Role of residues 104, 164, 166, 238 and 240 in the substrate profile of PER-1 beta-lactamase hydrolysing third-generation cephalosporins.
The class A beta-lactamase PER-1, which displays 26% identity with the TEM-type extended-spectrum beta-lactamases (ESBLs), catalyses the hydrolysis of oxyimino-beta-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-beta-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-beta-lactams in the Ala-166 mutant.
Bouthors
A T
AT
Laboratoire de Recherche Moléculaire sur les Antibiotiques, Faculté de Médecine Pitié-Salpêtrière, Université Pierre et Marie Curie, F-75634 Paris cedex 13, France.
Dagoneau-Blanchard
N
N
Naas
T
T
Nordmann
P
P
Jarlier
V
V
Sougakoff
W
W
D000595:Amino Acid Sequence; D001665:Binding Sites; D002511:Cephalosporins / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D002511:Cephalosporins; D011994:Recombinant Proteins; C108863:beta-lactamase PER-1; D001618:beta-Lactamases