Selection and characterization of amino acid substitutions at residues 237-240 of TEM-1 beta-lactamase with altered substrate specificity for aztreonam and ceftazidime.
Recently, natural variants of TEM-1 beta-lactamase with amino acid substitutions at residues 237-240 have been identified that have increased hydrolytic activity for extended-spectrum antibiotics such as ceftazidime. To identify the sequence requirements in this region for a given antibiotic, a random library was constructed that contained all possible amino acid combinations for the 3-residue region 237-240 (ABL numbering system) of TEM-1 beta-lactamase. An antibiotic disc diffusion method was used to select mutants with wild-type level activity or greater for the extended-spectrum cephalosporin ceftazidime and the monobactam aztreonam. Mutants that were selected for optimal ceftazidime hydrolysis contained a conserved Ala at position 237, a Ser for Gly substitution at position 238, and a Lys for Glu at position 240. Mutants selected for aztreonam hydrolysis exhibited a Gly for Ala substitution at position 237, a Ser for Gly substitution at position 238, and a Lys/Arg for Glu at position 240. The role of the A237G substitution in differentiating between ceftazidime and aztreonam was further investigated by kinetic analysis of the A237G, E240K, G238S:E240K, and A237G:G238S:E240K enzymes. The A237G single mutant and the G238S:E240K double mutant exhibited increases in catalytic efficiency for both ceftazidime and aztreonam. However, the triple mutant A237G:G238S:E240K, displayed a 12-fold decrease in catalytic efficiency for ceftazidime but a 3-fold increase for aztreonam relative to the G238S:E240K double mutant. Thus, the A237G substitution increases ceftazidime hydrolysis when present alone but antagonizes ceftazidime hydrolysis when it is combined with the G238S:E240K substitutions. In contrast, the A237G substitution acts additively with the G238S:E240K substitutions to increase aztreonam hydrolysis.
Cantu
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Structural and Computational Biology and Molecular Biophysics Program, Baylor College of Medicine, Houston, Texas 77030, USA.
Huang
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W
Palzkill
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D000595:Amino Acid Sequence; D001398:Aztreonam / Q000378:metabolism*; D002442:Ceftazidime / Q000378:metabolism*; D002511:Cephalosporins / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D008997:Monobactams / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D002511:Cephalosporins; D008997:Monobactams; D002442:Ceftazidime; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D001398:Aztreonam