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.

Bypass of genetic constraints during mutator evolution to antibiotic resistance

“…In this respect, it is worth mentioning the high proportion of PBP3 substitutions observed only once (22/34; electronic supplementary material, table S2), which points at the existence of many other possible mutations that just went unsampled [48]. This is consistent with the fact that more than 30 PBP3 substitutions not detected here have been described elsewhere to confer resistance to a variety of b-lactams, including CTX [42][43][44][45]49].…”

“…We identified 92 point mutations (34 unique), affecting 31 positions, accounting for a total of 39 different alleles (figure 3b and electronic supplementary material, tables S2 and S3). Some of these variants have been already associated with resistance to b-lactam antibiotics, either in laboratory strains of E. coli (A257V and N361S) [42,43] or in related species such as Salmonella enterica (P311S) [44] and Haemophilus influenzae (L369F) [45]. All the substitutions but one mapped within the transpeptidase module (D237-V577) of the PBP3 protein, clustered in the surroundings of the three catalytic motifs (STVK310, SSN361 and KTG496) [46].…”

Probing the Catalytic Activity of a Cell Division-Specific Transpeptidase In Vivo with β-Lactams

“…Hedge and Spratt selected for E. coli mutants resistant to cephalexin and obtained strains with lesions in the transpeptidase domain of PBP3 (18,19). Despite extensive efforts, only a few mutations that resulted in substantial cephalexin resistance were recovered, implying that very few amino acids in PBP3 can be changed to reduce the affinity for cephalexin without compromising transpeptidation.…”

Identification of a penicillin-binding protein 3 homolog, PBP3x, in Pseudomonas aeruginosa: gene cloning and growth phase-dependent expression

“…This probably resulted from the lower affinities of PBP3x (compared to PBP3) for the PBP3-targeted antibiotics, which could result from the nature of the various amino acid residues making up and surrounding the active binding site serine, since these are known to play roles in facilitating binding to the substrates (10,15,28). For example, it has been shown that replacement of threonine with proline in the second position of the SXXK motif of E. coli resulted in reduced affinity for cephalexin, a PBP3-targeted antibiotic (15). The amino acid sequences of the SXXK, SXN, and KTG motifs of P. aeruginosa PBP3 are STVK, SSN, and KSG, whereas for PBP3x they are SVIK, SSN, and KSG, respectively.…”