From Wyeth-Ayerst Research, Pearl River, New York 10965
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ABSTRACT |
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The crystal structure of the
metallo- Metallo--lactamase CcrA3 indicates that the active site of this
enzyme contains a binuclear zinc center. To aid in assessing the
involvement of specific residues in
-lactam hydrolysis and
susceptibility to inhibitors, individual substitutions of selected
amino acids were generated. Substitution of the zinc-ligating residue
Cys181 with Ser (C181S) resulted in a significant
reduction in hydrolytic activity; kcat values
decreased 2-4 orders of magnitude for all substrates. Replacement of
His99 with Asn (H99N) significantly reduced the hydrolytic
activity for penicillin and imipenem. Replacement of Asp103
with Asn (D103N) showed reduced hydrolytic activity for cephaloridine and imipenem. Deletion of amino acids 46-51 dramatically reduced both
the hydrolytic activity and affinity for all
-lactams. The metal
binding capacity of each mutant enzyme was examined using nondenaturing
electrospray ionization mass spectrometry. Two zinc ions were observed
for the wild-type enzyme and most of the mutant enzymes. However, for
the H99N, C181S, and D103N enzymes, three different zinc content
patterns were observed. These enzymes contained two zinc molecules, one
zinc molecule, and a mixture of one or two zinc molecules/enzyme
molecule, respectively. Two enzymes with substitutions of
Cys104 or Cys104 and Cys155 were
also composed of mixed enzyme populations.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactamases require zinc or another metal cofactor to
be enzymatically active and capable of hydrolyzing the amide bond of
the
-lactam ring (1-5). The metallo-
-lactamase CcrA hydrolyzes
almost all known
-lactams and is not effectively inactivated by the
marketed
-lactamase inhibitors clavulanic acid, sulbactam, and
tazobactam (6-8). The crystal structure of the metallo-
-lactamase CcrA3 indicates that the active site of this enzyme is at the edge of
the
-sandwich of the four-layer
/
/
/
molecule and contains two zinc atoms (1). Zn1 is coordinated to four ligands in a
tetrahedral geometry: His99,
His101, and His162, and a solvent
molecule defined as water 1 (Wat1).1 Zn2 ligates with
three amino acids (Asp103, Cys181, and
His223) and two water molecules (Wat1 and Wat2) (Fig.
1). The proposed mechanism of CcrA
enzymatic hydrolysis includes formation of a hydroxide ion from Wat1.
This hydroxide ion mounts a nucleophilic attack on the carbonyl carbon
atom of the
-lactam. Atomic absorption studies of the metal content
of CcrA (9) confirmed that CcrA binds two atoms of Zn2+ and
that the presence of both metal ions is required for full catalytic
activity.
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Fig. 1.
Stereoscopic representation of the atomic
model of the active site of CcrA3. The zinc- and metal-bound
solvent molecules are depicted. Amino acids that ligate with zinc
atoms, as well as Cys104, Lys184, and
Asn193, are indicated. W1, Wat1; W2,
Wat2.
In this work, we describe a set of CcrA derivatives harboring selected
amino acid substitutions of active-site and nearby residues (see Table
I) and the involvement of these individual amino acid residues in
enzymatic activity. Purified mutant proteins were used to analyze the
-lactam hydrolytic and inhibition properties conferred by each amino
acid substitution. The metal content of the CcrA wild-type and mutant
enzymes was determined using a novel approach, electrospray ionization
mass spectrometry (ESI/MS) utilizing nondenaturing and denaturing
conditions, and was then compared with the measurements obtained by a
classic approach, atomic absorption spectrophotometry (AAS). The
effects of the specific amino acid substitutions on metal content and
catalytic activity were analyzed.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains--
Escherichia coli strains
DH5 and BL21 (
DE3) (10) were used for gene construction and
protein production, respectively.
Antibiotics and Reagents-- Penicillin G and 1,10-phenanthroline were purchased from Sigma. Other reagents were obtained as indicated: cephaloridine from Lilly, imipenem from Merck, tazobactam from Lederle Laboratories, nitrocefin from Becton Dickinson Microbiology Systems (Hunt Valley, MD), and BRL 42715 from SmithKline Beecham (Worthing, United Kingdom).
Site-directed Mutagenesis--
All ccrA codon changes
were introduced into the respective genes ccrA1 and
ccrA3 using modified polymerase chain reaction (PCR)
techniques employing Taq polymerase (11). The enzymes CcrA1
and CcrA3 are both naturally occurring Bacteroides fragilis metallo--lactamases that differ at three amino acids, none of which
is located within the active-site cavity (10). For the Cys104 to Ser (C104S) and Cys155 to Val (C155V)
mutations, two sets of primers were used to amplify the
ccrA1 gene as overlapping 5' amino-terminal and 3'
carboxyl-terminal coding fragments with the desired mutation at the 3'
or 5' end, respectively. The amino-terminal and carboxyl-terminal
coding PCR products were melted, mixed, annealed, and amplified a
second time to give full-length ccrA1 containing the desired
mutation. These two mutations were constructed sequentially beginning
with the C155V encoding mutation (12). The remaining mutations were constructed using ccrA3 as the template gene (10). For the
Cys181 to Ser (C181S), Lys184 to Arg (K184R),
and Asn193 to Asp (N193D) mutations, one oligonucleotide
was constructed that covered the region encoding the codon to be
changed and harboring the new codon. A second oligonucleotide covering
the 3' terminal region of the gene was used as the partner primer in
the first PCR. A short 3' double-stranded DNA fragment was produced.
This 250-300-base pair double-stranded DNA fragment was used as one primer in combination with a 5' primer in the second PCR to produce the
carboxyl-terminal 650 base pairs of the gene. The resulting PCR product
was cloned into pUC119. A fragment from this clone was used to replace
the carboxyl-terminal region of ccrA, from the internal
KpnI site to the 3' end, into a pT7 expression vector containing a ccrA gene minus the signal sequence (6, 10, 12). The His99 to Asn (H99N), Asp103 to Asn
(D103N), Cys104 to Arg (C104R), and
46-51 mutations
were constructed in a similar manner, except that the amino-terminal
region of the gene was constructed first and used in combination with a
carboxyl-terminal primer in the second PCR to create the full-length
gene. The DNA sequence of all PCR products and the presence of the
expected mutation were confirmed by DNA sequence analysis.
Enzyme Purification--
All CcrA-encoding genes were under
transcriptional and translational control of a T7 promoter and
expressed in E. coli BL21 (DE3) cells from a kanamycin
resistance-conferring vector. Purification of all enzymes was performed
as described (6). Briefly, 2 liters of mid-log phase cells were induced
by the addition of 1 mM (final concentration)
isopropyl-
-D-thiogalactopyranoside for 2 h.
Overproduction of the
-lactamase resulted in the sequestering of the
-lactamase into inclusion bodies. The inclusion bodies were
harvested following disruption of the expressing cells using a French
press (15,000 p.s.i.) and centrifugation at 10,000 × g
for 10 min. The inclusion bodies were solubilized by resuspension in 8 M urea, 1 M NaCl, 10 mM HEPES, pH
7.4, and 100 µM ZnCl2 and then sequentially
dialyzed into 1 M NaCl, 10 mM HEPES, pH 7.4, and 100 µM ZnCl2, followed by 10 mM HEPES, pH 7.4, and 10 µM
ZnCl2. Material that precipitated during dialysis was
removed by centrifugation. Protein concentrations were determined using
the BCA assay (Pierce).
Enzyme Kinetics and Inhibition Study-- Hydrolysis rates for the purified enzymes were determined in 10 mM HEPES, pH 7.2, using a Beckman DU7400 or Gilford 250 spectrophotometer. The kinetic parameters kcat and Km were derived from the initial velocities obtained from six to eight substrate concentrations using the computer program ENZPACK (Biosoft/Elsevier). These parameters are presented as the mean value of at least duplicate experiments with the S.D. for each parameter being <20%. For the inhibition studies, enzyme and inhibitor were preincubated at 25 °C in a volume of 50 µl for 10 min before the addition of 50 µg/ml nitrocefin (final volume of 1000 µl). IC50 values were determined graphically.
Metal Content Determination Using ESI/MS-- Samples of the CcrA wild-type and mutant enzymes were exchanged with 10 mM ammonium acetate buffer prior to ESI/MS analysis by dilution and centrifugal filtration. Two ESI mass spectra were obtained for each protein under native state and denatured conditions. Denaturation of the proteins was performed by adding 40 µl of a 50:50 (v/v) water/acetonitrile solution containing 0.4% formic acid to 20 µl of the native protein in 10 mM ammonium acetate. The native or denatured protein solutions (0.5-3.0 nmol) were introduced into the ESI mass spectrometer at flow rates of 3-5 µl/min using a Harvard Model PHD 2000 syringe infusion pump. All mass spectra were acquired using an API 365 triple quadrupole mass spectrometer equipped with an atmospheric pressure ionization source and a PE-Sciex ionspray (pneumatically assisted electrospray) interface. A Power Macintosh 9600/300 computer was used for instrument control, data acquisition, and data processing. A mild set of ESI interface conditions was employed for the detection of noncovalent metal-protein complexes, i.e. low curtain gas (desolvation) pressure and a low orifice (declustering) potential (13-15). High purity nitrogen gas was used as the nebulizing gas. The mass scale of the spectrometer was calibrated with polypropylene glycol. The mass resolution of the spectrometer was tuned to give a constant peak width of 1 Da across the mass range of interest. Full scan mass spectra were acquired using a step size of 0.2 Da with a scan time of 7.5 s across the m/z range of 2000-3000 for the native protein samples and with a scan time of 6.8 s across the m/z range of 30-3000 for the denatured protein samples. Typically, 20-40 scans were acquired and added to yield a mass spectrum. Protein molecular masses were obtained by deconvoluting the multiply charged protein mass spectra using the PE-Sciex hyper mass program. The zinc content of each protein was derived from the mass difference between the native and denatured proteins.
Determination of Zinc Content Using Atomic Absorption
Spectrophotometry--
The wild-type and amino acid-substituted
enzymes were dialyzed against zinc-free 10 mM HEPES, pH
7.2, at 4 °C with multiple changes of dialysis buffer to remove zinc
from the sample buffer. Protein concentrations of the dialyzed samples
were determined using the BCA assay and adjusted to a final
concentration of ~1 mg/ml. The concentration of the zinc standard
covered the range from 0.2 to 1.0 ppm using five solutions. The enzyme
solutions were diluted 1:20 with H2O. The metal content of
the dialyzed CcrA wild-type and mutant enzymes was ascertained using an
Instrumentation Laboratory 551 atomic absorption spectrophotometer in
the flame mode using the zinc 213.9-nm line. The metal content values
reported for each sample are an average of readings from two
independent experiments.
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RESULTS |
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Construction of Amino Acid Substitutions and Enzyme
Preparation--
Using the described PCR techniques, eight mutant
genes were successfully constructed and confirmed by DNA sequence
analysis (Table I). These mutant genes
were placed into a T7 expression plasmid by replacing the
carboxyl-terminal region of a ccrA gene lacking a signal
sequence that was already present in the vector. The protein produced
following isopropyl--D-thiogalactopyranoside induction
accumulates in inclusion bodies, from which it is easily solubilized
and refolded (6). To ensure proper refolding, the enzymes were refolded
in a 30-fold excess of Zn2+. As an assessment of proper
folding, circular dichroism studies were performed on selected enzymes.
These studies revealed no structural differences when compared with the
wild-type enzyme (data not shown). The enzymatic activity of the
refolded enzymes paralleled the in vivo activity of the same
constructs. These observations strongly suggest that the enzymes used
in these studies have correctly and efficiently refolded. The purity
and molecular mass of all constructed proteins were confirmed by
SDS-polyacrylamide gel electrophoresis and ESI/MS.
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Hydrolytic Activity of Mutant Enzymes for the Major -Lactam
Classes--
The catalytic activities of the mutant enzymes were
compared with their respective wild-type enzymes for penicillin G
(PenG), cephaloridine (CLD), and imipenem (IMP) (Table
II). In general, substitution of amino
acid residues that ligate to one of the two zinc molecules (C181S,
D103N, or H99N) resulted in the most significant decrease in enzymatic
activity. Substitution of the Zn2-chelating residue Cys181
with Ser resulted in a significant reduction (greater than 2-3 orders
of magnitude) in hydrolytic activity for PenG, CLD, and IMP (Table II).
The affinity of the C181S enzyme for CLD and IMP was reduced 2-3-fold.
For PenG, the affinity was reduced 7-fold (increased
Km value). Replacement of His99 with Asn
significantly reduced the kcat values for PenG
(300-fold) and IMP (2500-fold), but only slightly reduced the
kcat for CLD (<4-fold). However, the
Km of the H99N enzyme for CLD increased 63-fold,
which resulted in a >200-fold reduced physiological efficiency
(kcat/Km value). The D103N
mutant showed reduced kcat values for CLD and
IMP as well as a 20-fold decrease in the affinity for PenG. The loop
mutant (deletion of amino acids 46-51, removing the disordered loop)
showed a dramatic reduction in both hydrolytic activity and affinity
for all the
-lactams tested. The most significant change observed
was for CLD, with a reduced physiological efficiency
(kcat/Km) of 4500-fold
compared with the wild-type enzyme (Table II). Substitution of
Lys184 with Arg and Asn193 with Asp resulted in
increased kcat and Km values
for PenG. Substitution of Cys104 with Arg had the least
effect on the kcat values for PenG and CLD, with
a slight effect on the kcat for IMP. The double
amino acid substitution C104S/C155V showed significantly increased
affinity for IMP.
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Inhibition Profiles of Mutant Enzymes--
Table
III shows the inhibitory activity of two
-lactamase inhibitors, the penicillanic acid sulfone tazobactam and
the penem BRL 42715, against all the mutant proteins in comparison with the wild-type enzyme. The IC50 values for the chelator
1,10-phenanthroline are also presented. Tazobactam showed weak
inhibitory activity against the wild-type metalloenzymes CcrA3 and
CcrA1, with IC50 values of 350-400 µM. A
>2-fold increase in the IC50 values for tazobactam was
observed for the mutant enzymes H99N and C181S and the
46-51
mutant. The penem inhibitor BRL 42715 showed significant inhibitory
activity against CcrA3 and CcrA1, with IC50 values of 0.05 and 0.09 µM, respectively. The H99N, C181S, and
46-51 enzymes were much less susceptible to inhibition by BRL 42715. For the
H99N, C181S, and
46-51 enzymes, the IC50 values
increased 4-5 orders of magnitude. The other mutant enzymes (D103N,
C104R, and N193D) were moderately less susceptible to inhibition by BRL 42715, exhibiting a 4-7-fold increase in IC50 values.
Interestingly, the H99N and C181S enzymes were more susceptible to the
chelator 1,10-phenanthroline than was the wild-type protein.
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Metal Content of the Mutant Proteins--
The metal content of
each substituted protein is summarized in Table
IV. Using ESI/MS with a triple quadrupole
mass spectrometer, two mass spectra were obtained for each enzyme, one
under native and the other under denaturing conditions (Fig.
2). The mass of the denatured form of
each enzyme matched very closely with the calculated mass of the
respective enzyme (Table IV), confirming the amino acid substitution.
The metal content measured by ESI/MS for each enzyme was determined
from the difference(s) in mass between the native and denatured forms.
The average mass of the divalent Zn2+ is 65.4. However, two
or more protons may be displaced upon protein binding, giving the bound
zinc an apparent molecular mass of 63.4. For the wild-type
metallo--lactamases CcrA3 and CcrA1, all enzyme molecules in the
sample coordinated two Zn2+ atoms. Three Zn2+
content patterns were observed for the enzymes in which the substituted amino acid coordinates one of the two Zn2+ atoms: H99N,
D103N, and C181S. Two Zn2+ atoms/enzyme molecule were
observed for H99N. For C181S, only one Zn2+ atom/enzyme
molecule was observed. The D103N enzyme preparation was composed of a
mixed population with either one or two zinc molecules/enzyme molecule.
The major component for the D103N enzyme, corresponding to 71% of the
enzyme population, held two Zn2+ atoms/enzyme molecule, and
29% of the enzyme content held only one Zn2+ atom. For
C104R, 32% of the native enzyme bound two Zn2+
molecules/enzyme molecule, and 68% bound one Zn2+
molecule. For the double mutant C104S/C155V, the majority of the
protein contained two Zn2+ atoms (83%), and the remaining
enzyme harbored one zinc atom. Substitutions of two other residues
(K184R and N193D) as well as the loop deletion did not result in
altered Zn2+ content. These mutant enzymes contained two
Zn2+ atoms/enzyme molecule.
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The metal content of the enzymes as measured by AAS is reported in
Table IV. The zinc content values reported by AAS generally corresponded well with the ESI/MS data. However, for H99N, only one
Zn2+ atom/molecule of protein was observed by AAS, whereas
ESI/MS data clearly indicated the presence of two zinc atoms/enzyme
molecule under native conditions. The reported zinc content of CcrA3 by AAS was 1.41 Zn2+ atoms/molecule of protein.
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DISCUSSION |
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The metallo--lactamases CcrA1 and CcrA3 from B. fragilis have a broad substrate profile and are refractory to
inhibition by the commercially available
-lactamase inhibitors (6,
7, 16). These enzymes require a metal cofactor to be enzymatically active (2, 3). Within the active site, there are two zinc ligands,
designated Zn1 and Zn2 (1). Using site-directed mutagenesis, eight
different ccrA genes have been constructed that encode
enzymes with amino acid substitutions in or near the active site. The hydrolytic activity, susceptibility to inhibitors, and metal content of
each mutant enzyme were investigated. The results indicate that many of
the substituted amino acids play a critical role in the enzymatic
function of the protein. The most dramatic effects were observed when
the enzyme harbored an amino acid substitution of one of the
zinc-ligating residues.
Among the most exciting findings of this study are the zinc content
determinations for the wild-type and mutant enzymes. Two different
methods were used to analyze the zinc content: ESI/MS and AAS. For most
of the enzymes, the zinc content was comparable using either of the two
methods. However, AAS tended to underestimate the zinc content of the
enzyme relative to that observed by ESI/MS. This may be the result of
the dialysis procedure that was required when performing AAS. Since
this method cannot discriminate between enzyme-bound zinc and zinc in
buffer solution, dialysis of the enzyme sample to remove extraneous
Zn2+ was required. Unfortunately, this process may
also remove weakly bound Zn2+ from the active site of the
enzyme, resulting in an underestimated Zn2+ content. This
is likely to be the case for the H99N enzyme. ESI/MS identified two
zinc molecules/enzyme molecule, whereas only one zinc molecule/enzyme
molecule was determined using AAS. This suggests that the H99N enzyme
binds Zn1 with much less affinity than the wild-type enzyme and that
this zinc is lost during dialysis. The lower IC50 value of
H99N for the Zn2+ chelator 1,10-phenanthroline (2 µM) compared with that of CcrA3 (15 µM)
supports the above hypothesis. Another unique advantage of using ESI/MS
to measure Zn2+ content is the ability to detect multiple
enzyme populations that have different Zn2+ contents within
the same sample. This is evident in the ESI/MS data for D103N, C104R,
and C104R/C155V, in which two enzyme populations containing one or two
zinc molecules/enzyme molecule were observed. This novel and powerful
application of ESI/MS has not been applied previously in studies of
metallo--lactamases.
The substitution of the Zn2-coordinating residue Cys181
with Ser resulted in dramatic changes in enzymatic function with
reduced hydrolytic activity for all the substrates tested (Table II)
and reduced susceptibility to the inhibitor BRL 42715 (Table III). ESI/MS indicated the presence of only one zinc atom/molecule of C181S
(Fig. 2, c and d). This might result from the
substitution of a negatively charged cysteine sulfur with a serine
hydroxyl or the inability of the two remaining ligands
(Asp103 and His223) to capture a
Zn2+ atom in the absence of the binding contribution of
Cys181. Recent x-ray crystallographic data (17) indicated
that, although the overall structure of the C181S enzyme remains the
same as the wild-type enzyme, with the side chain of Ser181
occupying the same spatial position as that of Cys181,
there is no electron density at the position of Zn2. Furthermore, in
C181S, the distance between the remaining Zn1 and Wat1 was extended. It
has been proposed that Wat1 exists as a hydroxide ion in the wild-type
enzyme and is responsible for carrying out the nucleophilic attack on
the -lactam ring. Based on the changes in the Wat1 coordination, it
is unlikely that Wat1 exists as a hydroxide ion in the C181S enzyme
(17). This is likely to be the major factor affecting the catalytic
activity of this enzyme. Interestingly, studies of the Bacillus
cereus metallo-
-lactamase II (2, 18) demonstrate the mono-zinc
form of the enzyme to be nearly as active as the di-zinc form.
Another substitution that affects Zn2 binding is the replacement of
Asp103 with Asn. ESI/MS data indicated that 29% of the
D103N molecules harbored one zinc atom, whereas 71% harbored two zinc
atoms (Fig. 2, e and f; and Table IV). However,
the enzymatic activity of D103N was <71% that of CcrA3. Thus, the
reduced hydrolytic activity for CLD and IMP and the decreased affinity
for PenG cannot be fully accounted for by assuming that the di-zinc
form of the enzyme is fully active. The carboxylate group of
Asp103 is predicted to function as a general base during
hydrolysis (17), forming interactions with both Zn2 and Wat1. The
decreased enzymatic activity may more accurately reflect the inability
of Asn to function as a general base to activate Zn2 and Wat1 as effectively as Asp. Substitution of Asp103 with Val has
been reported by Crowder et al. (9). For this enzyme, the
metal content (determined using AAS) was reported to be 0.43 ± 0.09. This is significantly less than the 1.2 zinc molecules/enzyme
molecule observed for the D103N enzyme using AAS, suggesting that the
valine-substituted enzyme is less effective at incorporating zinc into
the active site than the Asn substitution. The
kcat value for the Val enzyme, using the
cephalosporin nitrocefin as a substrate, decreased 6400-fold.
Similarly, the kcat value for the Asn enzyme,
using cephaloridine as a substrate, showed a 260-fold decrease. In
contrast to the cephaloridine kcat value, only a
2-fold decrease in the kcat value for penicillin
G was observed for the D103N enzyme. This minor effect suggests that the main function of Asp103 is to coordinate and activate
Zn2 and that it plays no direct role in the catalytic cycle as might be
predicted, and has been proposed, from the nitrocefin hydrolysis data
(9). Substitution of Asp103 clearly has an additional
effect on cephalosporin hydrolysis beyond the decreased zinc binding.
Both the Val and Asn substitutions may affect the active-site
architecture such that cephalosporin hydrolysis is more dramatically
affected than the hydrolysis of other classes of -lactams.
His99 is one of the three histidine residues that ligate to
Zn1 (Fig. 1). Kinetic and inhibition data (Tables II and III) indicated that His99 plays an active role in enzymatic function since
the physiological efficacy of H99N was reduced 2000-3000-fold for PenG
and IMP and >200-fold for CLD. Interestingly, H99N also had a 29-fold
higher hydrolytic activity for nitrocefin (a chromogenic cephalosporin) than PenG (data not shown). This indicated that some cephalosporins might fit in the structure of H99N better than five-membered ring -lactams. As discussed previously, two zinc molecules/enzyme molecule was observed using ESI/MS, whereas AAS identified only one
zinc molecule/enzyme molecule. Computer modeling predicted the distance
between Asn99 and Zn1 to be 4.5 Å (generated using the
coordinates from 1ZNB), compared with the distance between
His99 and Zn1 of 2.2 Å (1). Interestingly, the
metallo-
-lactamase CphA from Aeromonas hydrophila AE036
(19) has a naturally occurring Asn at residue 99. This enzyme shows
substrate specificity for carbapenems. Such substrate specificity was
not observed with the CcrA3 H99N mutant enzyme.
Although not directly ligated to either zinc molecule, substitutions of
Cys104 (C104R and C104S/C155V) resulted in proteins
harboring either one or two zinc atoms/enzyme molecule. The location of
Cys104 within the enzyme might play a critical role in this
phenomenon. Cys104 is at the active site and close to both
zinc molecules (Fig. 1). The sulfhydryl group on the cysteine might
contribute more stability to the protein structure than either arginine
or serine. Residue 104 is an arginine in the B. cereus
-lactamase II (20). Comparing the substrate profile for this enzyme
(21), the kcat value for PenG was 3.8-fold
higher than that for IMP. The C104R enzyme also showed stronger
hydrolytic activity for PenG (5.9-fold) than for IMP, compared with a
1.2-fold difference for CcrA3 (Table II). The double mutant C104S/C155V
contains the same residues at positions 104 and 155 as the
metallo-
-lactamase IMP-1 from Serratia marcescens (22).
IMP-1 has stronger hydrolytic activity for PenG than for IMP. The same
pattern was also seen for the C104S/C155V enzyme. The reduced
hydrolytic activity of
-lactamase II and IMP-1 for CLD (22, 23) was
not observed in either of the CcrA Cys104 substitutions and
may relate to differences in other regions of these enzymes.
Amino acids 46-51 form a "disordered loop" in CcrA3 (1). Removal
of these six amino acids significantly affected the activity against
PenG and CLD, the predominant effect being on substrate affinity
(increased Km) (Table II). Activity against IMP and
susceptibility to BRL 42715 were only marginally affected. The
crystallographic structure of CcrA3 indicates that residues 46-51
compromise the end of a hairpin loop that protrudes into the solvent
(1). This "flap" could adjust upon substrate binding to accommodate
a variety of -lactam side chain substitutions (1). Removal of the
loop may restrict substrate specificity, resulting in reduced affinity
for many
-lactams.
The data presented were obtained using the powerful technique of ESI/MS
to assess the Zn2+ content and its relation to enzymatic
activity. The Zn2+ content was differentially affected by
various amino acid substitutions. From these analyses, it becomes clear
that the zinc content was not the only factor contributing to the
observed changes in the enzymatic activity of these mutant enzymes.
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ACKNOWLEDGEMENTS |
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We are grateful to Niraja Bhachech for performing enzyme kinetic studies and to Ramaswamy Nilakantan for the preparation of Fig. 1. We also thank Elizabeth Glasfeld for critical reading of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: 205/214, Infectious
Disease, Wyeth-Ayerst Research, 401 N. Middletown Rd., Pearl River, NY
10965. Tel.: 914-732-4569; Fax: 914-732-2480; E-mail: RasmusB{at}war.wyeth.com.
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ABBREVIATIONS |
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The abbreviations used are: Wat, water; ESI/MS, electrospray ionization mass spectrometry; AAS, atomic absorption spectrophotometry; PCR, polymerase chain reaction; PenG, penicillin G; CLD, cephaloridine; IMP, imipenem.
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REFERENCES |
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