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Antimicrobial Agents and Chemotherapy, May 2008, p. 1897-1898, Vol. 52, No. 5
0066-4804/08/$08.00+0     doi:10.1128/AAC.01525-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Biochemical Characterization of SHV-55, an Extended-Spectrum Class A β-Lactamase from Klebsiella pneumoniae{triangledown}


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We biochemically characterized the Klebsiella pneumoniae extended-spectrum SHV-55 enzyme carrying the amino acid substitutions Tyr7->Phe (as in SHV-28) and Gly238->Ser and Glu240->Lys (both found in SHV-5) identified in a previous study (5). The SHV-55 extended-spectrum β-lactamase differed from SHV-5 only in the signal peptide region (1). The blaSHV-55 gene was obtained as described by Mendonça et al. (6), and transformants were selected on Luria broth agar supplemented with 30 µg of kanamycin/ml and 16 µg of amoxicillin/ml. SHV-55 was extracted and purified according to the previously described protocol (6). The Michaelis constant (Km) and catalytic activity (kcat) of purified extracts of SHV-55 were obtained by using a computerized microacidimetric method and a 702 SM Titrino pH-stat apparatus (Metrohm, Herisau, Switzerland) (3). The complete hydrolysis time courses were analyzed, and the kinetic progress curves were fitted by nonlinear least-squares regression. These kinetic parameters were determined and compared to those of the SHV-1 enzyme for 10 β-lactams (Table 1).


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  		TABLE 1. Kinetic constants of SHV-55 and SHV-1 β-lactamasesa

 
SHV-55 has a high affinity (Km, 5 to 10 µM) for penicillins, similar to that of SHV-5 (1) and higher than that of SHV-1 (Km, 11 to 31 µM). SHV-55 presented higher affinity values (Km, 9 to 58 µM) than SHV-1 (Km, 40 to 257 µM) for narrow-, extended-, and broad-spectrum cephalosporins and monobactams. This finding may be a consequence of the Gly238->Ser substitution present in the active sites of both SHV-55 and SHV-5, which pushes the β-strand out and away from the reactive Ser70 (2). This effect results in a slightly expanded active site that may improve binding and accommodate cephalosporins with bulky side chains (4). SHV-55 presented a higher affinity for cefotaxime than for ceftazidime (Kms, 21 and 58 µM, respectively), as did SHV-5 (1, 7). This finding is surprising because both enzymes possess the Glu240->Lys substitution, which increases hydrolytic activity against ceftazidime (8) due to the change in the electrostatic charge of the exposed group at position 240 (2). The enzymatic activities (kcats) of SHV-55 for penicillin G and amoxicillin were 84- and 45-fold lower, respectively, than those of SHV-1, and the catalytic efficiency (kcat/Km ratio) against penicillins was more than 10-fold higher for SHV-1 (kcat/Km ratio, 20 to 84 µM–1·s–1) than for SHV-55 (kcat/Km ratio, 2 to 5 µM–1·s–1). However, the enzyme activity and catalytic efficiency against extended- and broad-spectrum cephalosporins were higher for SHV-55 (kcat, 7 to 24 s–1, and kcat/Km ratio, 0.2 to 1 µM–1·s–1) than for SHV-1 (note, however, that the values for monobactam were undeterminable), although the catalytic efficiencies of the two enzymes against cephalothin were similar (kcat/Km ratios, 3.2 and 4.4 µM–1·s–1). This result may be due to the amino acid substitutions in SHV-55 causing conformational modifications in the active site. Fifty percent inhibitory concentrations (IC50s) indicated that SHV-55 was ninefold more susceptible to the inhibitor activity of clavulanate than SHV-1 (IC50s of clavulanate, 0.02 versus 0.17 µM).

In conclusion, these results confirmed the extended-spectrum activity of the SHV-55 enzyme, which is important due to the magnitude of extended- and broad-spectrum SHV β-lactamases described to date and not biochemically characterized, in spite of the ease of sequencing genes (http://www.lahey.org/studies).


   ACKNOWLEDGMENTS
 
This work was supported financially by the project grant POCTI/2001/ESP/43037 from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal, awarded to M. Caniça and by a grant from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, France. N. Mendonça and V. Manageiro were supported by grants BIC 03/2003-I from National Institute of Health Dr. Ricardo Jorge and SFRH/BD/32578/2006 from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal, respectively, and both were also the recipients of short-term research fellowship grants from the Federation of European Microbiological Societies (FEMS).


   FOOTNOTES
 
{triangledown} Published ahead of print on 3 March 2008. Back


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  1. Gutmann, L., B. Ferré, F. W. Goldstein, N. Risk, E. Pinto-Schuster, J. F. Acar, and E. Collatz. 1989. SHV-5, a novel SHV-type β-lactamase that hydrolyzes broad-spectrum cephalosporins and monobactams. Antimicrob. Agents Chemother. 33:951-956.[Abstract/Free Full Text]
  2. Huletsky, A., J. R. Knox, and R. C. Levesque. 1993. Role of Ser-238 and Lys-240 in the hydrolysis of third-generation cephalosporins by SHV-type β-lactamases probed by site-directed mutagenesis and three-dimensional modelling. J. Biol. Chem. 268:3690-3697.[Abstract/Free Full Text]
  3. Labia, R., J. Andrillon, and F. Le Goffic. 1973. Computerized microacidimetric determination of β-lactamase Michaelis-Menten constants. FEBS Lett. 33:42-44.[CrossRef][Medline]
  4. Matagne, A., J. Lamotte-Brasseur, and J.-M. Frère. 1998. Catalytic properties of class A β-lactamases: efficiency and diversity. Biochem. J. 330:581-598.[Medline]
  5. Mendonça, N., E. Ferreira, D. Louro, and M. Caniça. 2006. Occurrence of a novel SHV-type enzyme (SHV-55) among isolates of Klebsiella pneumoniae from Portuguese origin in a comparison study for extended-spectrum β-lactamase-producing evaluation. Diagn. Microbiol. Infect. Dis. 56:415-420.[CrossRef][Medline]
  6. Mendonça, N., V. Manageiro, F. Robin, M. J. Salgado, E. Ferreira, M. Caniça, and R. Bonnet. 2008. The Lys234Arg substitution in the enzyme SHV-72 is a determinant for resistance to clavulanic acid inhibition. Antimicrob. Agents Chemother. 52:1806-1811.[Abstract/Free Full Text]
  7. Mulgrave, L., and P. V. Attwood. 1993. Characterization of an SHV-5 related extended broad-spectrum beta-lactamase in Enterobacteriaceae from Western Australia. Pathology 25:71-75.[Medline]
  8. Randegger, C. C., A. Keller, M. Irla, A. Wada, and H. Hächler. 2000. Contribution of natural amino acid substitutions in SHV extended-spectrum β-lactamases to resistance against various β-lactams. Antimicrob. Agents Chemother. 44:2759-2763.[Abstract/Free Full Text]
Nuno Mendonça
Vera Manageiro
Antibiotic Resistance Unit
Department of Infectious Diseases
National Institute of Health Dr. Ricardo Jorge
Av. Padre Cruz
1649-016 Lisboa, Portugal

Richard Bonnet
Université Clermont 1
UFR Médecine
Laboratoire de Bactériologie
EA3844 Clermont-Ferrand, F-63001 France

Manuela Caniça*
Antibiotic Resistance Unit
Department of Infectious Diseases
National Institute of Health Dr. Ricardo Jorge
Av. Padre Cruz
1649-016 Lisboa, Portugal

* Phone and fax: 351.217519246, E-mail: manuela.canica{at}insa.min-saude.pt

Antimicrobial Agents and Chemotherapy, May 2008, p. 1897-1898, Vol. 52, No. 5
0066-4804/08/$08.00+0     doi:10.1128/AAC.01525-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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