Structure-function analysis of alpha-helix H4 using PSE-4 as a model enzyme representative of class A beta-lactamases.
We extracted maximum information for structure-function analysis of the PSE-4 class A beta-lactamase by random replacement mutagenesis of three contiguous codons in the H4 alpha-helix at amino acid positions Ala125, Thr126, Met127, Thr128 and Thr129. These positions were predicted to interact with suicide mechanism-based inhibitors when examining the PSE-4 three-dimensional model. Structure-function studies on positions 125-129 indicated that in PSE-4 these amino acids have a role distinct from those in TEM-1, in tolerating substitutions at Ala125 and being invariant at Met127. The importance of Met127 was suspected to be implicated in a structural role in maintaining the integrity of the H4 alpha-helix structure together, thus maintaining the important Ser130-Asp131-Asn132 motif positioned towards the active site. At the structural level, the H4 region was analyzed using energy minimization of the H4 regions of the PSE-4 YAM mutant and compared with wild-type PSE-4. The Tyr 125 of the mutant YAM formed an edge to face pi-pi interaction with Phe 124 which also interacts with the Trp 210 with the same interactions. Antibiotic susceptibilities showed that amino acid changes in the the H4 alpha-helix region of PSE-4 are particularly sensitive to mechanism based-inhibitors. However, kinetic analysis of PSE-4 showed that the two suicide inhibitors belonging to the penicillanic acid sulfone class, sulbactam and tazobactam, were less affected by changes in the H4 alpha-helix region than clavulanic acid, an inhibitor of the oxypenam class. The analysis of H4 alpha-helix in PSE-4 suggests its importance in interactions with the three clinically useful inhibitors and in general to all class A enzymes.
Savoie
A
A
Microbiologie Moléculaire et Génie des Protéines, Pavillon Charles-Eugène Marchand, Département de Biologie Médicale, Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada.
Sanschagrin
F
F
Palzkill
T
T
Voyer
N
N
Levesque
R C
RC
D002942:Circular Dichroism; D019818:Clavulanic Acid / Q000494:pharmacology; D003198:Computer Simulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry; D013329:Structure-Activity Relationship; D013407:Sulbactam; D000078142:Tazobactam; D018440:beta-Lactam Resistance; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid; D010397:Penicillanic Acid; C410213:beta-lactamase PSE-4; D001618:beta-Lactamases; D013407:Sulbactam; D000078142:Tazobactam