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

Abstract

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 Mendonca 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 ​(Table11). 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).