FEMS Microbiology Letters 160 (1998) 49^54. Received 27 December 1997; accepted 2 January 1998

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1 FEMS Microbiology Letters 160 (1998) 49^54 Substitution of Arg-244 by Cys or Ser in SHV-1 and SHV-5 L-lactamases confers resistance to mechanism-based inhibitors and reduces catalytic e ciency of the enzymes Panagiota Giakkoupi a, Eva Tzelepi a, Nicholas J. Legakis b, Leonidas S. Tzouvelekis a;b; * a Department of Bacteriology, Hellenic Pasteur Institute, 127 Vass. So as Ave., Athens 11521, Greece b Department of Microbiology, Medical School, University of Athens, Athens, Greece Received 27 December 1997; accepted 2 January 1998 Abstract The conserved residue Arg-244 was substituted by the smaller uncharged amino acids Cys and Ser in SHV-1 and SHV-5 L- lactamases by a PCR-based site-specific mutagenesis procedure. The mutant L-lactamases displayed decreased susceptibility to clavulanate and, to a lesser extent, to tazobactam and sulbactam. As shown in comparative MIC determinations, R244C and R244S enzymes retained a residual penicillinase activity while their activity towards cephalosporins was drastically diminished. The respective SHV-5 mutants were unable to hydrolyze oxyimino-l-lactams except aztreonam. The impaired catalytic activity of the mutant L-lactamases was mainly due to the lowering of affinity for L-lactam substrates. The above alterations were more pronounced in the R244C mutants. These results provide information on the mode of involvement of Arg-244 in (a) inactivation by L-lactamase inhibitors and (b) the proper positioning of L-lactams in the active site of SHV enzymes. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords: SHV L-lactamase; Clavulanate resistance; Arginine Introduction The most common plasmid-mediated L-lactamases in enterobacteria are the class A penicillinases TEM- 1 and SHV-1. During the last decade the pressure exerted by newer L-lactams and in particular oxyimino-cephalosporins has facilitated the selection and the wide spread of extended-spectrum mutant * Corresponding author. Tel.: +30 (1) ; Fax: +30 (1) ; Lstbact@hotmail.com TEM and SHV enzymes able to hydrolyze e ciently the latter antibiotics [1]. Also there have been recent studies reporting the emergence of class A penicillinase mutants resistant to mechanism-based inhibitors. Up to now, 16 naturally occurring inhibitor-resistant TEM-1-derived enzymes have been described. The substitutions of (a) Arg-244 by Ser or Cys (Ambler's numbering scheme [2]) and (b) Met-69 by Ile, Leu or Val alone or in combination with Asn-276CAsp, are considered responsible for resistance to inhibitors in TEM-1 mutants [3]. A Met-69CIle inhibitor-resistant laboratory mutant of OHIO-1 L-lactamase / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S (98)

2 50 P. Giakkoupi et al. / FEMS Microbiology Letters 160 (1998) 49^54 (an enzyme closely related to SHV-1) has also been described [4]. Both Met-69 and Arg-244 are conserved in class A L-lactamases of the TEM and SHV types. The e ect of Arg-244CCys or Ser substitutions in SHV type L-lactamases has not yet been studied. In the present work the e ect of Arg-244CSer or Cys substitutions on the SHV-1 penicillinase and its extended-spectrum derivative SHV-5 was examined. The latter L-lactamase is a Gly-238CSer, Glu- 240CLys double mutant of SHV-1 able to hydrolyze newer oxyimino-l-lactams. 2. Materials and methods 2.1. Bacterial strains, plasmids and cloning of L-lactamase genes Escherichia coli strains MPB-1 containing the plasmid pmpb1 that encodes SHV-1 L-lactamase [5] and BM694 containing the SHV-5 L-lactamase encoding plasmid paff2 [6] were used. E. coli XL1-Blue cells were used for transformation. The phagemid pbcsk(+) (Stratagene, La Jolla, CA) was used as the cloning vector. From both L-lactamase-encoding plasmids a 1.6-kb SmaI-ClaI fragment was puri ed from low-melting-point agarose and ligated into the multicloning site of the pbcsk(+). The ligates were used to transform E. coli XL1-Blue competent cells. L-Lactam-resistant clones were selected on LB agar containing ampicillin (50 mg l 31 ) plus chloramphenicol (20 mg l 31 ) Antibiotics and determination of susceptibility status Susceptibility to various L-lactam antibiotics was determined by an agar dilution method, with doubling dilutions of antibiotic incorporated into Mueller-Hinton agar and inoculated with 10 4 CFU/spot. The MIC values are expressed as mg l PCR-based site-directed mutagenesis Mutant L-lactamases were constructed by the `megaprimer' PCR-based site-directed mutagenesis method essentially as described previously [7]. A mutagenic primer and an external primer are used in the rst round of PCR to create the `megaprimer'. In the second round of PCR the `megaprimer' and an external primer are used. The resulting product were digested with SmaI and ClaI and subcloned into pbcsk(+). The mutagenic primer contained one single base mismatch to direct mutagenesis at codon 244 of the mature peptide (TGC for Arg-244CCys and AGC for Arg-244CSer). The mutagenic primers were 22 nucleotides long and the mismatch base was close to the center of the sequence. DNA sequencing was performed by the dideoxy chain termination method using the Sequence 2.0 kit (USB- Amersham, UK) L-Lactamase studies To obtain enzyme preparations containing the wild and mutant L-lactamases, the respective E. coli clones were grown overnight in Tryptone-soya broth. L-Lactamases were released after mild ultrasonic treatment of cells suspended in PBS (100 mm, ph 7). The extracts were clari ed by ultracentrifugation, desalted and concentrated by ultra ltration in Amicon tubes (Amicon, Germany). The protein content of the extracts was determined with the Bio-Rad Protein Assay kit (Bio-Rad, Germany). Kinetic parameters of L-lactam hydrolysis were evaluated by UV spectrophotometry at 37³C and ph 7 as described previously [8]. The maximum rate of hydrolysis (V max ) and K m values were determined by Lineweaver-Burk plots and are expressed as hydrolysis rates relative to that of penicillin G set at 100. The L-lactamase activity was quantitated using nitroce n as the substrate. Results were expressed as units of activity (1 U is the amount of enzyme hydrolysing 1 Wmol of substrate min 31 mg 31 of protein at ph 7 and 37³C). Inhibition pro les were determined using clavulanate, sulbactam and tazobactam [9]. The reporter substrate was nitroce n at a concentration of 100 WM. The amount of each enzyme was normalized to give 150 WM nitroce n hydrolyzed per min. The IC 50 values were determined after pre-incubating for 10 min the enzymes with various concentrations of inhibitor prior to the addition of nitroce n.

3 P. Giakkoupi et al. / FEMS Microbiology Letters 160 (1998) 49^ Results and discussion The MICs of L-lactams are presented in Table 1. The E. coli clones producing the mutant L-lactamases R244C and R244S that derived from SHV-1 were less resistant to amoxicillin and piperacillin than the clones producing the wild-type enzyme. A 2^4-fold reduction in the MIC values of the second generation cephalosporins cefaclor and cefprozil was observed; the MICs of oxyimino-cephalosporins and aztreonam were also reduced. The mutant SHV-1 L- lactamases conferred lower levels of resistance to piperacillin-tazobactam and ampicillin-sulbactam compared with SHV-1, whereas the MICs of amoxicillinclavulanate were essentially similar. The reduction in the MICs of penicillins in the clones producing SHV-5 mutants, as compared with the SHV-5-expressing clones, was less pronounced. The former clones also displayed slightly higher MICs to the penicillin-inhibitor combinations tested. The most drastic reduction was noticed in the MICs of oxyimino-cephalosporins. The extendedspectrum L-lactamase SHV-5 confers a high level of resistance to ceftazidime (`ceftazidimase' phenotype). In the clones expressing either R244C or R244S mutant enzymes, the MICs of ceftazidime were below the break points. A signi cant reduction was also observed in the MICs of cefotaxime and ceftriaxone. The lowering of resistance levels to aztreonam was less marked. The susceptibility testing was performed under isogenic conditions which permit the comparison of the activities of di erent L-lactamases [5]. It was therefore postulated that the replacement of Arg-244 by Cys or Ser impaired the catalytic e ciency of SHV-1 and SHV-5 L-lactamases. Determination of the respective kinetic constants showed that both substitutions signi cantly decreased the a nity for cephalosporins. A 2^3-fold reduction in hydrolysis rates was also noticed (Table 2). The di erences in the K m values for penicillin G indicate that hydrolysis of penicillins was a ected to a lesser extent. The introduced replacements rendered SHV-1 and SHV-5 L- lactamases resistant to mechanism-based inhibitors. The fold increase in IC 50 values for clavulanic acid was greater than for the penam sulfones sulbactam and tazobactam (Table 3). The fact that the mutant enzymes did not confer resistance to penicillin-inhibitor combinations was apparently due to the simultaneous weakening of the penicillinase activity. The two R244C mutant L-lactamases were more resistant to inhibitors than the respective R244S enzymes. It should also be noted that the decrease in hydrolytic e ciency was more pronounced in the Cys- than in the Ser-244 mutant L-lactamases. The involvement of Arg-244 in the inactivation Table 1 Susceptibility to L-lactam antibiotics of E. coli XL1-Blue clones expressing SHV-1, SHV-5 and the respective R244C and R244S mutant L-lactamases L-Lactam antibiotic MIC (mg l 31 ) for E. coli clones MIC (mg l 31 ) for E. coli clones SHV-1 R244C R244S SHV-5 R244C R244S Amoxicillin Amoxicillin-clavulanate (2:1) Ampicillin-sulbactam (2:1) Ticarcillin Piperacillin Piperacillin-tazobactam a Cefaclor Cephalothin Cefprozil Cefotaxime Ceftazidime Ceftriaxone Aztreonam a Inhibitor at xed concentration of 4 mg l 31.

4 52 P. Giakkoupi et al. / FEMS Microbiology Letters 160 (1998) 49^54 Table 2 Hydrolytic activities of SHV-1 (A) and SHV-5 (B) and mutant L-lactamases (R244C and R244S) against L-lactam antibiotics Substrate L-Lactamase SHV-1 R244C R244S V max a K m (WM) V max K m (WM) V max K m (WM) A Penicillin G Piperacillin Cephalothin Cefaclor ND b 5 72 Cefotaxime ND ND ND B Penicillin G Piperacillin Cephalothin Cefaclor Cefotaxime 20 9 ND ND a V max values are relative to that of penicillin G, which was set at 100. b Not determined. The hydrolysis rates were too low. process of class A L-lactamases by mechanism-based inhibitors has been recognized. It has been suggested that Arg-244 holds in place a structurally conserved water molecule (W673). The latter acts as a proton source for the formation of the acyclic intermediate of clavulanic acid which is a critical step towards irreversible inactivation [10]. The uncharged and shorter side chains of serine or cysteine may be unable to activate W673. Consequently the rate of L- lactamase inactivation becomes slower. The inactivation process with penam sulfones is dependent to a lesser extent on Arg-244 than with clavulanate [11], thus explaining the lower fold increase in the IC 50 values of tazobactam and sulbactam. The attenuation of catalytic e ciency caused by replacing Arg-244 with either Cys or Ser underlines the importance of the former amino acid residue in L-lactam hydrolysis. In class A L-lactamases Arg-244 is located on the L4 strand of the K/L domain. Its long side chain is directed towards the active site cavity and the positively charged guanidinium group is closed to the conserved KTG triad of the L strand [12]. As has been suggested in crystallographic studies and molecular modeling in the Bacillus licheniformis class A penicillinase, Arg-244 along with Arg- 274 and Lys-234 may form a positive eld which participates in the proper docking of the substrate in the active site [13]. It has also been suggested by others that Arg-244 forms a weak hydrogen bond with the C3 (C4) L-lactam carboxylate [14]. The above taken together with the signi cant increase of the K m values indicated that the ability of the mutant SHV enzymes to bind and align the substrates in the oxyanion cavity was impaired. The Table 3 Inhibition pro les of SHV-1 and SHV-5 and the respective R244C and R244S mutant L-lactamases L-Lactamase IC 50 (WM) of inhibitor Clavulanate Sulbactam Tazobactam SHV-1 WT R244C 12 s R244S 5.2 s SHV-5 WT R244C R244S

5 P. Giakkoupi et al. / FEMS Microbiology Letters 160 (1998) 49^54 53 suggested interaction between Arg-244 and the C3 (C4) carboxylate is supported by the results obtained with aztreonam. The data from the susceptibility testing indicated that both R244C and R244S mutants derived from SHV-5 were still able to hydrolyze the antibiotic e ciently whereas activity against cephalosporins was almost abolished. A di erence between aztreonam and oxyimino-cephalosporins is that the former has a sulfonic acid group bonded directly to the nitrogen of the lactam ring in place of the functionally equivalent carboxylate group. It is expected that the interaction of the side chain of Arg-244 with the sulfonic group is weaker than that with the respective carboxylate. Another amino acid of TEM-1 that interacts with the C3 (C4) carboxylate of L-lactams by forming a hydrogen bond is Ser-235 [15]. In SHV L-lactamases the same position is occupied by a threonine. From studies with TEM-1 L-lactamase there have been indications that Arg-244 is responsible for the proper positioning of Ser-235 which in turn interacts with the substrate's carboxylate contributing to L-lactam recognition and turnover. Interestingly, it appears that the role of Thr-235 is more critical for the hydrolysis of cephalosporins than penicillins [15,16]. Extrapolating these data to SHV L-lactamases, the maintenance of a signi cant part of the penicillinase activity in the R244C and R244S mutants can be explained. The extended-spectrum L-lactamases of the TEM and SHV types are more susceptible to inhibitors than the parental penicillinases [1,3]. As shown in a complex TEM mutant (TEM-50), Met-69CLeu and Asn-276CAsp o ered low-level resistance to inhibitors and weakened the extended-spectrum characteristics conferred by Lys for Glu-104 and Ser for Gly- 238 substitutions [17]. Similar results were obtained with a G238S/M69I OHIO-1 double mutant with extended-spectrum properties [18]. The results presented here extend these observations to SHV-5 L- lactamase and Arg-244. It is suggested that the substitutions of either Met-69 or Arg-244 can cause a decrease in the susceptibility to inhibitors but at the expense of hydrolytic activity against expanded-spectrum L-lactams. In that respect, such complex mutants may appear sporadically but they are not expected to prevail. Nevertheless, extensive amino acid rearrangements, like those recorded recently in a TEM L-lactamase [19], may give rise to inhibitorresistant enzymes with intact extended-spectrum characteristics. Acknowledgments We are grateful to L. Gutman and R. Hachler for providing E. coli strains producing SHV L-lactamases. We are indebted to Robert A. Bonomo for his valuable suggestions. References [1] Medeiros, A.A. (1997) Evolution and dissemination of L-lactamases accelerated by generations of L-lactam antibiotics. Clin. Infect. Dis. 24 (Suppl. 1), 19^45. [2] Ambler, R.P., Coulson, A.F.W., Frere, J.-M., Ghuysen, J.M., Joris, B., Forsman, M., Levesque, R.C., Tiraby, G. and Waley, S.G. (1991) A standard numbering scheme for the class A L-lactamases. Biochem. J. 267, 269^272. [3] Knox, J.R. (1995) Extended-spectrum and inhibitor-resistant TEM-type L-lactamases: mutations, speci city, and three-dimensional structure. Antimicrob. Agents Chemother. 39, 2593^2601. [4] Bonomo, R.A., Knox, J.R., Rudin, S.D. and Shlaes, D.M. (1997) Construction and characterization of an OHIO-1 L- lactamase bearing Met69Ile and Gly238Ser mutations. Antimicrob. Agents Chemother. 41, 1940^1943. [5] Nuesch-Inderbinen, M.T., Hachler, H. and Kayser, F.H. (1995) New system based on site-directed mutagenesis for highly accurate comparison of resistance levels conferred by SHV L-lactamases. Antimicrob. Agents Chemother. 39, 1726^ [6] Billot-Klein, D., Gutmann, L. and Collatz, E. (1990) Nucleotide sequence of the SHV-5 L-lactamase gene of a Klebsiella pneumoniae plasmid. Antimicrob. Agents Chemother. 34, 2439^2441. [7] Smith, K.D., Valenzuela, A., Vigna, J.L., Aalbers, K. and Lutz, C.T. (1993) Unwanted mutations in PCR mutagenesis: avoiding the predictable. PCR Methods Appl. 2, 253^257. [8] Prinarakis, E.E., Miriagou, V., Tzelepi, E., Gazouli, M. and Tzouvelekis, L.S. (1997) Emergence of an inhibitor-resistant L-lactamase (SHV-10) derived from an SHV-5 variant. Antimicrob. Agents Chemother. 41, 838^840. [9] Tzouvelekis, L.S., Gazouli, M., Prinarakis, E.E., Tzelepi, E. and Legakis, N.J. (1997) Comparative evaluation of the inhibitory activities of the novel penicillanic acid sulfone Ro 48^ 1220 against L-lactamases that belong to groups 1, 2b and 2be. Antimicrob. Agents Chemother. 41, 457^477. [10] Imtiaz, U., Billings, E., Knox, J.R., Manavathu, E.K., Lerner, S.A. and Mobashery, S. (1993) Inactivation of class A L-lactamase by clavulanic acid: the role of arginine 244 in a pro-

6 54 P. Giakkoupi et al. / FEMS Microbiology Letters 160 (1998) 49^54 posed nonconcerted sequence of events J. Am. Chem. Soc. 115, 4435^4442. [11] Imtiaz, U., Billings, E., Knox, J.R. and Mobashery, S. (1994) A structure-based analysis of the inhibition of class A L-lactamases by sulbactam. Biochemistry 33, 5728^5738. [12] Matagne, A. and Frere, J.-M. (1995) Contribution of mutant analysis to the understanding of enzyme catalysis: the case of class A L-lactamases. Biochim. Biophys. Acta 1246, 109^127. [13] Moews, P.C., Knox, J.R., Dideberg, O., Charlier, P. and Frere, J.-M. (1990) L-Lactamase of Bacillus licheniformis 749/C at 2 A î resolution. Protein Struct. Funct. Genet. 7, 156^171. [14] Zafaralla, G., Manavathu, E.K., Lerner, S.A. and Mobashery, S. (1992) Elucidation of the role of arginine 244 in the turnover processes of class A L-lactamases. Biochemistry 31, 3847^ [15] Imtiaz, U., Manavathu, E.K., Lerner, S.A. and Mobashery, S. (1993) Critical hydrogen bonding by serine 235 for cephalosporinase activity of TEM-1 L-lactamase. Antimicrob. Agents Chemother. 37, 2438^2442. [16] Fonze, E., Charlier, P., To'th, Y., Vermeire, M., Raquet, X., Dubus, A. and Frere, J.-M. (1995) TEM1 L-lactamase structure solved by molecular replacement and re ned structure of the S235A mutant. Acta Crystallogr. D51, 682^694. [17] Sirot, D., Recule, C., Chaibi, E.B., Bret, L., Croize, J., Chanal-Claris, C., Labia, R. and Sirot, J. (1997) A complex mutant of TEM-1 L-lactamase with mutations encountered in both IRT-4 and extended-spectrum TEM-15, produced by an Escherichia coli clinical isolate. Antimicrob. Agents Chemother. 41, 1322^1325. [18] Bonomo, R.A., Knox, J.R., Rudin, S.D. and Shlaes, D.M. (1997) Construction and characterization of an OHIO-1 L- lactamase bearing Met69Ile and Gly238Ser mutations. Antimicrob. Agents Chemother. 41, 1940^1943. [19] Perilli, M., Felici, A., Franceschini, N., De Santis, A., Pagani, L., Luzzaro, F., Oratore, A., Rossolini, G.M., Knox, J.R. and Amicosante, G. (1997) Characterization of a new TEM-derived L-lactamase produced in a Serratia marcescens strain. Antimicrob. Agents Chemother. 41, 2374^2382.