Protein Engineering, Vol. 12, No. 4, 313-318,
April 1999
© 1999 Oxford University Press
Site-directed mutagenesis of residues 164, 170, 171, 179, 220, 237 and 242 in PER-1 ß-lactamase hydrolysing expanded-spectrum cephalosporins
1 Laboratoire de Recherche Moléculaire sur les Antibiotiques, Université Pierre et Marie Curie (Paris VI), Faculté de Médecine Pitié-Salpêtrière and Faculté de Médecine Broussais-Hôtel Dieu, F-75634 Paris cedex 13 and 2 Laboratoire de Minéralogie-Cristallographie, CNRS URA 09, Université Pierre et Marie Curie (Paris VI), F-75252 Paris cedex 05, France
The class A ß-lactamase PER-1, which displays 26% identity with the TEM-type extended-spectrum ß-lactamases (ESBLs), is characterized by a substrate profile similar to that conferred by these latter enzymes. The role of residues Ala164, His170, Ala171, Asn179, Arg220, Thr237 and Lys242, found in PER-1, was assessed by site-directed mutagenesis. Replacement of Ala164 by Arg yielded an enzyme with no detectable ß-lactamase activity. Two other mutants, N179D and A164R+N179D, were also inactive. Conversely, a mutant with the A171E substitution displayed a substrate profile very similar to that of the wild-type enzyme. Moreover, the replacement of Ala171 by Glu in the A164R enzyme yielded a double mutant which was active, suggesting that Glu171 could compensate for the deleterious effect of Arg164 in the A164R+A171E enzyme. A specific increase in kcat 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. Finally, 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 relationships in PER-1, when compared with those previously described for the TEM-type ESBLs.
Keywords: ß-lactamase/expanded-spectrum cephalosporins/homology modelling/PER-1/serine enzyme
3 Present address: Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
4 To whom correspondence should be addressed. E-mail: sougakof{at}lmcp.jussieu.fr
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