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

Abstract

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 β‐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 β‐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 β‐lactam‐binding site in the three‐dimensional structure of the enzyme. Copyright © 1985, Wiley Blackwell. All rights reserved