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      Metallo-β-lactamases withstand low Zn II conditions by tuning metal-ligand interactions

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          Abstract

          A number of multiresistant bacterial pathogens inactivate antibiotics by producing Zn II-dependent β-lactamases. We show that metal uptake leading to an active dinuclear enzyme in the periplasmic space of Gram-negative bacteria is ensured by a cysteine residue, an unusual metal ligand in oxidizing environments. Kinetic, structural and affinity data show that such Zn II-Cys interaction is an adaptive trait tuning the metal binding affinity, thus enabling antibiotic resistance at restrictive Zn II concentrations.

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          Most cited references18

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          Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis.

          Intracellular zinc is thought to be available in a cytosolic pool of free or loosely bound Zn(II) ions in the micromolar to picomolar range. To test this, we determined the mechanism of zinc sensors that control metal uptake or export in Escherichia coli and calibrated their response against the thermodynamically defined free zinc concentration. Whereas the cellular zinc quota is millimolar, free Zn(II) concentrations that trigger transcription of zinc uptake or efflux machinery are femtomolar, or six orders of magnitude less than one atom per cell. This is not consistent with a cytosolic pool of free Zn(II) and suggests an extraordinary intracellular zinc-binding capacity. Thus, cells exert tight control over cytosolic metal concentrations, even for relatively low-toxicity metals such as zinc.
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            Metallo-beta-lactamases: novel weaponry for antibiotic resistance in bacteria.

            Metallo-beta-lactamases are broad-spectrum zinc enzymes, able to inactivate most clinically useful beta-lactam antibiotics. Their structural and functional diversity has thus far limited the understanding of their catalytic mechanism, therefore thwarting the rational design of a common inhibitor. On the basis of the recent availability of structures of enzyme-product complexes and novel mechanistic studies, here, we attempt to find minimal common elements in different members of this family. In contrast with other metalloenzymes, most of the substrate binding and catalytic power resides in the adequate positioning of one or two Zn(II) ions in the active site, empowered by an unusual flexibility.
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              The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold.

              The 3-D structure of Bacillus cereus (569/H/9) beta-lactamase (EC 3.5.2.6), which catalyses the hydrolysis of nearly all beta-lactams, has been solved at 2.5 A resolution by the multiple isomorphous replacement method, with density modification and phase combination, from crystals of the native protein and of a specially designed mutant (T97C). The current model includes 212 of the 227 amino acid residues, the zinc ion and 10 water molecules. The protein is folded into a beta beta sandwich with helices on each external face. To our knowledge, this fold has never been observed. An approximate internal molecular symmetry is found, with a 2-fold axis passing roughly through the zinc ion and suggesting a possible gene duplication. The active site is located at one edge of the beta beta sandwich and near the N-terminal end of a helix. The zinc ion is coordinated by three histidine residues (86, 88 and 149) and a water molecule. A sequence comparison of the relevant metallo-beta-lactamases, based on this protein structure, highlights a few well-conserved amino acid residues. The structure shows that most of these residues are in the active site. Among these, aspartic acid 90 and histidine 210 participate in a proposed catalytic mechanism for beta-lactam hydrolysis.
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                Author and article information

                Journal
                101231976
                32624
                Nat Chem Biol
                Nat. Chem. Biol.
                Nature chemical biology
                1552-4450
                1552-4469
                10 August 2012
                24 June 2012
                August 2012
                01 February 2013
                : 8
                : 8
                : 698-700
                Affiliations
                [1 ]Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Suipacha 531, S2002LRK Rosario, Argentina.
                [3 ]Laboratório Nacional de Luz Síncrotron, Rua Giuseppe Máximo Scolfaro 10000, Campinas, Sao Paulo, CEP 13083-100, Brazil.
                Author notes
                Correspondence should be addressed to JAC ( cricco@ 123456ibr.gov.ar ) and AJV ( vila@ 123456ibr.gov.ar )
                [2]

                Present address: Department of Pharmaceutical Sciences and UMXSS Crystallography Service, University of Maryland at Baltimore, Baltimore, MD 21201, USA.

                [4]

                Present address: Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, E-28040 Madrid, Spain.

                [*]

                These authors contributed equally to this paper.

                Article
                NIHMS397926
                10.1038/nchembio.1005
                3470787
                22729148
                5802a789-725b-4a53-83bf-e0ccb2beb422

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                History
                Funding
                Funded by: National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID
                Award ID: R01 AI100560 || AI
                Categories
                Article

                Biochemistry
                Biochemistry

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