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Antimicrobial Agents and Chemotherapy, April 2001, p. 1254-1262, Vol. 45, No. 4
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.4.1254-1262.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Biochemical Characterization of the FEZ-1
Metallo-
-Lactamase of Legionella gormanii ATCC
33297T Produced in Escherichia
coli
Paola Sandra
Mercuri,1
Fabrice
Bouillenne,1
Letizia
Boschi,2
Josette
Lamotte-Brasseur,1
Gianfranco
Amicosante,3
Bart
Devreese,4
Jozef
van
Beeumen,4
Jean-Marie
Frère,1
Gian Maria
Rossolini,2 and
Moreno
Galleni1,*
Centre d'Ingénierie des
Protéines, Université de Liège, B-4000
Liège,1 and Laboratorium voor
Eiwitbiochemie en Eiwitengineering, University of Gent, B-9000
Gent,4 Belgium, and Dipartimento di
Biologia Molecolare, Sezione di Microbiologia, Università di
Siena, I-53100 Siena, 2 and Dipartimento
di Scienze e Tecnologie Biomediche, Università di L'Aquila,
I-67100 Coppito, L'Aquila,3 Italy
Received 19 July 2000/Returned for modification 28 October
2000/Accepted 2 January 2001
 |
ABSTRACT |
The blaFEZ-1 gene coding for the
metallo-
-lactamase of Legionella (Fluoribacter) gormanii
ATCC 33297T was overexpressed via a T7 expression system in
Escherichia coli BL21(DE3)(pLysS). The product was purified
to homogeneity in two steps with a yield of 53%. The FEZ-1
metallo-
-lactamase exhibited a broad-spectrum activity profile, with
a preference for cephalosporins such as cephalothin, cefuroxime, and
cefotaxime. Monobactams were not hydrolyzed. The
-lactamase was
inhibited by metal chelators. FEZ-1 is a monomeric enzyme with a
molecular mass of 29,440 Da which possesses two zinc-binding sites. Its
zinc content did not vary in the pH range of 5 to 9, but the presence
of zinc ions modified the catalytic efficiency of the enzyme. A model
of the FEZ-1 three-dimensional structure was built.
 |
INTRODUCTION |
Metallo-
-lactamases (class B of
the molecular classification of Ambler [1] or group 3 according to the functional classification of Bush et al.
[6]) constitute a very heterogeneous family. Although
their primary structures exhibit low degrees of sequence isology
(38) (generally less than 43%), their three-dimensional structures show high degrees of similarity (7, 8, 9, 12,
35). All class B
-lactamases share five main characteristics (38): (i) they hydrolyze carbapenem compounds, (ii) they
do not interact with monobactams, (iii) they are inhibited by chelating agents such as dipicolinic acid and 1,10-o-phenanthroline,
(iv) they contain zinc ions as the naturally occurring cation, and (v)
they exhibit two metal-binding sites.
On the basis of structural analyses, these enzymes cluster into three
different groups: subclass B1 contains most known zinc
-lactamases
(for example, BcII from Bacillus cereus 569H
[18], CcrA [also named CfiA] from Bacteroides
fragilis [29], and the plasmid-encoded enzyme IMP-1
found in some isolates of Pseudomonas aeruginosa, Serratia
marcescens, and other gram-negative bacteria [19,
24]), subclass B2 includes the Aeromonas enzymes
(CphA [21], ImiS [37], and CphA2
[28]), and subclass B3 contains the tetrameric L1 enzyme
produced by Stenotrophomonas maltophilia (33,
36) and the Chryseobacterium meningosepticum GOB-1
(2) and the Legionella (Fluoribacter) gormanii
FEZ-1 (4) metallo-
-lactamases.
In the known crystal structures of metallo-
-lactamases, Zn-1 is
tetrahedrally coordinated to three histidines and a water molecule.
When present, Zn-2 interacts with three residues (a cysteine, an
aspartic acid, and a histidine in the case of BcII, IMP-1, and CcrA or
an aspartic acid and two histidines for L1) and two water molecules in
a trigonal pyramid. In the di-zinc form, one water molecule is bridged
between the metal ions.
The enzymes of subclass B1 are monomeric proteins. They possess a
broad-spectrum activity profile (10, 13, 14, 20, 26, 40)
and are inhibited by thiol compounds such as SB25566 (9, 16,
34). Interestingly, the mono- and di-zinc form of the BcII and
CcrA enzymes are nearly equally active. Kinetic and spectroscopic
studies indicated that for both forms a transient noncovalent
intermediate is formed during the hydrolysis of the substrate.
To date, no structure of a subclass B2 enzyme is available. For these
-lactamases, the optimal activity is observed with the mono-zinc
form. The second zinc ion behaves as a noncompetitive inhibitor
(17). Only carbapenems are efficiently hydrolyzed by these
enzymes (13), while all other
-lactams are poor
substrates. In addition, cephamycins and oxacephems behave as poor
inactivators of CphA (14, 27).
The enzymes belonging to subclass B3 can be either monomeric (GOB-1) or
multimeric (L1). Detailed kinetic studies performed on the L1 and
GOB-1 metallo-
-lactamases showed that the enzymes exhibit
broad-spectrum activity profiles (2, 10). In FEZ-1, all
the residues which interact with the zinc ions in L1 are conserved (4), while in GOB-1 the first histidine is replaced by a
glutamine residue (2). Interestingly, preliminary
biochemical studies of the L. gormanii FEZ-1
metallo-
-lactamase reveal a broad-spectrum activity profile, but
with a striking preference for cephalosporin compounds (4,
15).
In the work described here we produced the FEZ-1 metallo-
-lactamase
of L. gormanii ATCC 32197T in Escherichia
coli. The enzyme was purified to homogeneity, and a detailed
kinetic study was performed. The effects of various chelating agents,
pH, and zinc ion concentration on enzyme activity were tested. In
addition, a molecular model of the enzyme structure was built by
knowledge-based modeling methods.
(The results described here were presented in part at the 40th
Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 17 to 20 September 2000.)
 |
MATERIALS AND METHODS |
Antibiotics and other chemicals.
Choramphenicol, ampicillin
(
235 =
820 M
1
cm
1), cephalothin (
260 =
6,500
M
1 cm
1), cephaloridine
(
260 =
100,000 M
1
cm
1), cefoxitin (
260 =
7,000
M
1 cm
1), cefotaxime
(
260 =
7,500 M
1
cm
1), methicillin (
260 =
100
M
1 cm
1), carbenicillin
(
235 =
780 M
1
cm
1), cloxacillin (
260 = +140
M
1 cm
1), EDTA, pyridine-2,6-dicarboxylic
acid (dipicolinic acid), and 1,10-o-phenanthroline were
purchased from Sigma Chemical Co. (St. Louis, Mo.). Kanamycin was
purchased from Merck (Darmstadt, Germany). Isopropyl-
-D-thiogalactopyranoside (IPTG) was purchased
from Eurogentech (Liège, Belgium). Imipenem
(
300 =
9,000 M
1
cm
1) was a gift from Merck Sharp & Dohme Research
Laboratories (Rahway, N.J.). Meropenem (
298 =
6,500 M
1 cm
1) was a gift from ICI
Pharmaceuticals (Macclesfield, England). Biapenem
(
294 =
9,960 M
1
cm
1) and piperacillin (
235 =
820
M
1 cm
1) were gifts from Wyeth Lederle
(Tokyo, Japan). Nitrocefin (
482 = +15,000
M
1 cm
1) was purchased from Unipath Oxoid
(Basingstoke, United Kingdom). Benzylpenicillin
(
235 =
775 M
1 cm
1)
was a gift from Rhône-Poulenc (Paris, France). Cefuroxime
(
260 =
7,600 M
1
cm
1) and ceftazidime (
260 =
9,000
M
1 cm
1) were from Glaxo Group Research
(Greenford, United Kingdom). Temocillin (
235 =
660 M
1 cm
1), ticarcillin
(
235 =
660 M
1
cm
1), 6-aminopenicillanic acid (6-APA;

235 =
690 M
1 cm
1),
and clavulanic acid were gifts from SmithKline Beecham Pharmaceuticals (Brentford, United Kingdom). Cefepime (
260 =
10,000 M
1 cm
1) and aztreonam
(
320 =
700 M
1 cm
1)
were gifts from S.A. Bristol-Meyers Squibb (Brussels, Belgium). Tazobactam (
235 =
1,970 M
1
cm
1) was a gift from Wyeth-Ayerst Laboratories (West
Chester, Pa.). Moxalactam (
260 =
4,000
M
1 cm
1) and cefpodoxime
(
260 =
10,000 M
1
cm
1) were gifts from Sankyo Pharmaceuticals (Tokyo, Japan).
Bacterial strains and vectors.
Plamids pBLL/FEZ-1 and
pET24/FEZ-1 have been described previously (4). E. coli XL1-Blue (Stratagene Inc., La Jolla, Calif.) was used as the
host for recombinant plasmids during construction of the expression
vectors. E. coli BL21(DE3)(pLysS) (Novagen Inc., Madison,
Wis.) was used as the host for the T7-based expression vectors in
overexpression experiments. Plasmid pCR 2.1 (TA Cloning Kit; Invitrogen
BU, NV Leek, The Netherlands) was used to clone the PCR products. The
expression vector pET26b(+) (Novagen Inc.) was used for the
construction of the T7-based expression vector.
Construction of expression vector and preliminary expression
experiments.
The putative position of the signal peptidase
cleavage site in the FEZ-1 pre-
-lactamase was calculated with the
help of SignalP program (23), available under the server
page of the Centre for Biological sequence analysis
(http://www.cbs.dtu.dk).
To allow the removal of the predicted signal peptide amino acid
sequence, the NdeI or NcoI restriction site was
introduced into blaFEZ-1 after the signal peptide nucleotide
sequence. A BamHI restriction site was created after the
stop codon of the metallo-
-lactamase gene in order to eliminate all
unwanted DNA sequence. All these sites were generated by PCR. Primers
NdeILegi (5'-TCACATATGGCATATCCAATGCCAAATCCTTTTCCC-3') and
BamHILegi
(5'-CTGGGATCCTGAACAATTAGGCAGTTTCTTCTT-3') or primers NcoILegi
(5'-CAACCATGGCATATCCAATGCCAAATCCTTTTCCC-3') and BamHILegiwere the two
oligonucleotide primer pairs used for this purpose (newly introduced
restriction sites are underlined).
PCR conditions were as follows: incubation at 95°C for 5 min and 30 cycles of amplification (denaturation at 95°C for 1 min,
annealing at
55°C for 1 min, and extension at 72°C for 1 min).
The
tth DNA polymerase (Eurogentec, Seraing, Belgium) was used
for PCR. The PCR products (790 bp) were cloned into the pCR 2.1
vector
to obtain recombinant plasmids named pDML1807 (
NdeI
restriction
site) and pDML1808 (
NcoI restriction site).
These plasmids were
used to transform
E. coli XL1-Blue
competent cells. The nucleotide
sequences of the PCR-generated
fragments were verified in order
to rule out the presence of any
unwanted mutations. pDML1807 was
digested with the
NdeI and
BamHI restriction enzymes and pDML1808
was digested with the
NcoI and
BamHI restriction enzymes, and
then the
digested fragments were subcloned into the pET26b(+)
vector. The
corresponding plasmids, named pDML1809 and pDML1810,
respectively, were
introduced into
E. coli BL21(DE3)(pLysS) competent
cells. In
pDML1810 the gene coding for the mature form of FEZ-1
was fused in
frame with the nucleotide sequence of the PelB signal
peptide present
in the pET26b(+) vector. Since the amino acid
sequence of the
N-terminal part of FEZ-1 possesses a peculiar
proline-rich motif
(PMPNPFPP), a third expression vector based
on pET26b(+) was
constructed in which this proline-rich sequence
was removed by
introducing by PCR an
NcoI restriction site after
the
nucleotide sequence coding for the polyproline motif. The
primers used
in this case were
NcoILegi2
(5'-C
CCATGGCTGGAAACTTGTACTATGTAGGCACTGAT-3';
newly introduced restriction sites are underlined) and
BamHILegi.
This PCR product was cloned into the pCR 2.1 vector to yield recombinant
plasmid pDML1811. The
NcoI-
BamHI fragment isolated after digestion
of
pDML1811 was cloned in pET26b(+) to yield recombinant plasmid
pDML1812.
Also, in this case, the gene coding for the truncated

-lactamase was
fused to the PelB signal peptide
sequence.
In preliminary expression experiments, single colonies of
E. coli BL21(DE3)(pLysS) with pDML1809, pDML1810,
pDML1812, or pET24/FEZ-1
were used to inoculate 6 ml of Super broth
(SB) (
32) supplemented
with kanamycin (50 µg/ml) and
chloramphenicol (30 µg/ml). The
cultures were incubated overnight at
37 or 28°C with orbital shaking
at 250 rpm. A total of 2.5 ml of the
different cultures was added
to 100 ml of SB that had been preincubated
at 37 or 28°C. The
bacteria were grown to an optical density at 600 nm of 0.6, and
IPTG was added at a final concentration of 0.1 or 0.5 mM. Aliquots
(1 ml) of the different cultures were withdrawn at 0, 2, 4, 6,
and 10 h after induction. After centrifugation at
5,000 ×
g for
10 min, the bacterial pellet was
resuspended in 15 mM sodium cacodylate
buffer (pH 6.0) and sonicated
(three times for 30 s each time
at 12 W). The cells debris was
eliminated by centrifugation at
13,000 ×
g for 30 min.
A total of 20 µl of the different solutions
was loaded onto a sodium
dodecyl sulfate (SDS)-12% polyacrylamide
gel. The run was performed
at a constant voltage (120 V). The
protein concentration of each crude
extract was measured with
the help of the BCA (Pierce, Rockford, Ill.)
kit. The

-lactamase
activity of each preparation was determined by
measuring the initial
rate of hydrolysis of 100 µM
cefuroxime.
Production and purification of the zinc
-lactamase.
The
FEZ-1 enzyme was produced by E. coli BL21(DE3)(pLysS)
carrying pDML1810 in SB medium containing kanamycin and choramphenicol as the selecting agents during growth of the bacteria at 28°C under
orbital shaking. A total of 40 ml of an overnight preculture in SB was
used to inoculate 1 liter of fresh SB supplemented as described above
with kanamycin and choramphenicol. IPTG (final concentration, 0.5 mM)
was added at an absorbance value of 0.6 at 600 nm, and the culture was
continued for 6 h. Cells were harvested by centrifugation
(5,000 × g for 10 min at 4°C), and the pellet was
resuspended in 100 ml of 30 mM sodium cacodylate buffer (pH 6.0; buffer
A). The bacteria were disrupted with the help of cell disrupter
equipment (Basic Z model; Constant Systems Ltd., Warwick, United
Kingdom). Cell debris was removed by centrifugation (30,000 × g for 45 min at 4°C), and the supernatant was dialyzed
overnight against buffer A at 4°C. Thereafter, the crude extract was
loaded onto an S-Sepharose FF column (2.6 by 34 cm; Pharmacia, Uppsala, Sweden) equilibrated in buffer A. The column was washed with buffer A
until the A280 of the effluent was <0.1, and
the enzyme was eluted with a linear salt gradient (0 to 0.5 M) in five
column volumes. The active fractions were collected and concentrated on
a YM-10 membrane (Amicon, Beverly, Mass.) to a final volume of 5 ml.
The sample was loaded in a molecular sieve Sephacryl-100 (1.5 by 56 cm)
column equilibrated in 15 mM sodium cacodylate buffer (pH 6.0)
containing 0.2 M NaCl. The fractions that exhibited
-lactamase
activity were collected and their specific activities were monitored by
measuring the rate of hydrolysis of cefuroxime. The purity was also
checked by SDS-polyacrylamide gel electrophoresis (PAGE). The fractions
that exhibited constant specific activities were pooled (volume, 26 ml)
and concentrated to a final concentration of 1 mg/ml. The enzyme
preparation was stored at
20°C in 30 mM sodium cacodylate buffer
(pH 6.0).
Determination of quaternary structure of native
metallo-
-lactamase.
A total of 100 µl of FEZ-1 (1 mg/ml) was
loaded onto a molecular sieve (Superdex HR75; 1 by 30 cm; Pharmacia)
column equilibrated in 30 mM sodium cacodylate buffer (pH 6.0)-0.2 M
NaCl. The sample was eluted with the same buffer at a flow rate of 0.5 ml/min. The column was calibrated with lysozyme (14,400 Da), carbonic anhydrase (31,000 Da), ovalbumin (43,000 Da), and bovine serum albumin
(BSA; 66,200 Da) as standard proteins. The volume of the fractions was
1 ml. The retention volumes were measured by monitoring the
A280, and the retention volumes for the sample
containing
-lactamase were measured by determining the enzyme activity.
Determination of N-terminal sequence and molecular mass.
The
N-terminal sequence was determined with the help of a gas-phase
sequencer (Prosite 492 protein sequencer; Applied Biosystems, Foster
City, Calif.) The Mr of the enzyme was estimated
with an electrospray mass spectrometer (VG Bio-Q) upgraded with a
Platform source (Micromass, Altrincham, United Kingdom). The samples
(100 pmole) were suspended in 0.05% formic acid-50% acetonitrile in water and were injected into the source of the mass spectrometer with a
syringe pump (Harvard Instruments, South Natick, Mass.) at a flow rate
of 6 µl/min. The capillary was held at 2.7 kV, and the cone voltage
was set at 40 V. Fifteen scans covering 600 to 1,500 amu were
accumulated for 135 s and processed with the Masslynx software
delivered with the instrument. Calibration was performed with horse
heart myoglobin.
Determination of kinetic parameters.
Hydrolysis of the
antibiotics by FEZ-1 was followed by monitoring the variation in the
absorbance of the
-lactam solution in 15 mM sodium cacodylate buffer
(pH 6.0). All the measurements were made on a Uvikon 940 spectrophotometer connected to a personal computer via an RS232C
interface. The reactions were performed in a total volume of 500 µl
at 30°C. BSA (20 µg/ml) was added to diluted solutions of
-lactamase in order to prevent enzyme denaturation. The steady-state
kinetic parameters (Km and
kcat) were determined by analyzing the complete
hydrolysis time courses as described by De Meester et al.
(11) or by using the Hanes linearization of the
Michaelis-Menten equation.
Inactivation by chelating agents.
The loss of
-lactamase
activity was monitored in the presence of different concentrations of
EDTA, dipicolinic acid, and 1,10-o-phenanthroline. The
progressive inactivation of the enzyme was monitored by analyzing the
complete hydrolysis time course of 100 µM cefuroxime in 15 mM sodium
cacodylate (pH 6.0), used as a reporter substrate. The dependence of
the pseudo-first-order inactivation rate constant
(ki) upon the chelating agent concentration was determined.
Zn2+ dependence of FEZ-1 metallo-
-lactamase
activity.
Enzyme activity was measured by monitoring the initial
rates of hydrolysis of 100 µM cefuroxime, cefotaxime, imipenem,
nitrocefin, benzylpenicillin, and moxalactam in 15 mM sodium cacodylate
buffer (pH 6.0) containing 100 µM Zn2+. Kinetic
parameters were compared with those obtained under the same conditions
but without Zn2+.
pH dependence of FEZ-1 metallo-
-lactamase activity.
The
Km and kcat values were
calculated for cefuroxime, a good substrate of FEZ-1, in the following
buffers: 15 mM sodium acetate (pH 5.0), 15 mM sodium cacodylate (pH
6.0), 15 mM HEPES (pH 7.0), and 15 mM HEPES (pH 8.0).
Determination of Zn2+ content of FEZ-1 at different
pH values.
The pH dependence of the metal content of the
metallo-
-lactamase was measured by inductively coupled mass
spectrometry (ICPMS). One milliliter samples of FEZ-1 (0.88 mg/ml) were
dialyzed overnight at 4°C against 1 liter of the following buffers:
15 mM sodium acetate (pH 5.0), 15 mM sodium cacodylate (pH 6.0), 15 mM
HEPES (pH 7.0), and 15 mM HEPES (pH 8.0). The zinc concentration was determined in the protein sample and the dialyzing buffer by ICPMS, and
the Zn/enzyme molar ratio was calculated.
Molecular modeling.
The FEZ-1
-lactamase structural model
was based on the three-dimensional structure of the L1 enzyme from
S. maltophilia (35). The molecular model was
built with the Homology module of the Insight program (Molecular
Simulations, San Diego, Calif.) running on a Silicon Graphics
workstation. After model building, energy minimization was achieved
with the Discover module of the same package to avoid bad molecular
contacts. Finally, the geometric features were analyzed with the
Insight II program. The newly proposed BBL numbering of the class B
-lactamases was used (15a).
 |
RESULTS AND DISCUSSION |
Overexpression of FEZ-1
-lactamase in E. coli.
The production of FEZ-1 in E. coli BL21(DE3)(pLysS) was
tested with three different expression vectors (pDML1809, pDML1810, and pDML1812). pET24/FEZ-1, which was constructed previously
(4), was also included in these experiments for comparison.
With pDML1809, which encodes a FEZ-1 derivative from which the signal
peptide is deleted, no detectable production of metallo-

-lactamase
was found when the cultures were grown at 37 or 28°C in the presence
or the absence of 500 µM IPTG. With the other constructs, which
encode FEZ-1 forms that contain either the original signal peptide
(pET24/FEZ-1) or a PelB heterogeneous signal peptide fused with
two
different protein amino termini (pDML1810 and pDML1812),

-lactamase
activity was not detectable when the cultures were grown at 37°C
(in
the absence or the presence of IPTG) but was detectable in
cultures
grown at 28°C in the presence of 100 or 500 µM IPTG.
In these
cases, the production of the FEZ-1 enzyme seemed to be
optimal in the
presence of 500 µM IPTG. With pDML1810 and pDML1812,
the maximum
activity was reached after 6 h of induction, when
the specific
activities of the crude extracts against 100 µM cefuroxime
were 80 and 108 nmol · s
1 · mg of
protein
1, respectively (Fig.
1).

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FIG. 1.
Physical map of the different overproducing constructs.
The double-headed arrow delineates the nucleotide sequence of the
blaFEZ-1 gene. The nucleotide sequences
corresponding to the FEZ-1 signal peptide, the pelB
sequence, and the PMPNPFPP peptide are represented by boxes with light
dashes, boxes with heavy dashes, and black boxes, respectively. mgP,
milligrams of protein.
|
|
A different behavior was found with pET24/FEZ-1 which, as reported
previously (
4), yielded the maximum cell-associated

-lactamase activity 2 h after induction, with a specific
activity
against cefuroxime of 17 nmol · s
1
· mg of protein
1.
Replacement of the FEZ-1 signal peptide by the PelB leader sequence
therefore allowed the production of FEZ-1 in a soluble
form and in
larger amounts than those obtained with the endogenous
signal peptide.
Moreover, the highest level of enzyme production
was obtained when the
proline-rich sequence (PMPNPFPPF) close
to the FEZ-1 amino
terminus was also deleted. Fractionation of
the different cultures in
the soluble fraction (periplasm plus
cytoplasm) and the insoluble
fraction (membrane plus insoluble
material) showed that 30% of the
FEZ-1 was produced as insoluble
material. Nevertheless, in order to use
the more physiologically
relevant preparation of enzyme, it was decided
that strain
E. coli BL21(DE3)(pLysS)/pDML1810 would be used
for large-scale
production.
Purification of FEZ-1
-lactamase.
FEZ-1 overproduced in
E. coli BL21(DE3)(pLysS) was purified in two chromatography
steps as described in Materials and Methods. In the first purification
step, the enzyme was eluted from an S-Sepharose column at a salt
concentration of 0.2 M. At that stage, the purification yield of FEZ-1
was about 66% (Table 1) and other proteins with a lower molecular masses were revealed by SDS-PAGE (Fig.
2). To remove these contaminants the
enzyme preparation was loaded onto a Sephacryl-100 molecular sieve
column equilibrated with 30 mM sodium cacodylate (pH 6.0) containing
0.2 M NaCl. The active fractions were pooled and concentrated on an
Amicon YM-10 membrane to a final concentration of 1 mg/ml. Above this
concentration, a decrease in the specific activity of the preparation
was observed, a phenomenon that may be due to partial aggregation of
the
-lactamase. The final yield of the purification was 53%. Under
these conditions it was not necessary to perform the additional
cation-exchange chromatography step described by Fujii et al.
(15). The ability to overproduce the zinc
-lactamase in
E. coli facilitated the purification process and allowed
simplification of the protocol. The N-terminal sequence of the purified
FEZ-1 enzyme was AYPMPNPFPPF, as expected. Mass spectrometry
confirmed the homogeneity of the protein preparation (data not shown).
The Mr value was estimated to be 29,447 ± 7, which is very close to that deduced from the amino acid sequence
(Mr = 29,440).
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TABLE 1.
Purification of the FEZ-1 metallo- -lactamase of
L. gormanii produced by a 1-liter culture of E. coli BL21(DE3)(pLysS)/pDML1810
|
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FIG. 2.
SDS-PAGE after the different purification steps. Lane A,
molecular mass standards; lane B, crude extract of E. coli BL21(DE3)(pLysS)/pDML1810; lane C, protein pattern
after passage through an S-Sepharose column; lane D, protein pattern
after passage through a Sephacryl-100 molecular sieve. The arrow
indicates the Mr of FEZ-1.
|
|
The subclass B3 FEZ-1
-lactamase is a monomeric enzyme.
Calibration of the Superdex HR75 column yielded the following retention
volumes: lysozyme, 17.38 ml; carbonic anhydrase, 11.42 ml; ovalbumin,
10.08 ml; and BSA, 9.1 ml. The retention volume of FEZ-1 was 11.50 ml,
close to that of carbonic anhydrase (Fig. 3). Its molecular mass, determined by
molecular sieve chromatography on Superdex HR75, was thus approximately
30,000 Da, indicating that the native enzyme is a monomer, in agreement
with data previously obtained with crude extracts (4).
Therefore, metallo-
-lactamases of subclass B3 can be either
monomeric (FEZ-1, GOB-1) or multimeric (L1), a phenomenon which was not
observed for the metallo-
-lactamases of other subclasses.

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FIG. 3.
Elution pattern of the FEZ-1 -lactamase and carbonic
anhydrase on a Superdex HR75 molecular sieve column. The values on the
y axis represent the A280.
|
|
Kinetic parameters of the FEZ-1 enzyme.
The
kcat and Km values were
determined for a representative set of
-lactam antibiotics (Table
2). The results showed that the
-lactamase exhibited a broad-spectrum activity profile (Table 2),
although with notable differences for different substrates. It did not
significantly hydrolyze monobactams such as aztreonam and carumonam
(kcat/Km <0.0001
µM
1 s
1). The activity of the
-lactamase was not affected by a prolonged incubation (1 h at room
temperature) of the enzyme in the presence of both monobactams at a
concentration of 1 mM each. In addition, the rate of hydrolysis of
nitrocefin was not modified by the presence of a high concentration (1 mM) of aztreonam. Among the suicide substrates designed against
active-site serine
-lactamase tested, clavulanic acid (final
concentration, 1 mM) was not recognized and tazobactam behaved as a
substrate (Table 2).
FEZ-1 exhibited a higher level of activity against some cephalosporins
than against penicillins and carbapenems.
kcat/
Km values
for
cephalosporins varied from 0.004 to 6.6 µM
1
s
1. The best substrates for the
L. gormanii
metallo-

-lactamase
are cephalothin, cefuroxime, and cefotaxime (an
oxyiminocephalosporin).
The catalytic efficiency of FEZ-1 against these
drugs was higher
than 2 µM
1 s
1. On the
other hand, ceftazidime and cefepime were poorly hydrolyzed
(
kcat/
Km, <0.006
µM
1 s
1). The comparison of the kinetic
constants for the hydrolysis
of cephalothin and cefoxitin indicates
that the presence of an

-methoxy group at position C-7 negatively
affects the activity
of the metallo-

-lactamase. The decreased
catalytic efficiency
versus cefoxitin compared to the catalytic
efficiency of cephalothin
was accompanied by a 10-fold decrease in the
Km values, suggesting
that the

-methoxy group
at position C-7 has a remarkable detrimental
effect on hydrolysis but
also enhances recognition of the cephalosporin
substrate. This was
consistent with the high affinity exhibited
by the enzyme for
moxalactam. In fact, the
Km values for the
oxacephamycins
were significantly lower than those measured for all
other
substrates.
All the penicillin derivatives tested were poorly recognized by the
FEZ-1 metallo-

-lactamase. The measured
Km
values were
always high (>700 µM). Benzylpenicillin and cloxacillin
were the
best substrates. The presence of a small lateral chain (6-APA)
or the presence of a bulky substituent (as in piperacillin) at
position
C-6 somewhat decreased the catalytic efficiency. In addition,
the
presence of a charged C-6 substituent (as in ampicillin, carbenicillin,
and ticarcillin) did not significantly modify the enzyme
efficiency.
As observed for all the other class B

-lactamases, carbapenems were
well hydrolyzed by the
L. gormanii enzyme
(
kcat/
Km, >0.07
µM
1 s
1), with meropenem being the best
substrate due to its low
Km value.
The ratio between the
kcat/
Km values for
different antibiotics and the
kcat/
Km value for
imipenem calculated for different
class B

-lactamases underlines the
peculiar activity profile
of FEZ-1 (Table
3). CphA only hydrolyzed carbapenems
efficiently.
IMP-1, IMP-2, VIM-2, and L1 exhibited similar activities
toward
the different

-lactam families. CcrA and BcII had higher
levels
of activity against penicillins and cephalosporins than against
imipenem, while benzylpenicillin was the best substrate of BlaB.
Finally, some cephalosporin compounds were the best substrates
of
FEZ-1. It is interesting that cefoxitin behaved as a rather
good
substrate for FEZ-1, CcrA, ULA511, IMP-1, IMP-2, VIM-2, and
BlaB, a
poor substrate for BcII, and an inactivator for CphA.
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|
TABLE 3.
Values of
(kcat/Km for an
antibiotic)/(kcat/Km for
imipenem) for FEZ-1, BcII, CcrA, ULA511, IMP-1, IMP-2, VIM-2, BlaB,
and CphAa
|
|
The steady-state kinetic parameters were affected at pH 6 when
increasing Zn
2+ ion concentrations were added to the enzyme
(Table
4). The catalytic
efficiencies for
cefotaxime, imipenem, and nitrocefin increased
in the presence of zinc
ions, and the
kcat values for nitrocefin
and
cefotaxime were modified. The corresponding
Km
values were
not influenced by the presence of zinc ions. Even at a high
zinc
concentration (100 µM), the individual kinetic constant for
imipenem
could not be determined. In this case, the
Km value was larger
than 1 mM. A slight decrease
in the
Km value was found for cefuroxime.
Nevertheless, the catalytic efficiency of FEZ-1 against this antibiotic
was not significantly modified. The catalytic efficiency of the
FEZ-1

-lactamase against benzylpenicillin and moxalactam was
not affected
by the zinc concentration. In the case of moxalactam,
the
kcat and
Km value
increased by approximately the same amounts,
yielding unmodified values
of the catalytic efficiency in the
presence of an excess of zinc ions.
pH dependence of activity and zinc content of FEZ-1.
The pH
dependence of the zinc content of the metallo-
-lactamase of L. gormanii was measured by ICPMS the (Table
5). Other metallic ions such as Co(II),
Cd(II), Ca(II), Cu(II), Mn(II), Ni(II), and Fe(II) and Fe(II) or
Fe(III) were not found. In the absence of excess metal, between pH 6 and 8, two zinc ions were found per enzyme molecule. A similar zinc
content was found for the L1 enzyme (10). No measurements
of the zinc/enzyme molar ratios were made with the GOB-1 enzyme
(2). The presence of a glutamine residue as a putative
metal ligand may influence the zinc content of the latter enzyme. All
the metallo-
-lactamases studied have contained two zinc-binding
sites (8, 17, 20, 25). However, with the exception of
metallo-
-lactamases isolated from Aeromonas species and
when the mono-zinc species can be prepared, the presence of the second
metal ion does not strongly modify the catalytic efficiency of the
enzyme (17). The study of the interaction between
nitrocefin and the L1 enzyme showed that the second zinc ion might
participate in the stabilization of an intermediate (22).
The same phenomenon was observed for CcrA (38). In the mono-zinc form, the zinc ion participates in the formation of the
nucleophilic hydroxide and provides Lewis acid catalysis by polarization of the carbonyl of the
-lactam ring (5).
We can hypothesize that the mechanism of the FEZ-1 enzyme is
similar to that of L1. Finally, the pH dependence of the
steady-state kinetic parameters (kcat and
Km) of FEZ-1 with cefuroxime in the absence of
added zinc was measured. A time-dependent inactivation of the enzyme by
cefuroxime was observed at pH 5. The ki value was 3 × 10
3 s
1 with 100 µM
cefuroxime. The same phenomenon was observed even when the reaction was
performed in the presence of 100 µM Zn2+. The
Km and kcat values were
not strongly modified between pH 6 and 8, with maximum activity
detected at pH 6 (kcat = 320 s
1; Km = 48 µM). These data are
in good agreement with the results obtained for other
metallo-
-lactamases. For example, the maximum activities of the
B. cereus 569H (4) and IMP-1 (20)
enzymes were observed in the same pH range.
Inactivation by Zn-chelating agents.
All the chelating agents
tested behaved as strong inactivators. In all cases, a time-dependent
ki could be measured. The
ki values were found to be independent of the
chelator concentration and were similar for all three compounds (Table
6). These data indicated that at pH 6 the
chelators probably act by scavenging the free metal, and the
ki value might represent the rate of
dissociation of the protein-zinc ion complex. Interestingly, and in
contrast to all the subclass B1 and B2 enzymes (BlaB
[31], IMP-1 [20], BcII [unpublished
data], CfiA [unpublished data], and CphA [17]), in
which inactivation occurs via the formation of a transient enzyme-metal-chelator ternary complex, a similar behavior is observed with the L1 subclass B3 enzyme (3) and the
ki values are strikingly similar (from 1.7 × 10
2 to 3.8 × 10
2
s
1). In addition, the concentration needed to completely
inactivate FEZ-1 is far lower than the concentration needed to
completely inactivate the other enzymes tested (17, 20).
For example, EDTA and dipicolinic acid behaved as poor inactivators of
the IMP-1
-lactamase. IMP-1 could be inactivated by behaved the
enzyme in the presence of 10 mM EDTA or 300 µM dipicolinic acid
(20). It would be interesting to further analyze the
mechanisms of action of chelating agents against the other subclass B3
enzymes.
Structural features of the FEZ-1 enzyme.
Molecular modeling of
the FEZ-1 enzyme on the basis of the known structure of the L1
metallo-
-lactamase revealed some interesting features.
(i) Overall fold.
As already pointed out by Boschi et al.
(4), FEZ-1 and L1 could be aligned over the entire
sequence without the introduction of major gaps (Fig.
4). The major differences between L1 and
FEZ-1 are the absence of the N-terminal 310 helix region, a
two-residue insertion in the loop between
3 and
8, and a
six-residue insertion in the loop between
12 and the C-terminal
5
helix. The latter loop is already considerably longer in L1 than in the
subclass B1 enzymes, and it is constrained by an intramolecular
disulfide bridge that links the cysteine residues at positions 256 and
290 (35). Such a disulfide bridge between cysteine
residues can be predicted in FEZ-1 (Fig. 4).

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|
FIG. 4.
Comparison of the amino acid sequence of the mature zinc
-lactamase of L. gormanii (FEZ-1) with that of the
S. maltophilia zinc -lactamase (L1). The consensus
sequence indicates the identical amino acid residues. Residues involved
in zinc coordination are in boldface and are marked by asterisks. The
residues in boldface and labeled with a plus sign are residues which
may interact with the C-7 side chain of cefuroxime. Cysteine residues
in boldface are presumably involved in a disulfide bridge in the FEZ-1
metallo- -lactamase.
|
|
(ii) Zinc ligands.
The six residues known to be involved in
Zn2+ binding in the L1
-lactamase are conserved in the
FEZ-1 enzyme, i.e., His-116, His-118, and His-196 for the first
Zn2+ binding site and Asp-120, His-121, and His-263 for the
second one. The orientations of the zinc ligands are maintained by an extensive hydrogen bond network which is also very similar in the two
enzymes. However, the group that orients the first His is the Asp-220
side chain in L1 and the Ser-262 side chain via a water molecule in
FEZ-1, as in most subclass B1 enzymes.
(iii) Substrate binding site.
In the case of subclass B1
-lactamases (7, 8, 9), most investigators postulate
that the carbonyl oxygen of the
-lactam substrate lies in an
oxyanion hole formed by Zn-1 and the side chain of a conserved Asn
(Asn-233), while the substrate carboxylate moiety interacts with the
ammonium group of a conserved lysine residue (Lys 224). In L1, the side
chain of the Asn is 14 Å away from the substrate carbonyl
oxygen, and it is proposed that in this case the oxyanion is stabilized
by interaction with the side chain of Tyr-228 (35). This
tyrosine is conserved and could play the same role in FEZ-1. In turn,
the lysine that interacts with the
-lactam carboxylate in subclass
B1
-lactamases is replaced by Ser-223 in L1 and by a Gly residue in
FEZ-1. However, just after this Gly, an Asn residue is inserted in the
loop that connects
11 and
4. The side chain of this Asn-233 is
ideally placed to interact with the substrate carboxylate, especially
those of cephalosporins (Fig. 5). The
hydrophobic
substituent of the
-lactam substrate generally fits
in a hydrophobic pocket formed by the "flap" that connects
3 and
4 and by the loop between
3 and
8, which is considerably
longer in subclass B3 enzymes than in subclass B1 enzymes. In this
pocket, the hydrophobic residues Phe-156 and Ile-162 in L1 are replaced
by a Tyr and a Ser residue in FEZ-1, respectively. These substitutions
should influence the substrate specificity, with a facilitated
interaction between FEZ-1 and
-lactams that bear a less hydrophobic
side chain.

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|
FIG. 5.
Active site of the modeled structure of the FEZ-1 enzyme
showing the side chains of the amino acids discussed in the text. A
molecule of cefuroxime is docked in the catalytic cavity.
|
|
Conclusions.
The study of the FEZ-1 metallo-
-lactamase
underlines the heterogeneity of the biochemical properties of the class
B
-lactamases. The enzyme is monomeric and contains two zinc ions.
As shown for other metalloproteins, the enzyme was inactivated by Zn chelators.
The FEZ-1 enzyme exhibits a broad-spectrum activity profile and was at
least 1 order of magnitude more active against cephalosporins
than
against penicillins and carbapenems. Molecular modeling studies
suggested that the general conformation of the zinc-binding sites
are
very similar in the FEZ-1 and L1

-lactamases. However, the
side
chains of the Asn-225, Tyr-156, and Ser-162 residues, which
protrude in
the FEZ-1 active site, could probably be responsible
for the specific
substrate profile of the enzyme. Detailed structural
and mechanistic
studies are in progress in order to demonstrate
the functions of these
residues.
 |
ACKNOWLEDGMENTS |
This work was supported in part by a grant from the European
Union (grant ERB3512-IC15-CT98-0914) as part of the Training and
Mobility of Researchers Program and by the Belgian Program Pôles
d'Attraction Interuniversitaire initiated by the Belgian State, Prime
Minister's Office, Services Fédéraux des Affaires Economiques, Techniques et Culturelles (PAI P4/03). B.D. is a postdoctoral researcher of the Fonds voor Wetenschappelijk
Onderzoek-Vlaanderen.
The ICPMS measurements were performed by the Laboratoire de la
Santé et de l'Environnement, Institut Malvoz de la Province de
Liège, Liège, Belgium.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Centre
d'Ingénierie des Protéines, B6 Sart Tilman,
Université de Liège, B-4000 Liège, Belgium. Phone:
32-4-3663419. Fax: 32-4-3663364. E-mail: mgalleni{at}ulg.ac.be.
 |
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Antimicrobial Agents and Chemotherapy, April 2001, p. 1254-1262, Vol. 45, No. 4
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.4.1254-1262.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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