Role of the omega-loop in the activity, substrate specificity, and structure of class A beta-lactamase.

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TitleRole of the omega-loop in the activity, substrate specificity, and structure of class A beta-lactamase.
Publication TypeJournal Article
Year of Publication1998
AuthorsBanerjee, S, Pieper, U, Kapadia, G, Pannell, LK, Herzberg, O
JournalBiochemistry
Volume37
Issue10
Pagination3286-96
Date Published1998 Mar 10
ISSN0006-2960
KeywordsAmino Acid Sequence, Anti-Bacterial Agents, Base Sequence, beta-Lactamases, beta-Lactams, Binding Sites, Crystallography, X-Ray, DNA Primers, Electrochemistry, Kinetics, Models, Molecular, Protein Conformation, Protein Folding, Sequence Deletion, Staphylococcus aureus, Substrate Specificity
Abstract

The structure of class A beta-lactamases contains an omega-loop associated with the active site, which carries a key catalytic residue, Glu166. A 16-residue omega-loop deletion mutant of beta-lactamase from Staphylococcus aureus PC1, encompassing residues 163-178, was produced in order to examine the functional and structural role of the loop. The crystal structure was determined and refined at 2.3 A, and the kinetics of the mutant enzyme was characterized with a variety of beta-lactam antibiotics. In general, the wild-type beta-lactamase hydrolyzes penicillin compounds better than cephalosporins. In contrast, the deletion of the omega-loop led to a variant enzyme that acts only on cephalosporins, including third generation compounds. Kinetic measurements and electrospray mass spectrometry revealed that the first and third generation cephalosporins form stable acyl-enzyme complexes, except for the chromogenic cephalosporin, nitrocefin, which after acylating the enzyme undergoes hydrolysis at a 1000-fold slower rate than that with wild-type beta-lactamase. Hydrolysis of the acyl-enzyme adducts is prevented because the deletion of the omega-loop eliminates the deacylation apparatus comprising Glu166 and its associated nucleophilic water site. The crystal structure reveals that while the overall fold of the mutant enzyme is similar to that of the native beta-lactamase, local adjustments in the vicinity of the missing loop occurred. The altered beta-lactam specificity is attributed to these structural changes. In the native structure, the omega-loop restricts the conformation of a beta-strand at the edge of the active site depression. Removal of the loop provides the beta-strand with a new degree of conformational flexibility, such that it is displaced inward toward the active site space. Modeled Michaelis complexes with benzylpenicillin and cephaloridine show that the perturbed conformation of the beta-strand is inconsistent with penicillin binding because of steric clashes between the beta-lactam side chain substituent and the beta-strand. In contrast, no clashes occur upon cephalosporin binding. Recognition of third generation cephalosporins is possible because the bulky side chain substituents of the beta-lactam ring typical of these compounds can be accommodated in the space freed by the deletion of the omega-loop.

DOI10.1021/bi972127f
Alternate JournalBiochemistry
PubMed ID9521648
Grant ListR01-AI27175 / AI / NIAID NIH HHS / United States