Insights into the molecular basis for the carbenicillinase activity of PSE-4 beta-lactamase from crystallographic and kinetic studies.
PSE-4 is a class A beta-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 A 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 A for 263 Calpha atoms. Most carbenicillinases are unique among class A beta-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 A 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 beta-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 alpha-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.
Lim
D
D
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
Sanschagrin
F
F
Passmore
L
L
De Castro
L
L
Levesque
R C
RC
Strynadka
N C
NC
D000409:Alanine / Q000235:genetics; D001120:Arginine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010405:Penicillinase / Q000737:chemistry* / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001120:Arginine; D010405:Penicillinase; C410213:beta-lactamase PSE-4; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D008239:Lysine; D000409:Alanine