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INTRODUCTION |
-Lactamases are the primary cause of resistance to -lactam
antibiotics (1). These enzymes hydrolyze the -lactam moiety of
-lactam antibiotics, and by so-doing, render them inactive. There
are four classes of these enzymes, of which the class A is the largest
group (1). The active-site serine in the class A -lactamases
undergoes acylation by the substrate and the acyl-enzyme intermediate
is subsequently hydrolyzed to give substrate turnover (1,
2). Class A enzymes perform this task with their preferred substrates, penicillins, at the diffusion limit (3). Many of the
"parental" -lactamases of class A, such as the TEM-1
-lactamase, have undergone mutations that impart to them an increase
in the breadth of their substrate profile (1), as well as the ability to avoid being inhibited by the known clinical inhibitors. This is
currently a serious clinical challenge.
One new class A -lactamase, designated
NMC-A1 (4), and the highly
homologous Sme-1 (5) and IMI-1 (6) -lactamases, enjoy an unusually
broad substrate profile, which includes penicillins, cephalosporins,
and carbapenems (4, 7). Currently, carbapenem antibiotics such as
imipenem are considered antibiotics of last resort, and the advent of
enzymes that turn them over efficiently bodes poorly for the prospects
of continued clinical utility of these versatile antibacterials.
The x-ray structure of the NMC-A enzyme, and its comparison to that of
the classical class A enzyme (8, 9), showed that the carbapenamase
activity of the NMC-A -lactamase could be attributed to the
displacement of Asn-132. The subtle relocation of this residue in the
active site by a mere 1 Å, enlarges the substrate-binding site to
accommodate the 6
-hydroxyethyl substituent of carbapenems, allowing
the turnover process to take place (9). These observations were the
basis for the design of a novel inactivator for the NMC-A -lactamase, namely 6-hydroxypropylpenicillanate (9). The x-ray
structure determination of the complex of the NMC-A -lactamase and
the inhibitor illustrated that inactivation of the enzyme arose from
interactions between the protein and inhibitor that prevented the
approach of the hydrolytic water to the ester of the acyl-enzyme
intermediate (9).
We disclose herein inhibition of the NMC-A -lactamase by a set of
monobactam inhibitors. These are effective inactivators for this
enzyme, and their mechanism of action is distinct compared with that of
6-hydroxypropylpenicillanate, which was reported earlier (9). The
mechanistic implications of interactions of the NMC-A -lactamase and
one of the inhibitors of our design are addressed by the determination
of the crystal structure of the complex, as well as by computational
molecular dynamics simulations.
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EXPERIMENTAL PROCEDURES |
The NMC-A -lactamase was purified to homogeneity according to
a literature procedure (8). Cephaloridine was purchased from Sigma.
Syntheses of compounds 1 and 6 were reported
previously (10), and those for the remainder of the inhibitors are
given in the Supplementary Material available in the on-line version.
Spectrophotometric studies were performed on a Hewlett-Packard 8453 diode array instrument. Calculations were performed by the MS Excel program.
Kinetic Experiments--
A 1.0-ml assay mixture typically
consisted of 0.5 mM cephaloridine in 100 mM
sodium phosphate, pH 7.0. Hydrolysis of the -lactam ring of
cephaloridine was monitored at 290 nm (
290 = 2070 M
1 cm1) upon addition of the
enzyme (final concentration was typically 5 nM).
Inactivation experiments were performed as follows. An aliquot of the
stock solution of the inactivator (100 µM in
p-dioxane) was added to the NMC-A -lactamase (0.4 µM final concentration) in 100 mM sodium
phosphate, pH 7.0, at 4 °C (10% p-dioxane final). Portions (10 µl) were removed from the mixture at time intervals and
were diluted 100-fold into the assay mixture containing 0.5 mM cephaloridine. The enzyme activity was monitored until
cephaloridine was entirely consumed. The remaining enzyme activity was
calculated from the initial linear portion of the hydrolysis curve.
Rates of hydrolysis of the inactivated acyl-enzyme species
(krec), and the attendant recovery of activity,
were measured under conditions of excess substrate (0.5 mM
cephaloridine) (11). A solution of a given inactivator in
p-dioxane (typically 300 µM final
concentration) was mixed with the NMC-A -lactamase (0.7 µM final concentration). The mixture was incubated (30 min to 6 h, depending on the inhibitor) at room temperature until
residual enzyme activity was less than 2%. A 10-µl portion of this
mixture was added to the solution of cephaloridine (0.5 mM)
in 100 mM sodium phosphate, pH 7.0. Hydrolysis of
cephaloridine was monitored at 290 nm (290 = 2070 M1 cm1). The computation of the
rate constant was performed according to the method of Glick et
al. (12).
Michaelis-Menten parameters for turnover (Km and
kcat) for compounds 1-4
were evaluated by Lineweaver-Burk plots. The concentrations of the
compounds were varied from 2.5 to 15 µM. A portion of the
enzyme was added to a solution of the inhibitor to give a final
concentration of 4 nM for the enzyme in a total volume of
1.0 ml; hydrolysis was monitored at 240 nm (1, 240 = 11,480 M1
cm1; 2, 240 = 6140 M1 cm1; 3, 240 = 12,700 M1
cm1; 4, 240 = 15,100 M1 cm1).
Determination of the X-ray Structure of the Enzyme Inhibited by
Compound 6--
Crystals of NMC-A (8) were soaked in 2 µl of a
freshly prepared solution of the inhibitor containing 10 mM
of 6 in 19% polyethylene glycol 1500, 190 mM
MES, pH 5.25, and 10% (v/v) p-dioxane at 4 °C. After 15 min, crystals were mounted in cryoloops and were flash-frozen in a
stream of nitrogen gas cooled to 120 K. A 1.95-Å data set was
collected on the W32 wiggler beam line at LURE-DCI (Orsay, France),
tuned at a wavelength of 0.97 Å, and equipped with a large MarResearch
imaging plate. The crystal to detector distance was set to 270 mm. A
total of 60 frames (1.5° oscillation per frame and 10-min exposure)
were collected from a single NMC-A crystal. Data were processed with
MOSFLM (13) and CCP4 (14) packages (Table
I). The space group was
P21212 with cell parameters
a = 77.48 Å, b = 52.69 Å,
c = 67.10 Å. There is one molecule per asymmetric
unit. The structure was refined with the program X-PLOR, version 3.1 (15), applying a bulk solvent correction. A total of 5% of the
reflections were randomly selected in order to provide a test set for
the Rfree calculations (16). These reflections
were omitted during refinement, but were included in the electron
density map calculations. Models and electron density maps were
displayed with the program TURBO FRODO (17).
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Table I
X-ray diffraction data processing statistics given for the entire
resolution range and for the highest resolution shell
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Molecular replacement calculations, carried out with the AMoRe program
(18) using the 1.64-Å refined NMC-A -lactamase structure (8) as a
model, were performed to account for the 1.2-Å variation of the cell
parameters along the crystallographic a axis. Rigid body
refinement, performed between 8.0 and 3.0 Å (Rfactor = 0.36), was followed by molecular
dynamics refinement in the resolution range 31.2-1.95 Å, using a slow
cooling protocol starting from 3000 K, energy minimization, and
individual thermal factor refinement. Water molecules were added as
neutral oxygen atoms when they appeared as positive peaks above 4.0 
in the (Fobs 
Fcalc) exp(icalc) map in acceptable
hydrogen-bonding geometry. Hereafter, the simulated annealing was
performed from 500 K. The electron density maps revealed two
conformations for the inhibitor covalently bound in the active site to
Ser-70. The starting geometry of the inhibitor was obtained by
optimization using DISCOVER (MSI). The final model of the
enzymeinhibitor complex, composed of all protein atoms with the
exception of the solvent-exposed side chains of three lysine residues,
includes 266 crystallographic water molecules and three molecules of
MES buffer molecules. Alternate conformations were assigned to two side
chains. The average B factors were 11.7 Å2 for protein
atoms (10.5 Å2 and 12.9 Å2 for main chains
and side chains, respectively), 17.2 Å2 for Ser-70
acylated by 6, 23.1 Å2 for solvent atoms, and
58.8 Å2 for MES buffer atoms. The final crystallographic
R and Rfree values were 0.179 and
0.226, respectively.
Molecular Modeling and Dynamics Simulations--
The coordinates
of the native NMC-A -lactamase were obtained by removing the bound
inhibitor from the active site of the enzyme in the x-ray structure.
This native structure was utilized to construct an immediate
acyl-enzyme species for structure 11. The immediate
acyl-enzyme species refers to what would be generated after serine
acylation, whereby the ester carbonyl is ensconced in the oxyanion hole
with strong hydrogen bonds to the backbone amines of Ser-70 and
Ser-237. There is ample structural evidence for the existence of such a
species from our earlier work (19, 20). The model for the immediate
acyl-enzyme intermediate included the crystallographic waters and an
additional box of water molecules up to 8-Å thickness from the surface
of the enzyme. The model was energy-minimized and was utilized for the
molecular dynamics simulations as the starting conformation. Molecular
dynamics were performed using the Amber 5.0 software package (Oxford
Molecular Inc.) (21, 22), at constant pressure using periodic boundary conditions. Calculations were performed on a Silicon Graphics Octane
workstation, and the graphical analysis was performed using the SYBYL
molecular modeling software version 6.4 (23). The complete system was
warmed from 0 to 300 K in steps of 20 K per 5 ps and then
equilibrated at 300 K for 25 ps (a total of 100 ps). Snapshots of the
structures were collected from here on for every 0.2 ps for 128 ps, and
the resulting structures were analyzed.
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RESULTS AND DISCUSSION |
Kinetics--
Compounds 1-7 (see Scheme 1)
were tested for inhibition of the NMC-A
-lactamase. It was demonstrated (Table
II) that some of the compounds of this
group indeed inhibited the NMC-A -lactamase, such as we had reported
for the TEM-1 -lactamase previously (10). Although the behavior of
the two enzymes in inhibition by the monobactams shared some features,
there were significant differences. Surprisingly, compound
1, which is an excellent inhibitor of the TEM-1
-lactamase, failed to demonstrate any inhibition of the NMC-A
enzyme, but appears to be a good substrate
(kcat/Km = (5.7 ± 0.4) × 106 M1
s1). Compound 6 inhibits the TEM-1
-lactamase poorly (10). In contrast, compound 6 gave
rapid and irreversible loss of activity in the case of the NMC-A
enzyme. We could not detect any recovery of activity from inhibition by
this compound for which a rate constant could be measured (Table II).
Hence, the inhibited enzyme was used in analysis of structure by x-ray
diffraction (see below). Compound 3 is among the best
inhibitors of the TEM-1 -lactamase in terms of the rapidity of the
inactivation process, as well as the stability of inhibited species.
The NMC-A enzyme hydrolyzes this compound quite efficiently, although
some transient inhibition was observed. Finally, compound 7 demonstrated a biphasic pattern of inactivation, similarly to the case
of the TEM-1 enzyme (24).
X-ray Structure Determination and Refinement--
The structure of
the enzyme in the inhibited complex is identical to that of the native
protein (8). A global superimposition of both structures based on all
atoms led to a rms deviation of 0.17 Å.
Based on the first mechanistic report on this type of enzyme
inactivator (10), it is expected that Ser-70 acylation by inhibitor 6 would give rise to species 8, which would
eliminate the tosyl group to arrive at species 9 (see Scheme
2). The iminium species 9 may
tautomerize to enamine 10. In turn, 9 may undergo
hydrolysis of its iminium group to give the keto derivative
11, which also may exist in its tautomeric form
12.
The x-ray structure revealed that the catalytic Ser-70 is acylated by
the inhibitor, and two different binding modes of the same molecular
species were observed. The electron density indicated that the atoms of
the bound inhibitor were not in the same plane, an observation that
excludes structures 10 and 12. The structural
information is consistent with either the iminium species 9 or the keto species 11 as the entity resulting in enzyme
inhibition. However, we are inclined to favor the keto derivative
11 as the enzyme-bound species. We conclude so because the
imine moiety in 9 is fully exposed to the aqueous medium as
seen in the x-ray crystal structure, and it is likely to undergo
hydrolysis readily in the absence of any specific interactions with the
enzyme active site that would stabilize the iminium group. The
identical position for the hydroxyl group of Ser-130 in the structures
of the native enzyme and in the inhibited complex is suggestive of a
noncharged group, such as the ketone moiety of 11 in its vicinity.
As indicated earlier, the electron density is consistent with two bound
conformations for the inhibitor in the active site (Fig.
1). In conformation I (Fig. 1,
A and C), the oxygen atom of the ester is located
at 2.6 Å and 3.2 Å from the main chain nitrogen atoms of residues 70 and 237, respectively. The oxygen atom of the ketone group of
11 forms a hydrogen bond to the side chain of Ser-130 (2.7 Å). In conformation II of the bound inhibitor, the carbonyl oxygen
atom of the ester is at hydrogen-bonding distance to the hydroxyl group
of Ser-130 (3.0 Å), and the oxygen of the ketone is hydrogen-bonded to
the N
2 atom of Asn-132 (Fig. 1, B and C). The
water molecule implicated in the deacylation reaction is found in a
similar position as in the native enzyme structure. It has a full
occupancy according to the refinement criteria. However, the water
molecule does not seem to be in a good position to attack the ester
moiety, in either conformation observed in the x-ray crystal structure.
Conformation I of the complex is not positioned ideally within the
oxyanion hole, such as documented previously for other acyl-enzyme
intermediates (9, 20). So, despite the ester carbonyl being still
sequestered in the oxyanion hole, the hydrolytic water is held almost
within the plane formed by the ester moiety in the active site. In
other words, the angle of attack for the activated water (160° in
conformation I, at a distance of 3.2 Å) is not favorable (Fig.
1A). In conformation II, the ester carbonyl moiety is not
held in the oxyanion hole, and also the angle for attack of the
hydrolytic water is 148° at a distance of 3.3 Å (Fig.
1B). Thus, in neither conformation does the acylated enzyme
species appear to be in a position to undergo hydrolysis by the
promoted hydrolytic water. Furthermore, the occupancy of the energy
minima represented by conformations I and II of the inhibited enzyme
species must be high enough so that high resolution x-ray structure of
the complex could be solved and that the inhibited species does not
undergo hydrolysis allowing for the recovery of activity.
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Fig. 1.
Stereo views of the x-ray structure of the
NMC-A -lactamase inhibited by the monobactam
6. Electron density maps of the acyl-enzyme intermediate (species
11) in conformation I (A) and in conformation II
(B). In B, the oxyanion hole is occupied by a
water molecule ("W503"), shown here as a
sphere. In A and B, the inhibitor
molecule is shown in the ball-and-stick representation, and
the hydrolytic water molecule ("W520") is shown as a
sphere. C, schematic of the two different
conformations for species 11.
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Molecular Dynamics Simulations--
Recently we performed
molecular dynamics simulations to understand the dynamic nature of the
acyl-enzyme intermediate for imipenem, a carbapenem antibiotic, bound
to the active site of TEM-1 -lactamase (19). These studies revealed
that the ester carbonyl for the immediate acyl-enzyme intermediate for
the TEM-1 -lactamase moved out of the oxyanion hole in the
picosecond time scale. Furthermore, the ester carbonyl was capable of
returning into the oxyanion hole in a time-dependent
manner. We had proposed in our report that the simulations demonstrated
that the acyl-enzyme complexes of the -lactamases are not rigid
structures and would undergo dynamic motion in and out of the active
site oxyanion hole. Experimental demonstration of such a motion had not
been made prior to this work, as the alternative conformation needs to
be relatively stable to survive and be detected by various means, in
our case by x-ray structure determination. The existence of the two
conformations for the inhibitor 6 bound in the active site
of the NMC-A -lactamase illustrates the motion for the ester
carbonyl in and out of the oxyanion hole. It underscores the importance
of the dynamic aspect of these intermediary species to the catalytic
processes of -lactamases.
Molecular dynamics simulations were performed to better understand the
nature of the motions of the inhibited species in the active site of
the NMC-A -lactamase and to explain the observation of the two
conformations in the x-ray structure analyses. These simulations
started from the structure of the so-called immediate acyl-enzyme
intermediate, which is the direct product of active site serine
acylation of the enzyme by the -lactam entity.
The distances of the ester carbonyl oxygen from the backbone nitrogen
of Ser-70 and that of Ser-237 are plotted as a function of time (in
picoseconds) in Fig. 2A.
During the initial 135 ps of the simulations (including the warm-up and
equilibration periods), we observed that the hydrogen bond distances in
the oxyanion hole are maintained around 3 Å. At 133 ps, the hydrogen
bond between the carbonyl oxygen and the backbone nitrogen of Ser-70
broke (dark line in Fig. 2A). However, this
hydrogen bond formed again at 187 ps. The other hydrogen bond, that
between the carbonyl group of the ester moiety and the backbone
nitrogen of Ser-237 (gray line in Fig. 2A) broke and
reformed several times from 136-187 ps of the simulation period. At
approximately 187 ps, both hydrogen bonds were reformed, and the
conformation of the complex at this point was similar to that of the
immediate acyl-enzyme species (the initial starting conformation for
simulations). However, at approximately 203 ps of simulations
(i.e. 16 ps subsequent to its re-entry into the oxyanion
hole) the ester moiety showed motion out of the oxyanion hole. Both
hydrogen bonds with the oxyanion hole broke and reformed several times
from this point on to the end of the simulations.
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Fig. 2.
A, the distance versus time profiles for
the molecular dynamics simulations of the acyl-enzyme species of the
complex of compound 6 and the NMC-A -lactamase. The
black and gray lines show the distances between
the acyl carbonyl oxygen and the backbone amine of Ser-70 and that of
Ser-237, respectively. B, the conformational space sampled
by the C -C -O-C torsion angle in the
enzyme-inhibitor complex is shown as a function of time.
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We analyzed the structures of the snapshots that were collected during
the simulations, and the conformational space sampled by the
C-C-O-C torsion angle in the
enzyme-inhibitor complex was plotted as a function of time (Fig.
2B; the "C-C-O" portion
belongs to Ser-70 and the carbon attached to oxygen is that of the
inhibitor; see Fig. 1C). This torsion angle determines the
orientation of the ester moiety in the active site of the enzyme.
During the simulations, the above torsion angle assumes values close to
140° (for example, at 140 ps in Fig. 2B), where both the
oxyanion hole hydrogen bonds are lost. This geometry is close to
conformation II that was observed experimentally in the x-ray structure
(C-C-O-C torsion angle 142° in Fig. 1C). The other experimentally observed conformation
(conformation I) possesses a C-C-O-C
torsion angle of 73°, different than that for the immediate
acyl-enzyme species (93°; the starting point for simulations), which
should be prone to deacylation. Interestingly, we found that the
torsion angle corresponding to that for conformation I is sampled for a
reasonable duration of the simulation (Fig. 2B). We observe
in Fig. 2B that the ester moiety assumes various positions
in the active site, including the ones in the oxyanion hole. The two
conformations that were observed in the x-ray analysis can be
rationalized as two highly populated conformations of the acyl-enzyme
species. Any conceivable hydrolysis of these species depends on the
stability of the conformation(s) with ester oxygen in the oxyanion
hole, as well as the proper orientation for attack by the activated
hydrolytic water (see above). Consistent with the x-ray structure and
the dynamics simulations, which refute the possibility for deacylation
of the inhibited species, we did not see any detectable deacylation in
kinetics experiments either.
We have demonstrated in this manuscript that structurally simple
monobactam molecules can serve as very effective inhibitors for the
broad spectrum NMC-A -lactamase, and the x-ray structure of the
inhibited enzyme has shed light on the mechanism of inhibition. It is
interesting to note that the mere presence of a small portion of the
inhibitor in covalent liaison with the active site serine allows the
existence of two low-energy species, which do not undergo deacylation
with the attendant recovery of enzyme activity. Molecular dynamics
simulations have underscored the existence of considerable structural
flexibility for the inhibited enzyme species, a process that allowed
for the observations of the two species seen in the x-ray structure of
the inhibited enzyme as important entities in the course of
simulations. This structural dynamic nature is probably present in
other acyl-enzyme intermediates for -lactamases such as recently
documented for another -lactamase (25). Since simulations indicate
the possibility of such motion in picosecond time scale and the fact
that catalysis by these enzymes take place on millisecond time scale,
it is likely that such dynamic motion takes place with typical
substrates for these enzymes as well. However, structural properties of
substrates would prevent their acyl-enzyme species from being trapped
in energy minima that would result in enzyme inhibition, such as
demonstrated here in our report.
-Lactamase from Enterobacter cloacae by
Monocyclic 
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ABSTRACT
INTRODUCTION
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