N O Concha1, B A Rasmussen, K Bush, O Herzberg. 1. Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville 20850, USA.
Abstract
BACKGROUND: The metallo-beta-lactamase from Bacteroides fragilis hydrolyzes a wide range of beta-lactam antibiotics, and is not clinically susceptible to any known beta-lactamase inhibitors. B. fragilis is associated with post-surgery hospital infections, and there has been a recent report of plasmid-mediated dissemination of the enzyme. Effective inhibitors are therefore urgently needed. Knowledge of the three-dimensional structure will aid in the drug design effort. RESULTS: The crystal structure of the enzyme has been determined by using multiwavelength anomalous diffraction at the zinc absorption edge and refined to 1.85 A resolution. The structure is a four-layer alpha/beta/beta/alpha molecule. The active site, found at the edge of the beta sandwich contains a binuclear zinc center with several novel features. One zinc is tetrahedrally coordinated, the other has a trigonal bipyramidal coordination; a water/hydroxide molecule serves as a ligand for both metals. The residues that coordinate the two zincs are invariant in all metallo-beta-lactamases that have been sequenced, except for two conservative replacements. Despite the existence of the pattern for binuclear zinc binding, the reported structure of the Bacillus cereus enzyme contains only a single zinc. CONCLUSIONS: Structural analysis indicates that affinity for the penta-coordinated zinc can be modulated by neighboring residues, perhaps explaining the absence of the second zinc in the B. cereus structure. Models of bound substrates suggest that the active-site channel can accommodate a wide variety of beta-lactams. We propose that the zinc cluster prepares an hydroxide, probably the hydroxide that ligates both zincs, for nucleophilic attack on the carbonyl carbon atom of the beta-lactam. The resulting negatively charged tetrahedral intermediate implicated in catalysis is stabilized by an oxyanion hole formed by the side chain of the invariant Asn 193 and the tetrahedral zinc.
BACKGROUND: The metallo-beta-lactamase from Bacteroides fragilis hydrolyzes a wide range of beta-lactam antibiotics, and is not clinically susceptible to any known beta-lactamase inhibitors. B. fragilis is associated with post-surgery hospital infections, and there has been a recent report of plasmid-mediated dissemination of the enzyme. Effective inhibitors are therefore urgently needed. Knowledge of the three-dimensional structure will aid in the drug design effort. RESULTS: The crystal structure of the enzyme has been determined by using multiwavelength anomalous diffraction at the zinc absorption edge and refined to 1.85 A resolution. The structure is a four-layer alpha/beta/beta/alpha molecule. The active site, found at the edge of the beta sandwich contains a binuclear zinc center with several novel features. One zinc is tetrahedrally coordinated, the other has a trigonal bipyramidal coordination; a water/hydroxide molecule serves as a ligand for both metals. The residues that coordinate the two zincs are invariant in all metallo-beta-lactamases that have been sequenced, except for two conservative replacements. Despite the existence of the pattern for binuclear zinc binding, the reported structure of the Bacillus cereus enzyme contains only a single zinc. CONCLUSIONS: Structural analysis indicates that affinity for the penta-coordinated zinc can be modulated by neighboring residues, perhaps explaining the absence of the second zinc in the B. cereus structure. Models of bound substrates suggest that the active-site channel can accommodate a wide variety of beta-lactams. We propose that the zinc cluster prepares an hydroxide, probably the hydroxide that ligates both zincs, for nucleophilic attack on the carbonyl carbon atom of the beta-lactam. The resulting negatively charged tetrahedral intermediate implicated in catalysis is stabilized by an oxyanion hole formed by the side chain of the invariant Asn 193 and the tetrahedral zinc.
Authors: L Boschi; P S Mercuri; M L Riccio; G Amicosante; M Galleni; J M Frère; G M Rossolini Journal: Antimicrob Agents Chemother Date: 2000-06 Impact factor: 5.191
Authors: M Galleni; J Lamotte-Brasseur; G M Rossolini; J Spencer; O Dideberg; J M Frère Journal: Antimicrob Agents Chemother Date: 2001-03 Impact factor: 5.191
Authors: Hanna-Kirsti S Leiros; Pardha S Borra; Bjørn Olav Brandsdal; Kine Susann Waade Edvardsen; James Spencer; Timothy R Walsh; Orjan Samuelsen Journal: Antimicrob Agents Chemother Date: 2012-06-04 Impact factor: 5.191