(Received for publication, July 31, 1995; and in revised form, January 16, 1996)
From the
The E166Y and the E166Y/R164S TEM-1 -lactamase mutant
enzymes display extended spectrum substrate specificities. Electrospray
mass spectrometry demonstrates that, with penicillin G as substrate,
the rate-limiting step in catalysis is the hydrolysis of the E166Y
acyl-enzyme complex. Comparison of the 1.8-Å resolution x-ray
structures of the wild-type and of the E166Y mutant enzymes shows that
the binding of cephalosporin substrates is improved, in the mutant
enzyme, by the enlargement of the substrate binding site. This
enlargement is due to the rigid body displacement of 60 residues driven
by the movement of the
-loop. These structural observations
strongly suggest that the link between the position of the
-loop
and that of helix H5, plays a central role in the structural events
leading to extended spectrum TEM-related enzymes. The increased
-loop flexibility caused by the R164S mutation, which is found in
several natural mutant TEM enzymes, may lead to similar structural
effects. Comparison of the kinetic data of the E166Y, E166Y/R164S, and
R164S mutant enzymes supports this hypothesis.
Bacterial resistance to penicillins and cephalosporins
represents an increasing risk in the chemotherapy of Gram-negative
bacterial infections. This resistance often arises from the emergence
and the dissemination of the plasmid-encoded extended spectrum
-lactamases (EC 3.5.2.6) (Blazquez et al., 1995; Palzkill et al., 1995; Venkatachalam et al., 1994; Viadiu et al., 1995). Most of these proteins are derived from the Escherichia coli TEM enzyme, via the combination of a few
point mutations, which have led, so far, to 27 TEM-related enzymes
(Morosini et al., 1995). The parent TEM-1 is a very efficient
enzyme and hydrolyzes penicillin substrates through an acylation and a
deacylation step (Swarén et al., 1995).
Extended spectrum TEM-related enzymes hydrolyze third generation
cephalosporin substrates. Based on the kinetic data, two common
characteristics of these enzymes are (i) a severalfold reduction in the
catalytic turnover toward penicillins, and (ii) an increase in the
catalytic efficiency against cephalosporin substrates (Raquet et
al., 1994; Sowek et al., 1991).
At the molecular
level, it appears that high catalytic efficiencies against the third
generation cephalosporin oxyimino--lactams, such as cefotaxime and
ceftazidime, are primarily linked, in natural TEM mutants, to the
mutation of Arg
(Naumovski et al., 1992) or to
the mutation of Gly
(Jacoby et al., 1991). In
the x-ray structure of the TEM-1 enzyme (Brookhaven DataBank entry
1BTL) (Jelsch et al., 1993), Arg
forms two salt
bridge interactions. These are important for the conformation and the
stability of the
-loop region (residues 161-180). Some
residues from this loop define the active site topology, while others,
such as Glu
, are essential for catalysis. Site-directed
mutagenesis studies demonstrated the requirement for an acidic side
chain in position 166 (Delaire et al., 1991) and the
involvement of Glu
in the deacylation reaction (Adachi et al., 1991). Both the acid-base properties and the position
of this proton acceptor group are reflected in the value of the
deacylation rate constant (Swarén et
al., 1995).
Gly resides at the C-terminal edge of
strand S3, which borders the substrate binding site cavity, and is in
van der Waals contact with Asn
main-chain atoms in the
-loop. Detailed kinetic and mass spectrometric investigations on
the G238S mutant enzyme demonstrated a significant decrease of the
deacylation rate constant, likely related to a perturbation of the
deacylation machinery (Saves et al., 1995a).
The E166Y mutant displays one of the characteristic features of extended spectrum enzymes: similar activities toward penicillin and cephalosporin substrates (Delaire et al., 1991). The high resolution structure of this mutant shows unexpected structural differences compared with the wild-type enzyme. Comparison of the kinetic data of the E166Y, E166Y/R164S, and R164S (Sowek et al., 1991; Raquet et al., 1994) mutant enzymes suggests that these structural differences may play a key role in extending the substrate specificity of the TEM-related enzymes.
The overall structure of the E166Y -lactamase is very similar
to the 1.8-Å wild-type enzyme structure (Jelsch et al.,
1993). However, the E166Y mutation leads to significant structural
differences in two areas: the region 165-170 (WYPELN), which is
part of the
-loop, and residues 85-142, which move as a
rigid body. This region contains helices H3(109-111),
H4(119-128), and H5(132-142) and two coil regions
(86-108 and 112-118). It is part of the helical domain
(residues 62-212) of class A
-lactamases (secondary
structure assignment from Jelsch et al.(1993)).
Figure 1:
A, stereo view of
residues 70-73 and 165-170 in the wild-type (thin
lines) and E166Y (thick lines) structures. B,
stereo view of the substrate binding sites in the wild-type (thin
lines) and E166Y (thick lines) structures. The
crystallographic water molecules in the TEM-1 -lactamase are
represented by crosses. Those in the E166Y enzyme are
represented by dots. The location of the sulfate ion is also
represented. C, stereo view of the H5 helix (residues
132-142) and of residues 165-166 of the
-loop. The
hydrogen bonds between the Asn
side-chain atoms and the
main-chain atoms of residue 166, and between Thr
O-
and Trp
N
are shown by dotted lines. The
wild type is in thin lines, and the E166Y enzyme is in thick lines. The shifts in position for 166 C-
, 136
C-
and 140 O-
are, respectively, of 0.7, 0.4, and 0.6
Å.
These structural modifications within the
-loop affect the solvatation and the hydrogen bond network within
the substrate binding site. Four water molecules are lost, and two
water molecules and the sulfate ion are shifted compared with the
wild-type enzyme (Fig. 1B). The water molecule Wat297,
considered to be the nucleophile group in the deacylation step
(Strynadka et al., 1992; Jelsch et al., 1993,
Swarén et al., 1995), is shifted by 1.5
Å but maintains hydrogen bonds to Ser
nitrogen,
Ser
O-
, Asn
side-chain amide group, and
Wat391. The 1-Å motion of the sulfate ion has several
consequences: (i) it leads to the exclusion of Wat323 from the oxyanion
hole, and of Wat404, found in the vicinity of Ser
main-chain oxygen atom in the wild type structure, (ii) one of
its oxygen atoms is now at hydrogen bond distance (2.9 Å) from
Ala
nitrogen, and (iii) it provides more space in the
vicinity of the Ser
, Ser
, and Arg
side chains in the E166Y structure, where a new water molecule
(Wat518) is found. All other water molecules occupy nearly the same
positions in both structures (Fig. 1B). Solvent
molecules that are bound to residues of the
-loop move
accordingly and preserve their interactions, except for the two water
molecules (Wat422 and Wat472) that are expelled by the previously
described Asn
side-chain motion. However, the 1.0-Å
displacement of Pro
generates a cavity that is filled by
a new water molecule.
At the edges of
this moving domain, residues Arg and Thr
, at
the C termini of helices H2 and H5, respectively, and Glu
and Arg
, in the connecting loop between helices H2
and H3, are clustered within a 4-Å radius sphere (Fig. 2).
The movement of residue 141 induces the reorientation of the Arg
side chain and the expulsion of Wat380, which was bridging
Arg
N
-1, Glu
O
-1, and Thr
O-
in the wild-type structure. In the mutant enzyme,
Arg
N
-1 is now hydrogen-bonded to the Thr
main-chain oxygen atom.
Figure 2:
Stereo representation of the interactions
in the wild-type enzyme (thin and dotted lines) and
in the E166Y mutant enzyme (thick and hatched lines).
Arg and Thr
are at the C termini of helices
H2 and H5, respectively, and Glu
and Arg
are
in the loop connecting H2 and H3. Wat380 in the wild-type enzyme is
shown as a dot.
Figure 3:
A, electrospray mass spectra of the
reaction mixture of E166Y and penicillin G at 37 °C after 5 min of
reaction. The measured molecular mass from this peak distribution is
29,318 ± 3 Da, which corresponds to the covalent acyl-enzyme
E166Y/penicillin G intermediate. B, singly charged ions are
formed from low molecular mass molecules. Peaks labeled PG and PG + HO correspond
to protonated penicillin G (substrate) and to protonated penicilloic
acid (reaction product), respectively. The peak at m/z 309
corresponds to the decarboxylated (loss of 44 Da) penicilloic
acid.
The kinetic data of the E166Y
and the E166Y/R164S mutant enzymes are reported in Table 3. The
single E166Y mutation led to improved K values for
all substrates compared with the wild-type enzyme. However, compared
with the E166Y enzyme, the E166Y/R164S double mutant enzyme
discriminates between penicillin substrates (decreased K
values) and cephalosporin substrates (increased K
values). When compared with the wild-type enzyme, mutant proteins
bearing the E166Y mutation display highly reduced k
values, although they are higher for cephalosporin than for
penicillin substrates. These effects in K
and k
led to modified substrate spectra in both
mutant enzymes, a characteristic of extended spectrum TEM-related
enzymes, as exemplified by the R164S TEM-1 mutant (Table 3)
(Sowek et al., 1991; Raquet et al., 1994). Taken
together, these data suggest that the extended substrate spectra of
these mutants arise from structural events common to all of these
proteins.
The short range effect on the 165-170
-loop region is restricted by the preceding Arg
and
the following Glu
. Their side chains are engaged in two
salt bridges that are important for the conformation and the stability
of the
-loop and for the correct location of residue 166 within
the active site. The salt bridge between Arg
and
Asp
is buried and inaccessible to solvent molecules,
which increases the strength of this interaction.
Three hydrogen
bonds are exchanged between the Asn side-chain and
Glu
main-chain atoms and between Thr
O-
and Trp
N-
. Thus, the movement of the
165-170 region leads to the concerted relocation of
Asn
, Asn
, and Thr
(Fig. 1C). This rigid body motion of the H5 helix
drives the displacement of the whole 85-142 region, which
represents one-third of the enzyme helical domain (Fig. 4). This
movement requires only a few small main-chain dihedral angle rotations
and preserves all of the interactions that occur within this protein
domain.
Figure 4:
Stereo
view of the C- chain tracing of the E166Y protein structure. The
regions that move relative to the wild-type enzyme are in thick
lines. The bottleneck of the substrate binding site (between
Ser
O-
and Ser
O-
) is indicated
as well as penicillin G bound to the TEM-1 enzyme (from
Swarén et al.,
1995).
The 85-142 region bears residues that delineate one
side (residues 104-105, 130, 132) of the substrate binding
cavity. The active site bottleneck is found between the hydroxyl groups
of Ser and Ser
, and is precisely the
binding site of the thiazolidine ring of penicillins and of the
dihydrothiazine ring of cephalosporins (Fig. 4). The distance
between Ser
O-
and Ser
O-
is 5.4
Å in TEM-1 (Jelsch et al., 1993). The movement of
residues 85-142 in the E166Y enzyme increases this distance to
6.0 Å. As the K
of cefotaxime, measured by
competition procedures, is lowered 1500-fold compared with TEM-1
(Delaire et al., 1991), we suggest that the increased active
site aperture favors the better binding of cephalosporins. This is
because the dihydrothiazine ring has a larger steric volume than the
thiazolidine ring.
Within experimental errors, k is identical to
the k
value determined from steady-state kinetic
measurements, which prevents calculation of k
.
However, examination of the k
/K
values suggests slower acylation rates with the E166Y mutant
enzymes compared with the wild-type enzyme. The role of Lys
in the acylation reaction was recently described
(Swarén et al., 1995). Removal of the
negative charge provided by Glu
significantly decreases
the basicity of the unprotonated Lys
in the E166Y
Michaelis complex, in line with the decrease of k
suggested by the steady-state kinetic data (detailed
electrostatic calculations will be presented elsewhere).
ESMS
experiments using cephaloridin showed that there is no elimination of
the C-3` substituent of the substrate forming the acyl-enzyme complex.
Thus, the lower K values with the E166Y mutant
protein for cephalosporin substrates do not arise from a change in the
rate-limiting step resulting from a different kinetic pathway, as was
shown to occur with the PC1 enzyme (Faraci and Pratt, 1985, 1986). The
rate-limiting step for cephalosporin hydrolysis by the wild-type enzyme
is acylation (Saves et al., 1995a). A similar conclusion could
be drawn for the E166Y mutant, as ESMS shows that more than 90% of the
protein is found as free enzyme during the course of the reaction.
However, the detection of a small amount of acyl-enzyme complex would
suggest that the k
and k
values are of similar magnitude.
In the TEM-1 enzyme, the R164X (except for
lysine) mutation, by removing two out of the four salt bridges of the
-loop, will release some, but not all, of its conformational
constraints. This will likely affect the relative positions of the
partners involved in the deacylation step. A slight displacement of the
-loop residue Glu
will decrease the deacylation
rate of good TEM-1 substrates. Indeed, this rate is related both to the
direction of the electrostatic potential gradient between the
Glu
carboxylate and the ester carbonyl carbon of the
acyl-enzyme complex, and to its magnitude, which is very sensitive to
atomic positional differences (Swarén et
al., 1995). The reduction in k
is of
10-50-fold in the TEM-related enzymes bearing the Arg
mutation (R164S, R164H, R164S/E104K, and R164S/E240K) but is only
marginally affected in the E104K and E240K single mutants (Sowek et
al., 1991, Raquet et al., 1994, Petit et al.,
1995). This proposal is consistent with the situation observed in the
PC1
-lactamase, where a single salt bridge
(Arg
-Asp
) stabilizes the
-loop
conformation. The substantial disorder of the loop resulting from the
D179N mutation (Herzberg et al., 1991), led in that case to a
600-fold decrease in the k
value for penicillin
G.
The movement of the 85-142 region, in the E166Y mutant
enzyme structure, explains why binding is improved for cephalosporin
substrates compared with the wild-type enzyme. A similar movement of
the 85-142 region can be assumed to occur in the E166Y/R164S
mutant protein. Interestingly, the additional R164S mutation in the
E166Y enzyme has no effect with respect to the substrate spectrum (k/K
) for cephalosporin
substrates, whereas the single R164S mutation in TEM-1 led to major
kinetic differences. This paradox is explained if one assumes that the
R164S mutation allows structural perturbations similar to those
observed, and already achieved, in the E166Y protein. It explains why
the additional R164S mutation in the E166Y enzyme is kinetically silent
and offers a structural explanation of the consequences of the
Arg
mutation in the wild-type enzyme.
We propose that
the conformational constraints of the -loop, partly controlled by
residue 164, and the position of the 85-142 region are
interdependent in the TEM-1 enzyme. However, in the R164X TEM-1 enzymes, this structural link would only be kinetically
discernible when large substituents on the substrate molecule reach the
-loop residues, as is the case with third generation
cephalosporin substrates (Raquet et al., 1994). The release of
short contacts, achieved by the
-loop movement that drives the
85-142 region, accounts for the kinetic effects that are
consistently found in extended spectrum TEM-related enzymes bearing the
Arg
mutation. First, the improvement of the binding of
large cephalosporin substrates (i.e. ceftazidime) should
increase the catalytic efficiencies for such molecules relative to
penicillin substrates. k
/K
are, indeed, 1-3 orders of magnitude larger in the R164S
mutant than in the wild-type enzyme (Table 3). Second, the large k
/K
differences found in
TEM-1 within the cephalosporin substrates should level out in the
R164X mutant enzymes. Indeed, the k
/K
of cefaloridin versus ceftazidime is decreased from 3
10
in the TEM-1 enzyme to 33 in the R164S mutant enzyme. This
property is fulfilled in all TEM mutant proteins bearing the R164X mutation (Sowek et al., 1991; Raquet et al.,
1994).
Mutations that occur in the vicinity of the -loop
residues, such as G238S, were shown to drastically reduce the
deacylation rate constant (Saves et al., 1995a). Natural
mutants in this position also display extended substrate spectra, and
work is in progress that will further illustrate the involvement of the
-loop region in the molecular evolution of the TEM-1
-lactamase.