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Role of zinc content on the catalytic efficiency of B1 metallo beta-lactamases.

ABSTRACT: Metallo beta-lactamases (MbetaL) are enzymes naturally evolved by bacterial strains under the evolutionary pressure of beta-lactam antibiotic clinical use. They have a broad substrate spectrum and are resistant to all the clinically useful inhibitors, representing a potential risk of infection if massively disseminated. The MbetaL scaffold is designed to accommodate one or two zinc ions able to activate a nucleophilic hydroxide for the hydrolysis of the beta-lactam ring. The role of zinc content on the binding and reactive mechanism of action has been the subject of debate and still remains an open issue despite the large amount of data acquired. We report herein a study of the reaction pathway for binuclear CcrA from Bacteroides fragilis using density functional theory based quantum mechanics-molecular mechanics dynamical modeling. CcrA is the prototypical binuclear enzyme belonging to the B1 MbetaL family, which includes several harmful chromosomally encoded and transferable enzymes. The involvement of a second zinc ion in the catalytic mechanism lowers the energetic barrier for beta-lactam hydrolysis, preserving the essential binding features found in mononuclear B1 enzymes (BcII from Bacillus cereus) while providing a more efficient single-step mechanism. Overall, this study suggests that uptake of a second equivalent zinc ion is evolutionary favored.

SUBMITTER: Dal Peraro M

PROVIDER: S-EPMC2597527 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Role of zinc content on the catalytic efficiency of B1 metallo beta-lactamases.

Dal Peraro Matteo M Vila Alejandro J AJ Carloni Paolo P Klein Michael L ML

Journal of the American Chemical Society 20070217 10

Metallo beta-lactamases (MbetaL) are enzymes naturally evolved by bacterial strains under the evolutionary pressure of beta-lactam antibiotic clinical use. They have a broad substrate spectrum and are resistant to all the clinically useful inhibitors, representing a potential risk of infection if massively disseminated. The MbetaL scaffold is designed to accommodate one or two zinc ions able to activate a nucleophilic hydroxide for the hydrolysis of the beta-lactam ring. The role of zinc content ...[more]

PMID: 17305336

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Project description:BackgroundGenes conferring antibiotic resistance to groups of bacterial pathogens are cause for considerable concern, as many once-reliable antibiotics continue to see a reduction in efficacy. The recent discovery of the metallo β-lactamase blaNDM-1 gene, which appears to grant antibiotic resistance to a variety of Enterobacteriaceae via a mobile plasmid, is one example of this distressing trend. The following work describes a computational analysis of pathogen-borne MBLs that focuses on the structural aspects of characterized proteins.ResultsUsing both sequence and structural analyses, we examine residues and structural features specific to various pathogen-borne MBL types. This analysis identifies a linker region within MBL-like folds that may act as a discriminating structural feature between these proteins, and specifically resistance-associated acquirable MBLs. Recently released crystal structures of the newly emerged NDM-1 protein were aligned against related MBL structures using a variety of global and local structural alignment methods, and the overall fold conformation is examined for structural conservation. Conservation appears to be present in most areas of the protein, yet is strikingly absent within a linker region, making NDM-1 unique with respect to a linker-based classification scheme. Variability analysis of the NDM-1 crystal structure highlights unique residues in key regions as well as identifying several characteristics shared with other transferable MBLs.ConclusionsA discriminating linker region identified in MBL proteins is highlighted and examined in the context of NDM-1 and primarily three other MBL types: IMP-1, VIM-2 and ccrA. The presence of an unusual linker region variant and uncommon amino acid composition at specific structurally important sites may help to explain the unusually broad kinetic profile of NDM-1 and may aid in directing research attention to areas of this protein, and possibly other MBLs, that may be targeted for inactivation or attenuation of enzymatic activity.

Project description:Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central β-sheets in the conserved αβ/βα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of β-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of β-lactam-bound MBLs show that the binding of β-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides β-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.

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Role of zinc content on the catalytic efficiency of B1 metallo beta-lactamases.

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Role of zinc content on the catalytic efficiency of B1 metallo beta-lactamases.

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