Amino acid substitutions at Ambler position Gly238 in the SHV-1 beta-lactamase: exploring sequence requirements for resistance to penicillins and cephalosporins

Abstract

Site saturation mutagenesis of the 238 position in the SHV beta-lactamase was performed to identify the complete sequence requirements needed for the extended spectrum beta-lactamase (ESBL) phenotype. MICs (in micrograms per milliliter) in an isogenic background, Escherichia coli DH10B, demonstrated that the Gly238Ala mutation conferred the most resistance to penicillins and cephalosporins. The absolute increase in resistance was greatest against cefotaxime for the Gly238Ala mutant (0.06 to 8 micro g/ml). Except for the strain possessing the Gly238Pro beta-lactamase, ceftazidime MICs were also elevated. None of the mutant SHV beta-lactamases were expressed in as great an amount as the wild-type beta-lactamase. Kinetic analysis of the Gly238Ala mutant revealed that penicillin and cephalosporin substrates have a lower K(m) for the enzyme because of this mutation. Ampicillin and piperacillin MICs were inversely proportional to the side chain volume of the amino acid in cases larger than Ser, suggesting that steric considerations may be a primary requirement for penicillin resistance. Secondary structural effects explain increased resistance to oxyiminocephalosporins. Based upon this study, we anticipate that additional mutations of Gly238 in the SHV beta-lactamase will continue to be discovered with an ESBL (ceftazidime or cefotaxime resistant) phenotype.

FIG. 1.

Molecular representation of the SHV-1 β-lactamase-binding cavity generated by using coordinates for SHV-1, deposited in the Protein Data Bank, and WebLab ViewerLite 3.5. We performed site saturation mutagenesis of Gly238. Amino acids Ser70, Asn170, Ala237, and Glu240 are also shown.

FIG. 2.

Western blot and slot blot of SHV-1 β-lactamase and mutants at the 238 position. The Western blot and slot blot were probed with 1 μg of anti-SHV-1 antibody/ml and horseradish peroxidase-conjugated protein G (7). Single-letter designations for the Gly238 mutant β-lactamases are listed above each slot. SK− represents the strain E. coli DH10B with the vector pBCSK(−) without the SHV β-lactamase. (Inset) Western blot of SHV-1 and Gly238Ala variant of the SHV β-lactamase. Equal amounts of purified β-lactamase were loaded in each lane. Lane 1, SHV-1; lane 2, Gly238Ala. As evaluated by densitometry, SHV-1 and Gly238Ala are nearly equivalent.

FIG. 3.

Pulse-chase analysis of SHV-1 and Gly238Ala, -Leu, and -Ser mutant β-lactamases. Pulse-chase was terminated at 30 and 300 s. The β-lactamase was immunoprecipitated from solubilized E. coli DH10B cells and resolved by SDS-PAGE and autoradiographed. Lanes 1 to 4, 30-s cold methionine chase; lanes 5 to 8, 300-s cold methionine chase.