Role of the Ω-Loop in the Activity, Substrate Specificity, and Structure of Class A β-Lactamase,
- 20 February 1998
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 37 (10), 3286-3296
- https://doi.org/10.1021/bi972127f
Abstract
The structure of class A β-lactamases contains an Ω-loop associated with the active site, which carries a key catalytic residue, Glu166. A 16-residue Ω-loop deletion mutant of β-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 Å, and the kinetics of the mutant enzyme was characterized with a variety of β-lactam antibiotics. In general, the wild-type β-lactamase hydrolyzes penicillin compounds better than cephalosporins. In contrast, the deletion of the Ω-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 β-lactamase. Hydrolysis of the acyl-enzyme adducts is prevented because the deletion of the Ω-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 β-lactamase, local adjustments in the vicinity of the missing loop occurred. The altered β-lactam specificity is attributed to these structural changes. In the native structure, the Ω-loop restricts the conformation of a β-strand at the edge of the active site depression. Removal of the loop provides the β-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 β-strand is inconsistent with penicillin binding because of steric clashes between the β-lactam side chain substituent and the β-strand. In contrast, no clashes occur upon cephalosporin binding. Recognition of third generation cephalosporins is possible because the bulky side chain substituents of the β-lactam ring typical of these compounds can be accommodated in the space freed by the deletion of the Ω-loop.Keywords
This publication has 16 references indexed in Scilit:
- Site-directed Mutagenesis of Glutamate 166 in Two β-LactamasesPublished by Elsevier BV ,1997
- Molecular evolution of bacterial β-lactam resistanceCell Chemical Biology, 1996
- AMoRe: an automated package for molecular replacementActa Crystallographica Section A Foundations of Crystallography, 1994
- β‐lactamase TEM1 of E. coli Crystal structure determination at 2.5 Å resolutionFEBS Letters, 1992
- Refined crystal structure of β-lactamase from Staphylococcus aureus PC1 at 2.0 Å resolutionJournal of Molecular Biology, 1991
- Phosphocholine binding immunoglobulin Fab McPC603: An X-ray diffraction study at 2.7 ÅJournal of Molecular Biology, 1986
- Single amino acid mutations block a late step in the folding of β-lactamase from Staphylococcus aureusJournal of Molecular Biology, 1985
- Effects of sulphate and urea on the stability and reversible unfolding of β-lactamase from Staphylococcus aureus: Implications for the folding pathway of β-lactamaseJournal of Molecular Biology, 1985
- Penicillinase active sites: Labelling of serine‐44 in β‐lactamase I by 6β‐bromopenicillanic acidFEBS Letters, 1979
- Molecular architecture of the cephalosporins. Insights into biological activity based on structural investigationsJournal of the American Chemical Society, 1970