EVOLUTION OF AN ENZYME-ACTIVITY - CRYSTALLOGRAPHIC STRUCTURE AT 2-ANGSTROM RESOLUTION OF CEPHALOSPORINASE FROM THE AMPC GENE OF ENTEROBACTER-CLOACAE-P99 AND COMPARISON WITH A CLASS-A PENICILLINASE

The structure of the class C ampC beta-lactamase (cephalosporinase) fr om Enterobacter cloacae strain P99 has been established by x-ray cryst allography to 2-angstrom resolution and compared to a class A beta-lac tamase (penicillinase) structure. The binding site for beta-lactam ant ibiotics is generally more open than that in penicillinases, in agreem ent with the ability of the class C beta-lactamases to better bind thi rd-generation cephalosporins. Four corresponding catalytic residues (S er-64/70, Lys-67/73, Lys-315/234, and Tyr-150/Ser-130 in class C/A) li e in equivalent positions within 0.4 angstrom. Significant differences in positions and accessibilities of Arg-349/244 may explain the inabi lity of clavulanate-type inhibitors to effectively inactivate the clas s C beta-lactamases. Glu-166, required for deacylation of the beta-lac tamoyl intermediate in class A penicillinases, has no counterpart in t his cephalosporinase; the nearest candidate, Asp-217, is 10 angstrom f rom the reactive Ser-64. A comparison of overall tertiary folding show s that the cephalosporinase, more than the penicillinase, is broadly s imilar to the ancestral beta-lactam-inhibited enzymes of bacterial cel l wall synthesis. On this basis, it is proposed that the cephalosporin ase is the older of the two beta-lactamases, and, therefore, that a lo cal refolding in the active site, rather than a simple point mutation, was required for the primordial class C beta-lactamase to evolve to t he class A beta-lactamase having an improved ability to catalyze the d eacylation step of beta-lactam hydrolysis.