Crystal structures of the Bacillus licheniformis BS3 class A beta-lactamase and of the acyl-enzyme adduct formed with cefoxitin.
The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alpha-methoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 A resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges.
Fonzé
Eveline
E
Centre d'Ingénierie des Protéines, Institut de Physique B5 and Institut de Chimie B6, Université de Liège, B-4000 Sart Tilman, Belgium.
Vanhove
Marc
M
Dive
Georges
G
Sauvage
Eric
E
Frère
Jean-Marie
JM
Charlier
Paulette
P
D001407:Bacillus / Q000201:enzymology*; D020134:Catalytic Domain; D002440:Cefoxitin / Q000737:chemistry; D002513:Cephamycins / Q000737:chemistry; D018360:Crystallography, X-Ray; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D055672:Static Electricity; D001618:beta-Lactamases / Q000737:chemistry*
D002513:Cephamycins; D046911:Macromolecular Substances; D002440:Cefoxitin; D001618:beta-Lactamases