A Metallo-β-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and its Complex with Biapenem

Garau, Gianpiero; Bebrone, Carine; Anne, Christine; Galleni, Moreno; Frère, Jean‐Marie; Dideberg, Otto

doi:10.1016/j.jmb.2004.10.070

Cited by 217 publications

(457 citation statements)

References 23 publications

(27 reference statements)

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“…sobria with its native metal, zinc, in both the hydrolytically active, monozinc form (with and without bound product) and the inhibited, dizinc form. The results for 1-Zn ImiS confirm the first equivalent of zinc binds to the Zn 2 site, consistent with previous spectroscopic characterization of the Co IIsubstituted enzyme (79) and the crystal structure of CphA (82). EXAFS of the product-bound form of 1-Zn ImiS with imipenem indicates that the product is bound in a monodentate fashion, through a carboxylate ligand, suggesting that the product-bound species isolated here is the end-stage product complex implied but not observed in X-ray diffraction studies of CphA (82).…”

Section: Discussionsupporting

confidence: 90%

“…The results for 1-Zn ImiS confirm the first equivalent of zinc binds to the Zn 2 site, consistent with previous spectroscopic characterization of the Co IIsubstituted enzyme (79) and the crystal structure of CphA (82). EXAFS of the product-bound form of 1-Zn ImiS with imipenem indicates that the product is bound in a monodentate fashion, through a carboxylate ligand, suggesting that the product-bound species isolated here is the end-stage product complex implied but not observed in X-ray diffraction studies of CphA (82). EXAFS of 2-Zn ImiS (wild type) and 2-Zn ImiS (M146I) support three conclusions: (1) the second equivalent of zinc binds to a location remote from the active site; (2) the ligand set of the inhibitory metal ion includes the sulfur of M146; and (3) a histidine, most likely H118, is also coordinated by the inhibitory metal ion.…”

Section: Discussionsupporting

confidence: 90%

“…2-Zn ImiS extended X-ray absorption fine structure data support a binding site that is distant from the active site and contains both one sulfur donor and one histidine ligand. On the basis of the amino acid sequence of ImiS and the crystal structure of CphA [Garau et al (2005) J. Mol. Biol.…”

mentioning

confidence: 99%

“…The X-ray structure (82) and a previous extended X-ray absorption fine structure (EXAFS) study (77) of CphA are currently the only direct examinations of a B2 -lactamase in its catalytically active, monozinc form. A similar binding site, on the basis of cobalt substitution, has been predicted for ImiS from Aeromonas Veronii, which shares 98% amino acid sequence identity with CphA (79).…”

mentioning

confidence: 99%

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X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

et al. 2006

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X-ray absorption spectroscopy was used to investigate the metal-binding sites of ImiS from Aeromonas Veronii bV. sobria in catalytically active (1-Zn), product-inhibited (1-Zn plus imipenem), and inactive (2-Zn) forms. The first equivalent of zinc(II) was found to bind to the consensus Zn 2 site. The reaction of 1-Zn ImiS with imipenem leads to a product-bound species, coordinated to Zn via a carboxylate group. The inhibitory binding site of ImiS was examined by a comparison of wild-type ImiS with 1 and 2 equiv of bound zinc. 2-Zn ImiS extended X-ray absorption fine structure data support a binding site that is distant from the active site and contains both one sulfur donor and one histidine ligand. On the basis of the amino acid sequence of ImiS and the crystal structure of CphA [Garau et al. (2005) J. Mol. Biol. 345,[785][786][787][788][789][790][791][792][793][794][795], we propose that the inhibitory binding site is formed by M146, found on the B2-distinct R3 helix, and H118, a canonical Zn 1 ligand, proposed to help activate the nucleophilic water. The mutation of M146 to isoleucine abolishes metal inhibition. This is the first characterization of ImiS with the native metal Zn and establishes, for the first time, the location of the inhibitory metal site.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 90%

mentioning

confidence: 99%

mentioning

confidence: 99%

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X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

et al. 2006

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“…The presented GIM-1 structures imply that both loop 1 and loop 2 can adapt their conformations during catalysis. Other studies on MBLs have shown a complex interplay between sequence and structure, revealing that loop flexibility, protein stability, and particular mutations are important for biological function and efficiency (24)(25)(26). Some of the described effects are also ligand dependent.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structures of Pseudomonas aeruginosa GIM-1: Active-Site Plasticity in Metallo-β-Lactamases

Borra

Samuelsen

Spencer

et al. 2013

Antimicrob Agents Chemother

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Metallo-␤-lactamases (MBLs) have rapidly disseminated worldwide among clinically important Gram-negative bacteria and have challenged the therapeutic use of ␤-lactam antibiotics, particularly carbapenems. The bla GIM-1 gene, encoding one such enzyme, was first discovered in a Pseudomonas aeruginosa isolate from 2002 and has more recently been reported in Enterobacteriaceae. Here, we present crystal structures of GIM-1 in the apo-zinc (metal-free), mono-zinc (where Cys221 was found to be oxidized), and di-zinc forms, providing nine independently refined views of the enzyme. GIM-1 is distinguished from related MBLs in possessing a narrower active-site groove defined by aromatic side chains (Trp228 and Tyr233) at positions normally occupied by hydrophilic residues in other MBLs. Our structures reveal considerable flexibility in two loops (loop 1, residues 60 to 66; loop 2, residues 223 to 242) adjacent to the active site, with open and closed conformations defined by alternative hydrogen-bonding patterns involving Trp228. We suggest that this capacity for rearrangement permits GIM-1 to hydrolyze a wide range of ␤-lactams in spite of possessing a more constrained active site. Our results highlight the structural diversity within the MBL enzyme family.

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β‐Lactam Antibiotics

Testero

Fisher

Mobashery

2010

Burger's Medicinal Chemistry and Drug Discovery

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The β‐lactam classes of antibacterials are preeminent in the treatment of bacterial infection due to their unparalleled clinical efficacy and clinical safety. Following the discovery of the penicillins, successive β‐lactam drug discovery has added the cephalosporin, penem cephamycin, clavulanate, monobactam, nocardicin, and carbapenem subclasses. The driving force behind much of this era of discovery is the staggering ability of pathogenic bacteria to adapt previous generations of the β‐lactam by the acquisition and expression of resistance mechanisms. Although many factors contribute to β‐lactam resistance, alterations to the molecular targets of the β‐lactams (the penicillin binding proteins) and the use of enzymes (the β‐lactamases) capable of the hydrolytic deactivation of the β‐lactams are paramount. This review traces the historical development of β‐lactam drug discovery, with emphasis on the most recent progress in the medicinal chemistry, biochemistry, and microbiology of the β‐lactams leading to the discovery of new generation β‐lactam antibacterials effective against the Gram‐negative and ‐positive bacterial pathogens of current medical concern.

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A Metallo-β-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and its Complex with Biapenem

Cited by 217 publications

References 23 publications

X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

Crystal Structures of Pseudomonas aeruginosa GIM-1: Active-Site Plasticity in Metallo-β-Lactamases

β‐Lactam Antibiotics

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