Amino acid substitutions that reduce the affinity of penicillin-binding protein 3 of Escherichia coli for cephalexin

Eur J Biochem. 1985 Aug 15;151(1):111-21. doi: 10.1111/j.1432-1033.1985.tb09075.x.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / genetics*
  • Amino Acids
  • Bacterial Proteins*
  • Base Sequence
  • Binding Sites
  • Carrier Proteins*
  • Cephalexin / metabolism*
  • Chromosome Mapping
  • Drug Resistance, Microbial
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Escherichia coli Proteins*
  • Genes, Bacterial
  • Hexosyltransferases / genetics*
  • Hexosyltransferases / metabolism
  • Multienzyme Complexes / genetics*
  • Multienzyme Complexes / metabolism
  • Muramoylpentapeptide Carboxypeptidase*
  • Mutation
  • Penicillin-Binding Proteins
  • Peptidoglycan Glycosyltransferase*
  • Peptidyl Transferases / genetics*
  • Peptidyl Transferases / metabolism


  • Amino Acids
  • Bacterial Proteins
  • Carrier Proteins
  • Escherichia coli Proteins
  • FtsI protein, E coli
  • Multienzyme Complexes
  • Penicillin-Binding Proteins
  • Acyltransferases
  • Peptidyl Transferases
  • Hexosyltransferases
  • Peptidoglycan Glycosyltransferase
  • Muramoylpentapeptide Carboxypeptidase
  • Cephalexin