Substitution of Met-69 by Ala or Gly in TEM-1 beta-lactamase confer an increased susceptibility to clavulanic acid and other inhibitors.
In some inhibitor-resistant TEM-derived beta-lactamases, Met-69 is substituted by Leu, Ile or Val. Residue 69 is located in a region of strong structural constraints, at the beginning of H2 alpha-helix, and in the vicinity of B3 and B4 beta-strands. Analysis of the three-dimensional structure of TEM-1 beta-lactamase suggests that alteration of the substrate-binding site can be produced by changes of the size of residue 69 side chain. Met-69 was substituted by alanine or glycine in TEM-Bs beta-lactamase (a TEM-1-related enzyme) using site-directed mutagenesis. The minimum inhibitory concentrations of the mutants compared with the wild-type revealed an increased susceptibility to beta-lactamase inhibitor-beta-lactam combinations and to first-generation cephalosporins. Comparing the Met69Ala and Met69Gly beta-lactamases with TEM-Bs, K(m) constants of the mutants showed an increased affinity for most beta-lactams but the kcat for most substrates did not change substantially. Mutants also demonstrated lower IC50 for the three inhibitors (clavulanic acid, tazobactam and sulbactam). The two substitutions of the residue 69 by alanine and glycine had a noticeable effect on K(m) values of TEM-Bs beta-lactamase, and on affinity for beta-lactamase inhibitors.
Madec
Stéphanie
S
CNRS, UBO, MNHN, FRE 2125, 6 rue de l'Université, 29000, Quimper, France.
Blin
Claudine
C
Krishnamoorthy
Rajagopal
R
Picard
Bertrand
B
Chaibi
El Bachir
el B
Fouchereau-Péron
Martine
M
Labia
Roger
R
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D019818:Clavulanic Acid / Q000378:metabolism / Q000494:pharmacology*; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008715:Methionine / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation; D013379:Substrate Specificity; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid; D008715:Methionine; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D000409:Alanine; D005998:Glycine