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Antimicrobial Agents and Chemotherapy, March 1999, p. 543-548, Vol. 43, No. 3
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Structure-Function Studies of Ser-289 in the Class C beta -Lactamase from Enterobacter cloacae P99

Sonia Trépanier,1 James R. Knox,2 Natalie Clairoux,1 François Sanschagrin,1 Roger C. Levesque,1 and Ann Huletsky1,*

Département de Biologie Médicale, Pavillon Marchand, Université Laval, Ste-Foy, Québec, Canada G1K 7P4,1 and Department of Molecular and Cell Biology, The University of Connecticut, Storrs, Connecticut 06269-31252

Received 12 June 1998/Returned for modification 5 October 1998/Accepted 17 December 1998

Site-directed mutagenesis of Ser-289 of the class C beta -lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in beta -lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant beta -lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC beta -lactamase. However, replacement by Lys or Arg decreased by two- to threefold the kcat of four of the five beta -lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant beta -lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.

* Corresponding author. Mailing address: Département de Biologie Médicale, Pavillon Marchand, Université Laval, Ste-Foy, Québec, Canada G1K 7P4. Phone: (418) 656-2131, ext. 2669. Fax: (418) 656-7176. E-mail: ann.huletsky{at}rsvs.ulaval.ca.

Antimicrobial Agents and Chemotherapy, March 1999, p. 543-548, Vol. 43, No. 3
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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