain.
-lactamase. Substitutions of the
asparagine by an arginine, a lysine, a cysteine, an isoleucine, a
leucine, a phenylalanine, a proline, or a tyrosine lead to
-lactamases that are not active enough to confer resistance to
penicillin antibiotics such as amoxicillin or ticarcillin. The other
substitutions, except for glycine and serine substitutions, confer
resistance to penicillins, but the transformed strains are very
sensitive to all cephalosporins. This very low activity of the variant
enzymes can be explained by a poor suppressional context at the
position studied. However, the activity of the N276S and N276G variants
is sufficient to confer resistance to cephalosporins, although the
efficiency of the serine and glycine suppressors is not very high
(Normanly et al., 1990). It thus appears that residue 276
cannot be substituted by most amino acids without a marked decrease of
the hydrolytic activity.
-lactamase against penicillins and first and second generation
cephalosporins, because growth is not inhibited around the antibiotic
discs. The most striking effect of the N276D substitution is the marked
resistance to clavulanic acid combinations. The inhibitor is not
efficient against the N276D
-lactamase and does not inhibit growth
when it is used in combination with amoxicillin or ticarcillin.
Kinetic Characterization of the N276D Mutant
The
N276D mutant was purified to homogeneity and kinetically characterized.
The effect of the substitution on the hydrolysis of substrates is not
drastic (Table 4) with catalytic efficiencies 11.4-50.4% of
those of wild type TEM-1. The K constants
for all the substrates are decreased, but the N276D hydrolyzes
penicillin substrates at faster rates than the wild type. On the
contrary, the hydrolysis rate of cephalosporins is decreased to
19.5-37.2% of that of TEM-1. However, there is no major change in
substrate profile on the ground of the catalytic efficiencies,
indicating that the N276D mutation does not alter the substrate binding
site topology.
, leading to an inhibitory
efficiency of only 4.3% of that of the wild type enzyme. The decrease
in binding constant, combined with a better hydrolysis of clavulanic
acid with a turnover of 250, changes the suicide inhibitor into a poor
substrate.
ESMS Analysis of the Inhibited Enzymes
ESMS allows
the visualization of proteins and their complexes with various
substrates or inhibitors and determination of their molecular masses.
The wild type enzyme (28,947 Da) and the N276D mutant (28,948 Da) were
incubated in the same conditions with clavulanic acid. With an
inhibitor-to-enzyme molar ratio of 150, after 3 h of incubation, all
the molecules of TEM-1 enzyme are complexed (Fig. 1A),
whereas a large amount of the N276D mutant remains free (Fig. 1B) and active against amoxicillin, as shown by
spectrophotometric measurement. For TEM-1 and for the N276D mutant, the
mass of the major complex observed (29,028 ± 8 Da and 29,025
± 13 Da for TEM-1 and the N276D mutant, respectively)
corresponds to the addition of 80 Da to the mass of the free
enzyme. This additional mass is lower than the molecular mass of
clavulanic acid (199 Da), indicating that only a cleaved moiety of the
inhibitor molecule is covalently bound in the major inactivated enzyme
complex. The presence of small peaks at higher masses (Fig. 1)
may be due to minor forms of the enzyme-inhibitor complexes, consistent
with earlier characterization from isoelectric focusing (Charnas et
al., 1978).
Modeling and Electrostatic Calculations
Modeling
of the mutation was straightforward, and energy minimization by X-PLOR
(Brnger, 1992) brought the Arg N
-2 and the Asp
O
-1 atoms 2.7 Å
from each other. The electrostatic potential maps corresponding to the
wild type and to the mutant enzyme were computed using DelPhi, and they
showed a decrease of the positive potential in the binding area of the
penicillin carboxylate group. This translates into a 4.4 kcal/mol
relative decrease of the electrostatic binding energy between this
carboxylate group and the enzyme. Desolvation effects were not taken
into account in these calculations because they could reasonably be
assumed to be of equivalent magnitude in both wild type and N276D
enzymes.
-lactamase has been described by Brun et al.(1994). The sequence analysis of this TEM-1-derived
-lactamase shows two substitutions: a leucine for a methionine at
position 69 and an asparagine for an aspartic acid at position 276. The
mutation at position 69 has already been shown to induce resistance to
clavulanic acid (Zhou et al., 1994), and the natural mutant
enzymes TEM-32, TEM-33, and TEM-34 contain an isoleucine, a leucine, or
a valine at this position, respectively (Table 1). Zhou et
al.(1994) proposed that this substitution was sufficient to
explain clavulanic acid resistance of the TEM-35 enzyme and that the
N276D substitution had no contribution to this phenomenon. The
construction and analysis of the N276D mutant of the TEM-1
-lactamase indicate that this is not the case.
-lactam
antibiotics are identical to those obtained with the TEM-1 expressing
strain. However, this mutation induces clavulanic acid resistance. It
is known that residue 276 is not directly involved in substrate binding
and catalysis, but these results emphasize the fact that it indirectly
influences the progress of catalysis. Interestingly, as this manuscript
was under way, Bonomo et al.(1995) described a N276G mutant in
OHIO-1, another class A
-lactamase that is also highly resistant
to clavulanic acid, whereas our TEM-1-derived N276G mutant seems only
moderately resistant.
-hydroxyl of serine 130
by the iminium group of the acylated intermediate, which results in the
covalent binding of only a part of the inhibitor molecule linked on one
side to serine 70 and on the other side to serine 130. This hypothesis
is consistent with our ESMS results, which demonstrate that only part
of the clavulanic acid molecule is bound to the major inactivated
enzyme complex. The proposed mechanism involves a deprotonation of
Ser
-bound clavulanate via a water molecule. This water
molecule, which is a proton donor in the stepwise sequence of events of
the inactivation process, interacts with the side chain of arginine 244
and with the carboxylate of valine 216 in the wild type enzyme
structure. Positional difference of this water molecule as a
consequence of the N276D mutation, as well as of the R244S mutation
that leads to IRT enzymes, could impair the efficiency of the
inactivation process. This would explain why, under identical
experimental conditions, free N276D mutant enzyme is observed by ESMS,
whereas the wild type enzyme is totally engaged in inactivated
complexes. The fact that the same major molecular complex is observed
by mass spectrometry for both enzymes also suggests that the
inactivation process leading to this species is similar for the wild
type and the N276D mutant. From an evolutionary point of view, it is
interesting to note that the Gram-positive enzyme PC1 is highly
sensitive to clavulanic acid, although it has an aspartic acid at
position 276 (as in Bacillus licheniformis and several other
Gram-positive enzymes). In the Staphylococcus aureus enzyme, a
full clavulanic acid molecule is bound to the active site, thus
suggesting another inhibition mechanism (Chen and Herzberg, 1992).
) and to the increased turnover of
clavulanic acid (Table 5), which then behaves as a poor substrate
instead of as a suicide inhibitor. In the three-dimensional structure
of TEM-1 (Jelsch et al., 1993), asparagine 276 is accessible
to bulk solvent and found at hydrogen bond distance to arginine 244 (Fig. 2). According to the structure and to the kinetic data (Table 4), the N276D mutation was not anticipated to induce
significant structural changes. Indeed, energy minimization of the
modeled mutant enzyme only induces a slight reorientation of the
Arg
and Asp
side chains. The Arg
N
-2 and Asp
O
-1 are now 2.7 Å from
each other, which strongly suggests that a salt-bridge interaction is
formed. The electrostatic potential map computed for the N276D mutant
enzyme showed that the altered charge distribution on the Arg
and Asp
side chains leads to a decrease of the
positive potential in the binding site of the substrate carboxylate
compared with the wild type enzyme. The electrostatic binding energy
provided by this carboxylate, found 2.7 Å from Arg
in the x-ray structure of the acyl-enzyme complex (Strynadka et al., 1992), was calculated to be decreased by 4.4 kcal/mol
in the N276D protein mutant, a value that corresponds to the strength
of this interaction (Hendsch and Tidor, 1994).
N
-2 and the Asp
O
-1 atoms is 2.7 Å. Dotted lines indicate short distance interactions (less than
2.9 Å). The water molecule, found at hydrogen bond distance of
the Arg
N
-2 and main chain Val
oxygen
atoms in the TEM-1 x-ray structure (Jelsch et al., 1993) and
involved in the inactivation process (Imtiaz et al., 1993), is
shown.
for all substrates and in
turn for the decrease of the catalytic efficiencies. The fact that the
decrease of affinity is more marked with penicillins, with a 10-fold
increase in K
for ticarcillin for
instance, than with cephalosporins is consistent with the proposal that
penicillins have a stronger interaction with arginine 244 than
cephalosporins (Zafaralla et al., 1992).
for
penicillin substrates.
-lactamases has never been found alone and is
always associated with the M69L substitution, known to confer
clavulanic acid resistance by itself, it appears that an aspartic acid
at position 276 plays a significant role in the resistance to
clavulanic acid inhibition. The electrostatic interaction of the
aspartic acid with arginine 244 and the possible displacement of the
water molecule involved in the inactivation process would be
responsible for such effects.
We thank Arlette Savagnac for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.