Insights into the Molecular Basis for the Carbenicillinase Activity of PSE-4 β-Lactamase from Crystallographic and Kinetic Studies

PSE-4 is a class A β-lactamase produced by strains of Pseudomonas aeruginosa and is highly active for the penicillin derivative carbenicillin. The crystal structure of the wild-type PSE-4 carbenicillinase has been determined to 1.95 Å resolution by molecular replacement and represents the first structure of a carbenicillinase published to date. A superposition of the PSE-4 structure with that of TEM-1 shows a rms deviation of 1.3 Å for 263 Cα atoms. Most carbenicillinases are unique among class A β-lactamases in that residue 234 is an arginine (ABL standard numbering scheme), while in all other class A enzymes this residue is a lysine. Kinetic characterization of a R234K PSE-4 mutant reveals a 50-fold reduction in k cat/K m and confirms the importance of Arg 234 for carbenicillinase activity. A comparison of the structure of the R234K mutant refined to 1.75 Å resolution with the wild-type structure shows that Arg 234 stabilizes an alternate conformation of the Ser 130 side chain, not seen in other class A β-lactamase structures. Our molecular modeling studies suggest that the position of a bound carbenicillin would be shifted relative to that of a bound benzylpenicillin in order to avoid a steric clash between the carbenicillin α-carboxylate group and the conserved side chain of Asn 170. The alternate conformation of the catalytic Ser 130 in wild-type PSE-4 may be involved in accommodating this shift in the bound substrate position.