©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Evolution of a Class C -Lactamase Extending Its Substrate Specificity (*)

(Received for publication, November 10, 1994; and in revised form, January 9, 1995)

Michiyoshi Nukaga Shin Haruta Kyoko Tanimoto Keiko Kogure Kazuo Taniguchi Mami Tamaki Tetsuo Sawai (§)

From the Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, 1-33, Yayoi-cho, Inage-ku, Chiba 263, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Enterobacter cloacae GC1, a clinical strain isolated in 1992 in Japan, was found to produce a chromosomal class C beta-lactamase with extended substrate specificity to oxyimino beta-lactam antibiotics, significantly differing from the known E. cloacae beta-lactamases such as the P99 beta-lactamase. The 1560 nucleotides including the GC1 beta-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 beta-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 beta-lactamases produced by the chimeric genes from the GC1 and P99 beta-lactamase genes indicated that the extended substrate specificity is entirely attributed to the 3-amino acid insertion. Two mutant beta-lactamases were prepared from P99 beta-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 beta-lactamase are the newly inserted residues, and this phenomenon is independent of the characteristics of the amino acids inserted.


INTRODUCTION

beta-Lactamases (EC 3.5.2.6) are enzymes responsible for bacterial resistance to beta-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) . beta-Lactamases, except class B enzymes belonging to metallo-beta-lactamases, are the serine enzymes that have serine as their active site participating in the formation of an acyl-enzyme intermediate with a beta-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 beta-lactam bond from hydrolysis by the serine beta-lactamases. After the introduction of oxyimino beta-lactams into clinical medicine in the early 1980s, R plasmid-mediated resistance to oxyimino beta-lactams as well as to usual beta-lactams occurred in clinical isolates of Klebsiella pneumoniae and other enteric bacteria(4) . This developed resistance is attributed to the extended substrate specificity beta-lactamases of class A, which originated from TEM- or SHV-type beta-lactamases by the replacement of 1-4 amino acids in their ancestral enzymes.

Most class C beta-lactamases are produced in Gram-negative bacteria as chromosomal beta-lactamases. Distinct from the case of class A beta-lactamases, a class C beta-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 beta-lactamase of Citrobacter freundii GN346, we isolated the mutant enzymes with extended substrate specificity to oxyimino beta-lactams by an amino acid substitution on a loop structure between alpha-8 and alpha-9 helices of the class C beta-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 beta-lactamase capable of hydrolyzing oxyimino beta-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 beta-lactams in nature. In this paper, we report the properties of GC1 beta-lactamase.


EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids

E. cloacae GC1 is a clinical isolate found to be highly resistant to oxyimino beta-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 beta-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 beta-lactam susceptibility of cells bearing the cloned beta-lactamase gene and as host cells for enzyme preparation in order to avoid contamination by the ampC beta-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 beta-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 times yeast extract/tryptone (2 times YT) broth and YT agar (12) were employed, respectively. For the beta-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 beta-lactams.

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). [alpha-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 beta-Lactamase Gene and DNA Sequencing

Chromosomal DNA of the E. cloacae cells was prepared according to the procedure of Owen and Borman(13) . A 1.6-kb (^1)DNA fragment containing the beta-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 beta-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(2), 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 beta-lactamase activity. A 1.6-kb DNA fragment with the beta-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 beta-lactamase genes in pCS100 and pCS900 were amplified by the use of the PCR method. The resulting DNAs carrying beta-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 beta-lactamase genes was absent.

Site-directed Mutagenesis of the P99 beta-Lactamase

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 beta-lactamase was performed using oligonucleotides listed in Table 1. Those primers were synthesized using a Cyclone Plus DNA/RNA synthesizer (Milligen Biosearch Co.).

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(2), 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 beta-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.

beta-Lactamase Purification and beta-Lactamase Assay

E. coli AS226-51 cells carrying pTTQ18K with the beta-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-beta-D-galactopyranoside for 12 h. Crude beta-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 times 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 beta-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: beta-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(m) and K(i), 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 beta-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.


RESULTS

Properties of E. cloacae GC1

E. cloacae GC1 showed higher resistance to typical beta-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 beta-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 beta-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 beta-lactamase was estimated to have an isoelectric point higher than 8.5, which is an indication that it belongs to a chromosomal class C beta-lactamase.

Cloning and Sequencing of the GC1 beta-Lactamase Gene

A 1.6-kb DNA fragment containing the beta-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 beta-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 beta-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 beta-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 beta-lactamases of E. cloacae, the E. coli beta-lactamase, and the C. freundii beta-lactamase. These alignments indicated that valine at position 16 is common in the E. cloacae beta-lactamases and that proline at position 88 is also common in many class C beta-lactamases. The most remarkable feature of the GC1 enzyme is an insertion sequence, Ala-Val-Arg, suggesting a duplicative mutation on the chromosomal gene.


Figure 1: DNA sequence of the E. cloacae GC1 beta-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 alpha-helices and beta-strands, respectively, the indication being made on the basis of the sequence alignment of the enzyme with the C. freundii class C beta-lactamase(24) .




Figure 2: Comparison of the deduced amino acid sequence, in part, of the E. cloacae GC1 beta-lactamase with those of other class C beta-lactamases. The sequence alignment of the GC1 beta-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 beta-Lactamase and Its Kinetic Properties

The observations in the preceding genetic experiments suggested that the GC1 beta-lactamase is a naturally occurring mutant of the chromosomal beta-lactamase. The duplicative mutation may confer on the enzyme a unique substrate specificity extending to oxyimino beta-lactams. The GC1 beta-lactamase and the P99 beta-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 beta-lactamases for six beta-lactams are summarized in Table 2, and chemical structures of the beta-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 beta-lactams (cefuroxime, ceftazidime, and aztreonam). The k value of the GC1 enzyme for cephalothin, a favorable substrate for class C beta-lactamases, was 88% that of the P99 enzyme. Although the K(m) 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 beta-lactamases. On the other hand, the catalytic activity of the GC1 enzyme for oxyimino beta-lactams, unfavorable substrates for usual class C beta-lactamases, was significantly higher than that of the P99 beta-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(m) or K(i) values for those unfavorable substrates. This result is very similar to the observation in the cases of the mutant beta-lactamases of C. freundii, which extended its substrate spectrum toward oxyimino beta-lactams by substitution of Glu with other amino acids(6) .




Figure 3: Structures of the beta-lactams used in this study. Three classical beta-lactams are represented by benzylpenicillin, ampicillin, and cephalothin. Three oxyimino beta-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 beta-Lactamase Genes and Their Phenotypes

To confirm that the duplicative mutation on the loop structure is the sole reason for the extended substrate spectrum found in the GC1 beta-lactamase, two chimeric beta-lactamase genes were constructed as illustrated in Fig. 4. A KpnI restriction enzyme site in the E. cloacae beta-lactamase genes was utilized as a recombination site, and the resulting recombinant plasmids were termed pCS102 and pCS902. The chimeric beta-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 beta-lactamases. The chimeric beta-lactamase gene on pCS102 corresponds to a modified GC1 beta-lactamase gene in which the duplicative mutation is eliminated. MIC levels of E. coli AS226-51 cells harboring the plasmids were examined to five beta-lactams, and the extracts from the cells were assayed for their activity toward the beta-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 beta-lactamase toward oxyimino beta-lactams.


Figure 4: Graphical illustration of the hybrid genes from the GC1 and P99 beta-lactamase genes. pCS100 and pCS900 carry the GC1 and P99 beta-lactamase genes, respectively. The boxed areas denote the cloned DNA fragments, and the large boxes represent the beta-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 beta-Lactamase and Its Effect on the Activity to Oxyimino beta-Lactams

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 beta-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 beta-lactams, and the cell extracts were assayed for their hydrolytic activity to the beta-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.




DISCUSSION

The work described here shows for the first time an extended specificity class C beta-lactamase capable of hydrolyzing oxyimino beta-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 beta-lactamase(5, 6) . Oxyimino beta-lactams such as cefuroxime, ceftazidime, and aztreonam act as progressive inhibitors of a class C beta-lactamase of C. freundii, and a minimum scheme for hydrolysis of the beta-lactam ring of oxyimino beta-lactams by the class C beta-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 alpha-8 and alpha-9 helices of the C. freundii beta-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 beta-lactamases in nature. The sequence alignment between E. cloacae and C. freundii beta-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 beta-lactamase, a counterpart of Glu in the C. freundii enzyme, for lysine certainly changes the P99 enzyme to an extended substrate specificity enzyme. (^2)These observations indicate that the structure, known as a hot spot for the extended substrate specificity mutation, is common among class C beta-lactamases.

Recently, the crystalline structure of the P99 beta-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 beta-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 beta-lactamase, corresponding to the AVR sequence of the P99 and GC1 beta-lactamase.



The GC1 beta-lactamase showed lower affinity for oxyimino beta-lactams than the P99 enzyme as can be seen by higher K(m) or K(i) 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 beta-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 beta-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 beta-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 beta-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 beta-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 beta-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.^2 On the other hand, Sowek et al.(29) reported that TEM-1 beta-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 beta-lactamases.


FOOTNOTES

*
This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan and the Foundation for Life Science Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-43-290-2928; Fax: 81-43-255-1574.

(^1)
The abbreviations used are: kb, kilobase pair; bp, base pair(s); PCR, polymerase chain reaction; MIC, minimum inhibitory concentration.

(^2)
M. Nukaga, S. Haruta, K. Taniguchi, T. Yamashita, and T. Sawai, unpublished observation.


ACKNOWLEDGEMENTS

We are grateful to A. F. Ehrhardt for providing E. cloacae P99.


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