Site-directed mutagenesis of residues 164, 170, 171, 179, 220, 237 and 242in PER-1 beta-lactamase hydrolysing expanded-spectrum cephalosporins

The class A P-lactamase PER-1, which displays 26% identity with the TEM-typ e extended-spectrum P-lactamases (ESBLs), is characterized by a substrate p rofile similar to that conferred by these latter enzymes, The role of resid ues Ala164, His170, Ala171, Asn179, Arg220, Thr237 and Lys242, found in PER -I, was assessed by site-directed mutagenesis, Replacement of Ala164 by Arg yielded an enzyme with no detectable P-lactamase activity. Two other mutan ts, N179D and A164R+N179D, were also inactive. Conversely, a mutant with th e A171E substitution displayed a substrate profile very similar to that of the wild-type enzyme. Moreover, the replacement of Ala171 by Glu in the A16 4R enzyme yielded a double mutant which was active, suggesting that Glu171 could compensate for the deleterious effect of Arg164 in the A164R+A171E en zyme. A specific increase in k(cat) for cefotaxime was observed with H170N, whereas R220L and T237A displayed a specific decrease in activity towards the same drug and a general increase in affinity towards cephalosporins. Fi nally, the K242E mutant displayed a kinetic behaviour very similar to that of PER-1, Based on three-dimensional models generated by homology modelling and molecular dynamics, these results suggest novel structure-activity rel ationships in PER-I, when compared with those previously described for the TEM-type ESBLs.