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(Received for publication, November 10, 1994; and in revised form, January 9, 1995) From the
Enterobacter cloacae GC1, a clinical strain isolated in
1992 in Japan, was found to produce a chromosomal class C
Most class C We have been conducting studies among
clinical isolates of C. freundii, Enterobacter
cloacae, and Escherichia coli and in the process
discovered a new strain, E. cloacae GC1, which produces a
class C
Enzymes and enzyme kits for DNA technology were
purchased from Stratagene (La Jolla, CA), Takara Shuzo Co. (Kyoto,
Japan), Toyobo Co. (Osaka, Japan), and Wako Junyaku Co. (Tokyo, Japan).
[
For the primary PCR reactions, the following
combinations of oligonucleotides listed in Table 1were employed:
primer 1, 207AAAr; primer 2, 207AAA; primer 1, 211AAAr; and primer 2,
211AAA. Mutation was performed in a 100-µl reaction mixture
comprising 0.1-0.3 µg of template DNA (pCS900), 200
µM each of dNTP, 0.5 µM each of
oligodeoxynucleotide, 2.5 units of Taq DNA polymerase, and the PCR
buffer composed of 10 mM Tris HCl, pH 9.0, 50 mM KCl,
3.5 mM MgCl The secondary PCR
reaction for relocation of the mutation was performed with 0.3 µg
of each first PCR products in a 100-µl reaction mixture, the
composition of which was similar to that used in the first reaction
except for the lack of the primer oligodeoxynucleotides. Amplification
involved 5 cycles of 1 min of denaturation at 93 °C, 2 min of
annealing at 45 °C, and 2 min of extension at 72 °C. After
primers 1 and 2 (50 pmol each) were added to the reaction mixture, 25
cycles of the PCR reaction were continued under the same conditions
except that the annealing temperature was increased to 55 °C. The
synthesized DNA was purified by the same procedure as mentioned above
and digested with AflII. The resulting 200-nucleotide DNA
fragment was replaced with the corresponding region of the P99
Figure 1:
DNA sequence of the E. cloacae GC1
Figure 2:
Comparison of the deduced amino acid
sequence, in part, of the E. cloacae GC1
Figure 3:
Structures of the
Figure 4:
Graphical illustration of the hybrid genes
from the GC1 and P99
The work described here shows for the first time an extended
specificity class C Recently, the
crystalline structure of the P99
Figure 5:
Stereoview of the polypeptide segment
including the active site serine and the HVP sequence of the C.
freundii
The GC1 Joris et al.(25) demonstrated seven conserved boxes in the amino acid
sequences of all the serine
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 5729-5735
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Lactamase Extending Its Substrate
Specificity (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-lactamase with extended substrate specificity to oxyimino
-lactam antibiotics, significantly differing from the known E.
cloacae -lactamases such as the P99 -lactamase. The 1560
nucleotides including the GC1 -lactamase gene were sequenced, and
the amino acid sequence of the mature enzyme comprising 364 amino acids
was deduced. A comparison of the amino acid sequence with those of
known E. cloacae -lactamases revealed the duplication of
three amino acids at positions 208-213, i.e. Ala-Val-Arg-Ala-Val-Arg. This duplication was attributed to a
tandem duplication of a 9-nucleotide sequence. The chimeric
-lactamases produced by the chimeric genes from the GC1 and P99
-lactamase genes indicated that the extended substrate specificity
is entirely attributed to the 3-amino acid insertion. Two mutant
-lactamases were prepared from P99 -lactamase by
site-directed mutagenesis, i.e. an Ala-Ala-Ala sequence was
inserted before or after the native Ala-Val-Arg at positions
208-210. These mutant enzymes revealed that the Ala-Val-Arg
located from positions 211 to 213 in the GC1 -lactamase are the
newly inserted residues, and this phenomenon is independent of the
characteristics of the amino acids inserted.
-Lactamases (EC 3.5.2.6) are enzymes responsible for
bacterial resistance to -lactam antibiotics and are classified
into four classes, A, B, C, and D according to the homology of the
primary amino acid sequence(1, 2) . -Lactamases,
except class B enzymes belonging to metallo--lactamases, are the
serine enzymes that have serine as their active site participating in
the formation of an acyl-enzyme intermediate with a
-lactam(3) . According to the traditional grouping based
on substrate specificity, class A and D enzymes are penicillinases, and
class C enzymes are the so-called cephalosporinases. The introduction
of an oxyimino group into the side chain at position 7 of the
cephalosporin nucleus or at position 3 of the monobactam nucleus is the
major means of protecting a -lactam bond from hydrolysis by the
serine -lactamases. After the introduction of oxyimino
-lactams into clinical medicine in the early 1980s, R
plasmid-mediated resistance to oxyimino -lactams as well as to
usual -lactams occurred in clinical isolates of Klebsiella
pneumoniae and other enteric bacteria(4) . This developed
resistance is attributed to the extended substrate specificity
-lactamases of class A, which originated from TEM- or SHV-type
-lactamases by the replacement of 1-4 amino acids in their
ancestral enzymes.-lactamases are produced in
Gram-negative bacteria as chromosomal -lactamases. Distinct from
the case of class A -lactamases, a class C -lactamase with
the extended substrate specificity has not yet been found from a
clinical isolate. On the other hand, during the course of our
investigation of the functional amino acids in the active site of a
class C -lactamase of Citrobacter freundii GN346, we
isolated the mutant enzymes with extended substrate specificity to
oxyimino -lactams by an amino acid substitution on a loop
structure between 
-8 and -9 helices of the class C
-lactamase(5, 6) . The finding of such a mutant
enzyme suggested to us the appearance of a naturally occurring mutant
enzyme with broad specificity.-lactamase capable of hydrolyzing oxyimino -lactams.
The extended substrate specificity was confirmed because of duplication
of the 3-amino acid sequence on the loop structure. This is an example
of molecular evolution of a bacterial enzyme against new -lactams
in nature. In this paper, we report the properties of GC1
-lactamase.
Bacterial Strains and Plasmids
E. cloacae GC1 is a clinical isolate found to be highly resistant to oxyimino
-lactams such as ceftazidime and aztreonam in the latter half of
1992 in Japan. E. cloacae P99 is a typical strain of this
species producing a chromosomal class C -lactamase of this species (7) and was kindly provided by Dr. A. F. Ehrhardt of Creighton
University, Omaha. E. coli TG1(8) , a derivative of
K12, was employed for DNA technology. E. coli AS226-51(9) , an ampD mutant of C600, which
also has a deletion mutation in ampC, was used for measuring
the -lactam susceptibility of cells bearing the cloned
-lactamase gene and as host cells for enzyme preparation in order
to avoid contamination by the ampC -lactamase of E. coli. Plasmids pHSG398 (10) and M13mp18 (11) were used
as the cloning vector and the vector for DNA sequencing, respectively.
pTTQ18 carrying a Tac promoter was purchased from Amersham
Corp. and employed for preparing a high expression vector for the
-lactamase production.Media, Chemicals, and Enzymes
Nutrient broth
(Eiken Chemical Co., Tokyo, Japan) was used as the culture medium for
chromosomal DNA preparation. For transformation and transfection, 2
yeast extract/tryptone (2 YT) broth and YT agar (12) were employed, respectively. For the -lactamase
preparation, bacteria were grown in heart infusion broth (Eiken
Chemical Co., Tokyo, Japan). Heart infusion agar (Eiken Chemical Co.,
Tokyo, Japan) was used for measuring bacterial susceptibility to
-lactams.-
P]dCTP was purchased from Amersham Corp.
The antibiotics used in this study were kindly provided by the
following pharmaceutical companies: benzylpenicillin, ampicillin, and
kanamycin from Meiji Seika Kaisha Ltd., Tokyo, Japan; cephalothin from
Shionogi & Co., Ltd., Osaka, Japan; cefuroxime and ceftazidime from
Nippon Glaxo Ltd., Tokyo, Japan; cefoxitin from Daiichi Pharmaceutical
Co., Tokyo, Japan; and aztreonam from Eisai Co., Tokyo, Japan.Cloning of the
Chromosomal DNA of the E. cloacae cells was
prepared according to the procedure of Owen and Borman(13) . A
1.6-kb (-Lactamase Gene and DNA
Sequencing
)DNA fragment containing the -lactamase gene
was cloned from the chromosomal DNA fractions of E. cloacae GC1 by the polymerase chain reaction (PCR) method (14, 15) . PCR primers were designed with reference to
the known nucleotide sequence of the P99 -lactamase
gene(7) , and the primers listed in Table 1were
employed. PCR was performed using a MiniCycler (MJ Research Inc.) in a
100-µl reaction mixture comprising 0.3 µg of template DNA, 200
µM each of dNTP, 0.5 µM of each primer, and
PCR buffer (10 mM Tris-HCl, pH 9.0, 50 mM KCl, 3.5
mM MgCl
, 0.1% Triton X-100, 0.01% bovine serum
albumin). The reaction was initiated with 2.5 units of Taq DNA
polymerase, and the amplified DNA fragments were digested with BamHI and then ligated into the BamHI site of
pHSG398. The recombinant plasmid, termed pCS100, was transformed into E. coli AS226-51, and the positive transformants were
selected with reference to their ceftazidime resistance and
-lactamase activity. A 1.6-kb DNA fragment with the
-lactamase gene was cloned from the chromosomal DNA of P99 into
pHSG398 in a similar manner as above, and the recombinant plasmid was
termed pCS900. Those 1.6-kb DNA fragments were sequenced by the dideoxy
chain termination method (16) using a Bca BEST(TM) Dideoxy
sequencing kit or AmpliTaq(TM) sequencing kit (Takara Shuzo Co.).
Construction of High Expression Vector
A high
expression vector, pTTQ18K, was prepared from pTTQ18 by replacement of
the ampicillin resistance gene on the original vector with a kanamycin
resistance gene derived from pUC4K. Using primer 2 and primer ECPTTQ (Table 1), the -lactamase genes in pCS100 and pCS900 were
amplified by the use of the PCR method. The resulting DNAs carrying
-lactamase genes were digested with EcoRI and BamHI, and then the EcoRI-BamHI fragment was
inserted into EcoRI-BamHI site of pTTQ18K. The 750-bp
DNA fragment was prepared by the digestion with KpnI and XbaI of the recombinant plasmid, and the fragment was
substituted for the counterpart in pCS100 and pCS900, respectively. The
resulting plasmids were termed pCS101 and pCS901, respectively. It was
confirmed by direct sequencing that an unwanted mutation in the
-lactamase genes was absent.Site-directed Mutagenesis of the P99
Site-directed mutagenesis was carried out using
the overlap extension method, a modification of the PCR method,
developed by Higuchi et al.(17) and Ho et
al.(18) . Insertion of 3 alanine residues at amino acid
position 207 or 211 of the P99 -Lactamase-lactamase was performed using
oligonucleotides listed in Table 1. Those primers were
synthesized using a Cyclone Plus DNA/RNA synthesizer (Milligen
Biosearch Co.)., 0.1% Triton X-100, and 0.01% bovine
serum albumin. Amplification involved 25 cycles of 1 min of
denaturation at 93 °C, 2 min of annealing at 55 °C, and 2 min
of extension at 72 °C. After the PCR reaction, 2 µl of 10
mM each of dNTP and 2.5 units of Klenow were added into the
reaction mixture, followed by 10 min of incubation at 37 °C. The
synthesized DNA was purified by agarose gel electrophoresis and the
Geneclean II kit (BIO 101, Inc., Vista, CA).-lactamase gene on pCS900. About 200 bp around the AflII
restriction site in the mutant gene was entirely sequenced to confirm
the desired exchange in the nucleotide sequence by the chain
termination method.
E. coli AS226-51 cells carrying pTTQ18K
with the -Lactamase Purification and -Lactamase
Assay-lactamase gene were grown overnight in heart infusion
broth containing a sublethal concentration of kanamycin (30 µg/ml)
at 37 °C. The preculture was diluted with a 40-fold volume of fresh
medium followed by growth at the same temperature under aeration until
the midlogarithmic phase and induction with 1 mM isopropyl-1-thio--D-galactopyranoside for 12 h.
Crude -lactamase was prepared by disruption of the cells with a
French press in 50 mM sodium phosphate buffer, pH 7.0,
followed by centrifugation for 1 h at 40,000 g and 4
°C after removal of cell debris. The crude enzyme was purified
according to the procedure used for purification of the C. freundii -lactamase(19) . The enzyme was purified to
homogeneity by ion exchange chromatography on a CM-Sephadex C-50 column
in 10 mM sodium phosphate buffer (pH 6.0) with a 0-0.6 M linear NaCl gradient followed by gel filtration on a
Sephadex G-75 column in 100 mM sodium phosphate buffer (pH
7.0); its purity was confirmed by SDS-polyacrylamide gel electrophores:
-Lactamase activity was assayed by the microiodometric method of
Novick (20) with slight modification and by a UV
spectrophotometric method(21) . One unit of enzyme was defined
as the amount of enzyme that hydrolyzed 1 µmol of substrate in 1
min at pH 7.0 and 30 °C. The kinetic parameters, K
and K
, were determined by procedures
reported previously(22) .Isoelectric Focusing
Isoelectric focusing was
carried out with an Atto model SJ-1071 apparatus (Atto Co., Tokyo,
Japan) and a gel plate containing 5% ampholine (pH 3.5-9.5). The
enzyme protein on the gel plate was detected by staining with Coomassie
Brilliant Blue.Antibiotic Susceptibility Testing
Bacterial
susceptibility to -lactams was measured by the serial agar
dilution method (23) and expressed as the minimum inhibitory
concentration (µg/ml) of a drug.Nucleotide Sequence Accession Number
The
nucleotide sequence data reported in this paper will appear in the
GSDB, DDBJ, EMBL, and NCBI nucleotide sequence data bases with the
following accession number D44479.
Properties of E. cloacae GC1
E. cloacae GC1 showed higher resistance to typical -lactams, including
oxyimino cephalosporins and monobactam such as ceftazidime and
aztreonam, than did E. cloacae P99, the latter of which is
known to produce high amounts of an inducible class C -lactamase
of this bacterial species. MIC levels of ceftazidime and aztreonam to
GC1 were 800 and 400 µg/ml, respectively, which were 8-63
times that to P99. E. cloacae GC1 produced a constitutive
-lactamase, and its specific activity in the cell extract was 2.2
units/mg of bacterial protein, using cephalothin as the substrate,
which is about half that of the cell extract from the P99 cells fully
induced by an inducer, cefoxitin (50 µg/ml). However, the extract
from the GC1 cells exhibited a high hydrolytic activity to cefuroxime,
about 100 times that of the extract from the P99 cells. The GC1
-lactamase was estimated to have an isoelectric point higher than
8.5, which is an indication that it belongs to a chromosomal class C
-lactamase.Cloning and Sequencing of the GC1
A 1.6-kb DNA fragment containing the -Lactamase Gene-lactamase gene was
cloned from the chromosomal DNA fractions of E. cloacae GC1
and E. cloacae P99 by the PCR method. The 1.6-kb DNA fragments
were ligated into the BamHI site of pHSG398. The recombinant
plasmids carrying the GC1 gene and the P99 gene were termed pCS100 and
pCS900, respectively. E. coli AS226-51 cells harboring
pCS100 showed markedly higher resistance (4-16 times higher) to
oxyimino -lactams than the bacterial cells harboring pCS900 (data
not shown). The 1560-bp segment of the 1.6-kb DNA fragment in pCS100
was completely sequenced (Fig. 1). The sequence region was found
to contain an open reading frame starting from nucleotide position
390-1544, and the amino acid sequence composed of 364 amino acids
was deduced. When the amino acid sequence was compared with that of the
P99 -lactamase(7) , 359 of the 364 amino acids were found
to be identical to the corresponding residues of the P99 enzyme. On the
basis of the P99 amino acid sequence, the observed differences between
the two -lactamases are as follows: Ile
Val,
Ala
Pro, and
Ala
-Val
-Arg
Ala-Val-Arg-Ala-Val-Arg. These differences in amino acid sequences were
reconfirmed by direct sequencing of the GC1 DNA by the aid of the PCR
method. Fig. 2shows the alignment of the three regions with
those of three known -lactamases of E. cloacae, the E. coli -lactamase, and the C. freundii -lactamase. These alignments indicated that valine at
position 16 is common in the E. cloacae -lactamases and
that proline at position 88 is also common in many class C
-lactamases. The most remarkable feature of the GC1 enzyme is an
insertion sequence, Ala-Val-Arg, suggesting a duplicative mutation on
the chromosomal gene.
-lactamase gene and flanking regions as well as the
predicted amino acid sequence for the enzyme. The nucleotides are
numbered from the BamHI site. The position of the N-terminal
amino acid of the mature enzyme is designated as position 1 of the
amino acid sequence. The amino acid sequence from -20 to -1
is assumed to be the signal peptide. The active site serine at position
64 is indicated by an arrowhead. Amino acids in the
duplicative region are indicated by thick letters. The stop
codon is indicated by an asterisk. The shaded and underlined regions in the sequence indicate -helices and
-strands, respectively, the indication being made on the basis of
the sequence alignment of the enzyme with the C. freundii class C -lactamase(24) .
-lactamase with
those of other class C -lactamases. The sequence alignment of the
GC1 -lactamase is performed with the enzymes of E. cloacae P99(7) , E. cloacae Q908R(7) , E.
cloacae MHN1(7) , C. freundii GN346(9) ,
and E. coli K12(25) . The three variable regions are boxed, and the gap between the GC1 enzyme and other class C
enzymes is indicated with hyphens.
Purification of the GC1
The observations in the preceding genetic
experiments suggested that the GC1 -Lactamase and Its Kinetic
Properties-lactamase is a naturally
occurring mutant of the chromosomal -lactamase. The duplicative
mutation may confer on the enzyme a unique substrate specificity
extending to oxyimino -lactams. The GC1 -lactamase and the
P99 -lactamase as the reference enzyme were extracted from the E. coli cells carrying the high expression vectors and
completely purified. The kinetic parameters of the E. cloacae -lactamases for six -lactams are summarized in Table 2, and chemical structures of the -lactams are shown
in Fig. 3. Catalytic activity, expressed as k
, was determined for traditional cephalosporin
(cephalothin), two traditional penicillins (benzylpenicillin and
ampicillin), and three oxyimino -lactams (cefuroxime, ceftazidime,
and aztreonam). The k value of the GC1 enzyme
for cephalothin, a favorable substrate for class C -lactamases,
was 88% that of the P99 enzyme. Although the K value of the GC1 enzyme for cephalothin was somewhat higher than
that of the P99 enzyme, no significant difference in catalytic activity
toward the favorable substrate was observed between the two E.
cloacae -lactamases. On the other hand, the catalytic
activity of the GC1 enzyme for oxyimino -lactams, unfavorable
substrates for usual class C -lactamases, was significantly higher
than that of the P99 -lactamase. Examples of the striking
difference can be seen in the case of cefuroxime. It should be
emphasized that the increase in the catalytic activity was accompanied
by an increase in the K or K values for those unfavorable substrates. This result is very
similar to the observation in the cases of the mutant -lactamases
of C. freundii, which extended its substrate spectrum toward
oxyimino -lactams by substitution of Glu
with other
amino acids(6) .
-lactams used in
this study. Three classical -lactams are represented by
benzylpenicillin, ampicillin, and cephalothin. Three oxyimino
-lactams include two oxyiminocephalosporins (cefuroxime and
ceftazidime) and a monobactam with an oxyimino group in the side chain
(aztreonam).
Construction of Hybrid Genes from the GC1 and P99
To confirm that the
duplicative mutation on the loop structure is the sole reason for the
extended substrate spectrum found in the GC1 -Lactamase Genes and Their Phenotypes-lactamase, two
chimeric -lactamase genes were constructed as illustrated in Fig. 4. A KpnI restriction enzyme site in the E.
cloacae -lactamase genes was utilized as a recombination
site, and the resulting recombinant plasmids were termed pCS102 and
pCS902. The chimeric -lactamase gene on pCS902 has the duplicative
mutation but has the same amino acids at positions 16 and 88 as those
found in the common E. cloacae -lactamases. The chimeric
-lactamase gene on pCS102 corresponds to a modified GC1
-lactamase gene in which the duplicative mutation is eliminated.
MIC levels of E. coli AS226-51 cells harboring the
plasmids were examined to five -lactams, and the extracts from the
cells were assayed for their activity toward the -lactams (Table 3). It was evident from the experimental facts that the
duplicative mutation of the Ala-Val-Arg is solely attributed to the
extended substrate spectrum of the GC1 -lactamase toward oxyimino
-lactams.
-lactamase genes. pCS100 and pCS900 carry the
GC1 and P99 -lactamase genes, respectively. The boxed areas denote the cloned DNA fragments, and the large boxes represent the
-lactamase structural genes. The three variable regions in Fig. 2are shown by open or shaded regions, and
a widely shaded region denotes the AVRAVR sequence. pCS102 and pCS902
carry the hybrid genes, which were constructed by recombination at the Kpnl restriction site between position 88 and the AVR
region.
Insertion of Ala-Ala-Ala before or after the Ala-Val-Arg
Sequence of the P99
It was thought to be of interest to
identify which is the newly inserted Ala-Val-Arg and whether an
Ala-Val-Arg sequence is essential for the unique phenotype or not. Two
mutant enzymes were prepared on the basis of P99 -Lactamase and Its Effect on the Activity to
Oxyimino -Lactams-lactamase, i.e. the mutant enzymes with an Ala-Ala-Ala insertion before
Ala (AAA-Ala) and after Arg (Arg-AAA). These mutant genes were prepared by
means of a modified PCR method(17, 18) , and the
plasmids carrying the mutant genes were termed pCS903 and pCS904,
respectively. E. coli AS226-51 cells harboring these
plasmids were compared in terms of their MIC levels to five
-lactams, and the cell extracts were assayed for their hydrolytic
activity to the -lactams (Table 4). It is evident from the
experimental results that only an insertion after Arg causes the phenotype that exhibits an extended substrate spectrum
and that this phenomenon is independent of the characteristics of the
amino acids inserted.
-lactamase capable of hydrolyzing oxyimino
-lactams, produced by a clinical isolate. It should be emphasized
that this finding was predicted from the preceding investigations on
the in vitro mutants of the C. freundii -lactamase(5, 6) . Oxyimino -lactams
such as cefuroxime, ceftazidime, and aztreonam act as progressive
inhibitors of a class C -lactamase of C. freundii, and a
minimum scheme for hydrolysis of the -lactam ring of oxyimino
-lactams by the class C -lactamase was proposed(22) .
This reaction sequence includes two kinds of acyl-enzyme intermediates, i.e. an unstable complex and a stable complex, and the
rate-limiting step in the sequence was identified as that which
converts the stable acyl-enzyme complex into an unstable one. When
glutamic acid at position 219 located on the loop structure between
-8 and -9 helices of the C. freundii -lactamase
was substituted for lysine, the reaction rate at the rate-limiting step
for aztreonam hydrolysis became 2000 times that of the wild-type
enzyme. A similar phenomenon was observed in the mutant enzyme with the
substitution of aspartic acid at position 217 for lysine(26) ,
and position 217 is located on the loop structure as well as position
219(24) . These observations on the in vitro mutant
enzymes suggest the existence of the extended substrate specificity
class C -lactamases in nature. The sequence alignment between E. cloacae and C. freundii -lactamases indicated
that the loop structure corresponds to a region of the E. cloacae enzyme from Val
to Asn
(Asn
in the GC1 enzyme). The inserted Ala-Val-Arg in the GC1 enzyme
just locates on the loop structure. It was also confirmed that the
substitution of glutamine at position 219 of the P99 -lactamase, a
counterpart of Glu in the C. freundii enzyme,
for lysine certainly changes the P99 enzyme to an extended substrate
specificity enzyme. ()These observations indicate that the
structure, known as a hot spot for the extended substrate specificity
mutation, is common among class C -lactamases.-lactamase was
reported(27) , and the proposed structure for the active site
space shows that Ala, Val, and Arg are not the functional residues and are unable to interact
directly with a substrate adapted to the active site hollow. The
Pro-Val-His at positions 208-210 of the C. freundii -lactamase corresponds to the Ala-Val-Arg in the P99 enzyme.
The stereoview of the Pro-Val-His in the enzyme structure was
constructed as reported previously (28) (Fig. 5), and
the actual distance between the 3 residues and the functional residues
such as Ser
and Lys
is similar to that of the
P99 enzyme.
-lactamase, corresponding to the AVR sequence of the
P99 and GC1 -lactamase.
-lactamase showed lower affinity for
oxyimino -lactams than the P99 enzyme as can be seen by higher K or K values (Table 3), and the P99 mutant enzymes with the 3-amino acid
insertion at position 211 showed similar kinetic characteristics to
those of the GC1 enzyme. These results obtained from the E. cloacae mutant enzymes are the same as those found between the E219K
mutant and the wild-type C. freundii -lactamase(5) . This kinetic feature common between
the in vitro mutant and the GC1 enzyme suggests that the high
activity of the GC1 enzyme toward oxyimino -lactams may be
attributable to an acceleration of the rate-limiting step, as mentioned
above. The Ala-Ala-Ala insertion after position 210 of the P99
-lactamase showed almost the same extended substrate specificity
as that found in the GC1 enzyme; however, the insertion before position
208 did not lead to the same effect. This fact is of interest in
connection with the relationship between the alteration in the
molecular configuration and the extended substrate specificity and may
be understood if we assume that Ala-Val-Arg is relatively fixed in the
molecular configuration of the enzyme and that the amino acid insertion
after Ala-Val-Arg causes an effective stretching of the loop structure
toward the active site center. As shown in Fig. 5, His has a possibility to interact with Thr
and
Gln
. The basic amino acid in the three amino acids may
act as a fixed point of the loop structure. Confirmation of this
assumption is under way.-lactamases and the penicillin-binding
proteins, and box V was found to locate on the loop structure in
question. One of the significant differences in molecular structure
found between class A and C -lactamases is directionality of a
peptide comprising the loop structure. This region in class A enzyme
extends in a direction reverse of that in class C enzyme(27) .
A 3-amino acid sequence of TEM-1 class A -lactamase, corresponding
to the Ala-Val-Arg in the E. cloacae enzyme, was estimated to
be Glu-Ala-Ile at positions 171-173 by superpositioning of the
two enzymes. We constructed a duplicative mutant of the 3-amino acid
sequence; however, the resulting TEM-1 mutant does not show the
extended substrate specificity. On the other hand, Sowek et al.(29) reported that TEM-1 -lactamase
greatly increased its hydrolytic activity toward ceftazidime by
replacement of Arg
with serine. They suggested that this
position is distant from the active site cleft and that this phenomenon
was by an indirect effect of the substitution on the active site. It is
tempting to speculate that the loop structure acts as the hot spot for
modification of substrate specificity of both class A and C
-lactamases.
))
We are grateful to A. F. Ehrhardt for providing E. cloacae P99.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 H. Mammeri, L. Poirel, and P. Nordmann Extension of the hydrolysis spectrum of AmpC {beta}-lactamase of Escherichia coli due to amino acid insertion in the H-10 helix J. Antimicrob. Chemother., September 1, 2007; 60(3): 490 - 494. [Abstract] [Full Text] [PDF]
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M. C. de la Prieta, M. V. Francia, A. Seoane, and J. M. G. Lobo Characterization of defective {beta}-lactamase genes in Yersinia enterocolitica J. Antimicrob. Chemother., September 1, 2006; 58(3): 661 - 664. [Abstract] [Full Text] [PDF]
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Y. Doi, J.-i. Wachino, M. Ishiguro, H. Kurokawa, K. Yamane, N. Shibata, K. Shibayama, K. Yokoyama, H. Kato, T. Yagi, et al. Inhibitor-Sensitive AmpC {beta}-Lactamase Variant Produced by an Escherichia coli Clinical Isolate Resistant to Oxyiminocephalosporins and Cephamycins Antimicrob. Agents Chemother., July 1, 2004; 48(7): 2652 - 2658. [Abstract] [Full Text] [PDF]
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 M. Nukaga, S. Kumar, K. Nukaga, R. F. Pratt, and J. R. Knox Hydrolysis of Third-generation Cephalosporins by Class C {beta}-Lactamases: STRUCTURES OF A TRANSITION STATE ANALOG OF CEFOTAXIME IN WILD-TYPE AND EXTENDED SPECTRUM ENZYMES J. Biol. Chem., March 5, 2004; 279(10): 9344 - 9352. [Abstract] [Full Text] [PDF]
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X. Kong, Z. Li, X. Gou, S. Zhu, H. Zhang, X. Wang, and J. Zhang A Monomeric L-Aspartase Obtained by in Vitro Selection J. Biol. Chem., June 28, 2002; 277(27): 24289 - 24293. [Abstract] [Full Text] [PDF]
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S. B. Vakulenko, D. Golemi, B. Geryk, M. Suvorov, J. R. Knox, S. Mobashery, and S. A. Lerner Mutational Replacement of Leu-293 in the Class C Enterobacter cloacae P99 {beta}-Lactamase Confers Increased MIC of Cefepime Antimicrob. Agents Chemother., June 1, 2002; 46(6): 1966 - 1970. [Abstract] [Full Text] [PDF]
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A. Philippon, G. Arlet, and G. A. Jacoby Plasmid-Determined AmpC-Type {beta}-Lactamases Antimicrob. Agents Chemother., January 1, 2002; 46(1): 1 - 11. [Full Text] [PDF]
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Z. Zhang, Y. Yu, J. M. Musser, and T. Palzkill Amino Acid Sequence Determinants of Extended Spectrum Cephalosporin Hydrolysis by the Class C P99 beta -Lactamase J. Biol. Chem., November 30, 2001; 276(49): 46568 - 46574. [Abstract] [Full Text] [PDF]
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C. Arpin, R. Labia, C. Andre, C. Frigo, Z. El Harrif, and C. Quentin SHV-16, a {beta}-Lactamase with a Pentapeptide Duplication in the Omega Loop Antimicrob. Agents Chemother., September 1, 2001; 45(9): 2480 - 2485. [Abstract] [Full Text] [PDF]
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A. Raimondi, F. Sisto, and H. Nikaido Mutation in Serratia marcescens AmpC {beta}-Lactamase Producing High-Level Resistance to Ceftazidime and Cefpirome Antimicrob. Agents Chemother., August 1, 2001; 45(8): 2331 - 2339. [Abstract] [Full Text] [PDF]
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D. Girlich, T. Naas, S. Bellais, L. Poirel, A. Karim, and P. Nordmann Heterogeneity of AmpC Cephalosporinases of Hafnia alvei Clinical Isolates Expressing Inducible or Constitutive Ceftazidime Resistance Phenotypes Antimicrob. Agents Chemother., November 1, 2000; 44(11): 3220 - 3223. [Abstract] [Full Text]
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S. Iyobe, H. Kusadokoro, J. Ozaki, N. Matsumura, S. Minami, S. Haruta, T. Sawai, and K. O'Hara Amino Acid Substitutions in a Variant of IMP-1 Metallo-beta -Lactamase Antimicrob. Agents Chemother., August 1, 2000; 44(8): 2023 - 2027. [Abstract] [Full Text]
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D. Girlich, T. Naas, S. Bellais, L. Poirel, A. Karim, and P. Nordmann Biochemical-Genetic Characterization and Regulation of Expression of an ACC-1-Like Chromosome-Borne Cephalosporinase from Hafnia alvei Antimicrob. Agents Chemother., June 1, 2000; 44(6): 1470 - 1478. [Abstract] [Full Text]
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S. Trépanier, J. R. Knox, N. Clairoux, F. Sanschagrin, R. C. Levesque, and A. Huletsky Structure-Function Studies of Ser-289 in the Class C beta -Lactamase from Enterobacter cloacae P99 Antimicrob. Agents Chemother., March 1, 1999; 43(3): 543 - 548. [Abstract] [Full Text]
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N. Matsumura, S. Minami, and S. Mitsuhashi Sequences of Homologous beta -Lactamases from Clinical Isolates of Serratia marcescens with Different Substrate Specificities Antimicrob. Agents Chemother., January 1, 1998; 42(1): 176 - 179. [Abstract] [Full Text]
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F. Hayes, B. Hallet, and Y. Cao Insertion Mutagenesis as a Tool in the Modification of Protein Function. EXTENDED SUBSTRATE SPECIFICITY CONFERRED BY PENTAPEPTIDE INSERTIONS IN THE Omega -LOOP OF TEM-1 beta -LACTAMASE J. Biol. Chem., November 14, 1997; 272(46): 28833 - 28836. [Abstract] [Full Text] [PDF]
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