title: Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase.
abstract: The structure of the class C ampC beta-lactamase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-A resolution and compared to a class A beta-lactamase (penicillinase) structure. The binding site for beta-lactam (penicillinase) structure. The binding site for beta-lactam antibiotics is generally more open than that in penicillinases, in agreement with the ability of the class C beta-lactamases to better bind third-generation cephalosporins. Four corresponding catalytic residues (Ser-64/70, Lys-67/73, Lys-315/234, and Tyr-150/Ser-130 in class C/A) lie in equivalent positions within 0.4 A. Significant differences in positions and accessibilities of Arg-349/244 may explain the inability of clavulanate-type inhibitors to effectively inactivate the class C beta-lactamases. Glu-166, required for deacylation of the beta-lactamoyl intermediate in class A penicillinases, has no counterpart in this cephalosporinase; the nearest candidate, Asp-217, is 10 A from the reactive Ser-64. A comparison of overall tertiary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral beta-lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two beta-lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C beta-lactamase to evolve to the class A beta-lactamase having an improved ability to catalyze the deacylation step of beta-lactam hydrolysis.
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269-3125.
authors.forename: E
authors.initials: E
authors.lastname: Lobkovsky
authors.forename: P C
authors.initials: PC
authors.lastname: Moews
authors.forename: H
authors.initials: H
authors.lastname: Liu
authors.forename: H
authors.initials: H
authors.lastname: Zhao
authors.forename: J M
authors.initials: JM
authors.lastname: Frere
authors.forename: J R
authors.initials: JR
authors.lastname: Knox
mesh_terms: D001665:Binding Sites; D005075:Biological Evolution; D002510:Cephalosporinase / Q000648:ultrastructure*; D018360:Crystallography, X-Ray; D016972:Enterobacter cloacae / Q000201:enzymology*; D008024:Ligands; D010405:Penicillinase / Q000648:ultrastructure; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012694:Serine
chemical_list: D008024:Ligands; D012694:Serine; D002510:Cephalosporinase; D010405:Penicillinase