Amino acid substitutions that reduce the affinity of penicillin-binding protein 3 of Escherichia coli for cephalexin.
The location of amino acid substitutions that allow an enzyme to discriminate between the binding of its normal substrate and a substrate analogue may be used to identify regions of the polypeptide that fold to form the substrate binding site. We have isolated a large number of cephalexin-resistant mutants of Escherichia coli in which the resistance is due to the production of altered forms of penicillin-binding protein 3 that have reduced affinity for the antibiotic. Using three mutagens, and a variety of selection procedures, we obtained only five classes of mutants which could be distinguished by their patterns of cross-resistance to other beta-lactam antibiotics. The three classes of mutants that showed the highest levels of resistance to cephalexin were cross-resistant to several other cephalosporins but not to penicillins or to the monobactam, aztreonam. The penicillin-binding protein 3 gene from 46 independent mutants was cloned and sequenced. Each member of the five classes of cephalexin-resistant mutants had the same amino acid substitution in penicillin-binding protein 3. The mutants that showed the highest levels of resistance to cephalexin had alterations of either Thr-308 to Pro, Val-344 to Gly, or Asn-361 to Ser. The Thr-308 to Pro substitution had occurred within the beta-lactam-binding site since the adjacent residue (Ser-307) has been shown to be acylated by benzylpenicillin. The Asn-361 to Ser change occurred in a region that showed substantial similarity to regions in both penicillin-binding protein 1A and 1B and may also define a residue that is located within the beta-lactam-binding site in the three-dimensional structure of the enzyme.
D000217:Acyltransferases / Q000235:genetics*; D000596:Amino Acids; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D002506:Cephalexin / Q000378:metabolism*; D002874:Chromosome Mapping; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000235:genetics* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D002267:Muramoylpentapeptide Carboxypeptidase; D009154:Mutation; D046915:Penicillin-Binding Proteins; D046928:Peptidoglycan Glycosyltransferase; D010458:Peptidyl Transferases / Q000235:genetics* / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C478704:FtsI protein, E coli; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D000217:Acyltransferases; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D046928:Peptidoglycan Glycosyltransferase; D002267:Muramoylpentapeptide Carboxypeptidase; D002506:Cephalexin