Abstract
beta-Lactamases inactivate beta-lactam antibiotics by catalyzing the hydrolysis of the amide bond in the beta-lactam ring. The plasmid-encoded class A TEM-1 beta-lactamase is a commonly encountered beta-lactamase. It is able to inactivate penicillins and cephalosporins but not extended-spectrum antibiotics. However, TEM-1-derived natural variants containing the G238S amino acid substitution display increased hydrolysis of extended-spectrum antibiotics. Two models have been proposed to explain the role of the G238S substitution in hydrolysis of extended-spectrum antibiotics. The first proposes a direct hydrogen bond of the Ser238 side chain to the oxime group of extended-spectrum antibiotics. The second proposes that steric conflict with surrounding residues, due to increased side chain volume, leads to a more accessible active site pocket. To assess the validity of each model, TEM-1 mutants with amino acids substitutions of Ala, Ser, Cys, Thr, Asn, and Val have been constructed. Kinetic analysis of these enzymes with penicillins and cephalosporins suggests that a hydrogen bond is necessary but not sufficient to achieve the hydrolytic activity of the G238S enzyme for the extended-spectrum antibiotics cefotaxime and ceftazidime. In addition, it appears that the new hydrogen bond interaction is to a site on the enzyme rather than directly to the extended-spectrum antibiotic. The data indicate that, for the G238S substitution, a combination of an optimal side chain volume and hydrogen bonding potential results in the most versatile and advantageous antibiotic hydrolytic spectrum for bacterial resistance to extended-spectrum antibiotics.
Highlights
Bacterial resistance to -lactam antibiotics, such as penicillins and cephalosporins, is primarily mediated by the production of -lactamases, which catalyze the hydrolysis of -lactam antibiotics to inactive products [1]
These results indicate that a hydrogen-bonding amino acid at position 238 is necessary but not sufficient to increase the hydrolysis of extended-spectrum cephalosporins to an extent that is clinically relevant (i.e. G238S)
The “hydrogen bond to substrate” model for the role of residue 238 would predict that, out of the residues tested in this study, only Ser, Thr, and Asn would provide increases in catalytic efficiency of the TEM enzyme for extended-spectrum antibiotics while the “steric conflict” model would predict a direct correlation between an increase in the side chain volume of residue 238 and an increase in catalytic efficiency of the enzyme for extended-spectrum antibiotics
Summary
Materials—All enzymes were purchased from New England Biolabs, except T7 DNA polymerase, which was purchased from U. Ampicillin, benzylpenicillin, cephaloridine, cephalothin, and cefotaxime were purchased from Sigma. E. coli SB646 (⌬fhuA ⌬ptr ⌬degP ⌬ompT ⌬prc::kan) is a proteasedeficient strain that was used for expression and purification of mutants G238S, G238C, G238T, G238N, and G238V. Oligonucleotide primers used for mutagenesis and DNA sequencing were synthesized by the oligonucleotide synthesis facility at Genentech, Inc., at Stanford University Medical School, and at Genosys Biotechnologies, Inc. Single-stranded plasmid DNA was prepared for sequencing as described previously [11]. The ranges of antibiotic concentrations (2-fold increases) tested were: 128 –5000 g/ml for ampicillin and benzylpenicillin (concentrations increased 2-fold between 128 and 4096 g/ml; 3000 and 5000 g/ml concentrations were added for increased sensitivity), 8 –1024 g/ml for cephaloridine and cephalothin, 0.03– 4 g/ml for cefotaxime, and 0.12–16 g/ml for ceftazidime. Enzyme Kinetics—The kinetics of TEM-1 -lactamase and the residue 238 mutants were determined with ampicillin, benzylpenicillin, cephaloridine, cephalothin, cefotaxime, and ceftazidime. The values reported are based on velocity measurements at 25, 50, and/or 100 M substrate concentrations
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