The class A β-lactamase PER-1, which displays 26% identity with the TEM-type extended-spectrum β-lactamases (ESBLs), catalyses the hydrolysis of oxyimino-β-lactams such as cefotaxime (CTX), ceftazidime (CAZ) and aztreonam (AZT). Molecular modelling was used to identify in PER-1 the amino acid residues corresponding to those found at positions 104, 164, 238 and 240 in the TEM-type ESBLs, which are critical for hydrolysis of oxyimino-β-lactams. The function of these residues in PER-1 was assessed by site-directed mutagenesis. In this enzyme, residue 104 could be either a glutamine, an asparagine or a threonine. The Gln → Gly mutation did not significantly affect the catalytic efficiency, while Asn → Gly and Thr → Glu resulted in a marked decrease in catalytic activity, probably due to the alteration of a hydrogen bond network connecting the putative Asn-104 residue to Asn-132 and Glu-166. Replacement of Ala-164 by Arg in PER-1 resulted in a mutant with no detectable activity, thus suggesting that Ala-164 is important for catalysis and stability of PER-1. Conversely, Ser-238 → Gly and Gly-240 → Glu had little effect on kcat and Km values. Finally, the replacement of the catalytic residue Glu-166 by an alanine resulted in a complete loss of activity for CTX and a marked decrease of kcat for CAZ and AZT. These results suggest that Glu-166 is an important residue in PER-1. However, residues other than Glu-166 could contribute in maintaining residual activity towards oxyimino-β-lactams in the Ala-166 mutant.

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