(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
-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.
Most class C -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.
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 -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.
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).
[-
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.
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, 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).
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
-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.
Figure 1:
DNA sequence of the E. cloacae GC1 -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) .
Figure 2:
Comparison of the deduced amino acid
sequence, in part, of the E. cloacae GC1 -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.
Figure 3:
Structures of the -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).
Figure 4:
Graphical illustration of the hybrid genes
from the GC1 and P99 -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.
The work described here shows for the first time an extended
specificity class C -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.
Recently, the
crystalline structure of the P99 -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.
Figure 5:
Stereoview of the polypeptide segment
including the active site serine and the HVP sequence of the C.
freundii -lactamase, corresponding to the AVR sequence of the
P99 and GC1
-lactamase.
The GC1 -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.
Joris et al.(25) demonstrated seven conserved boxes in the amino acid
sequences of all the serine -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.