BINDING OF CEPHALOTHIN AND CEFOTAXIME TO D-ALA-D-ALA-PEPTIDASE REVEALS A FUNCTIONAL BASIS OF A NATURAL MUTATION IN A LOW-AFFINITY PENICILLIN-BINDING PROTEIN AND IN EXTENDED-SPECTRUM BETA-LACTAMASES

Two clinically-important beta-lactam antibiotics, cephalothin and cefo taxime, have been observed by X-ray crystallography bound to the react ive Ser62 of the D-alanyl-D-alanine carboxypeptidase/ transpeptidase o f Streptomyces sp. R61. Refinement of the two crystal structures produ ced R factors for 3 sigma (F) data of 0.166 (to 1.8 Angstrom) and 0.17 0 (to 2.0 Angstrom) for the cephalothin and cefotaxime complexes, resp ectively. In each complex, a water molecule is within 3.1 and 3.6 Angs trom of the acylated beta-lactam carbonyl carbon atom, but is poorly a ctivated by active site residues for nucleophilic attack and deacylati on. This apparent lack of good stereochemistry for facile hydrolysis i s in accord with the long half-lives of cephalosporin intermediates in solution (20-40 h) and the efficacy of these beta-lactams as inhibito rs of bacterial cell wall synthesis. Different hydrogen binding patter ns of the two cephalosporins to Thr301 are consistent with the low cef otaxime affinity of an altered penicillin-binding protein, PBP-2x, rep orted in cefotaxime-resistant strains of Streptococcus pneumoniae, and with the ability of mutant class A beta-lactamases to hydrolyze third -generation cephalosporins.