Inactivation of <i>Aeromonas hydrophila</i> metallo‐β‐lactamase by cephamycins and moxalactam

Zervosen, Astrid; Hernandez-Valladares, Maria; Devreese, Bart; Prosperi-Meys, Christelle; Adolph, Hans-Werner; Mercuri, Paola Sandra; Vanhove, Marc; Amicosante, Gianfranco; Beeumen, Jozef Van; Frère, Jean‐Marie; Galleni, Moreno

doi:10.1046/j.1432-1327.2001.02298.x

Cited by 31 publications

(27 citation statements)

References 30 publications

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“…This mass increase of 381 Da corresponds well with the mass of hydrolyzed cefuroxime after departure of the 3Ј leaving group -NH 2 COOH. Such as inactivation mechanism is in good agreement with the reported inactivation of Aeromonas hydrophila metallo-␤-lactamase CphA by cefoxitin (33). In the latter case, a disulfide bridge is possibly formed between the only cysteine of CphA and the dihydrothiazine sulfur atom of cefoxitin or between the cysteine and the exomethylene group of the hydrolyzed cefoxitin.…”

Section: Kinetic Properties Of the Asn 225supporting

confidence: 89%

Probing the Specificity of the Subclass B3 FEZ-1 Metallo-β-lactamase by Site-directed Mutagenesis

Mercuri

Garcia-Saez

Vriendt

et al. 2004

Journal of Biological Chemistry

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The subclass B3 FEZ-1 ␤-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the ␤-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Metallo-␤-lactamases are bacterial enzymes that hydrolyze antibiotics of the ␤-lactam family. They are classified as class B (1) or group 3 (2) ␤-lactamases. In the last decade, the discovery of an increasing number of new metallo-␤-lactamases resulted in a subdivision into three molecular subclasses: B1, B2, and B3. Thereafter, a standard numbering scheme (3) was adopted that identifies conserved residues involved in the catalytic activity. Subclass B3 ␤-lactamases are broad spectrum enzymes that require one or two Zn (II) ions for activity (4). These enzymes are produced by various environmental species, of which some can cause opportunistic infections (such as S. maltophilia (5) and F. gormanii (6)), whereas others are not pathogenic such as Janthinobacterium lividum (7) and Caulobacter crescentus (8, 9). The structures of several subclass B1 ␤-lactamases have been solved by x-ray crystallography (BcII (10, 11), CcrA (12), BlaB (13), and IMP-1 (14)). To date, no structure of a subclass B2 enzyme is available. In subclass B3, the crystal structures of the metallo ␤-lactamases L1 from S. maltophilia (15) and FEZ-1 from F. gormanii (16) have been solved. Comparison of the tertiary structures of the different enzymes highlighted similar organizations of the secondary structure elements; they all contain an ␣␤␤␣ sandwich with two central ␤-sheets and ␣-helices on the external faces (10). The active site with the binuclear zinc center is located at the bottom of the ␤-sheet core. Zn1 is tetrahedrally coordinated by three histidines, His 116 , His 118 , His 196 , and a water molecule. In the subclass B1 enzymes, Zn2 is coordinated by His 263 , Asp 120 , Cys 221 , and two water molecules to form a trigonal bipyramid. In subclass B3 ␤-lactamases, a Ser residue replaces Cys 221 , and His 121 is the third ligand of Zn2. Our studies were performed on the FEZ-1 ␤-lactamase. FEZ-1 is a monomeric enzyme. The sequence of the mature protein is easily aligned with that of the L1 enzyme with 33% of isology (17). The two subclass B3 ␤-lactamases exhibit a broad activity spectrum against ␤-lactam antibiotics, but FEZ-1 shows a preference for cephalosporins (18), whereas the L1 ␤-lactamase seems to be more active against penicillins (19,20). Comparison of the x-ray structures reveals similar zinc binding sites in...

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Section: Kinetic Properties Of the Asn 225supporting

confidence: 89%

Probing the Specificity of the Subclass B3 FEZ-1 Metallo-β-lactamase by Site-directed Mutagenesis

Mercuri

Garcia-Saez

Vriendt

et al. 2004

Journal of Biological Chemistry

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“…This may reflect an inability of CphA to transfer a proton to the bound intermediate in the crystal. We see no evidence of inactivation as was seen previously for CphA with moxalactam, where the 3′ leaving group reacted with the active-site cysteinate (87). Inhibitory Zn Site.…”

Section: -Zn Imissupporting

confidence: 56%

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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“…Both studies agree in a bridging role of the metal-bound sulfur of the inhibitor, whereas the carboxylate group of the inhibitors binds to an accessible amino acid, thus stabilizing the complex. Other known inhibitory compounds are 2,3-(S,S)-disubstituted succinic acids for IMP-1 (6) or moxalactam and cefoxitin for CphA (7). The latter compounds lead to irreversible inactivation of the enzyme by the hydrolyzed reaction products.…”

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confidence: 99%

Coordination Geometries of Metal Ions in D- or L-Captopril-inhibited Metallo-β-lactamases

Heinz

Bauer²,

Wommer

et al. 2003

Journal of Biological Chemistry

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D-and L-captopril are competitive inhibitors of metallo-␤-lactamases. For the enzymes from Bacillus cereus (BcII) and Aeromonas hydrophila (CphA), we found that the mononuclear enzymes are the favored targets for inhibition. By combining results from extended x-ray absorption fine structure, perturbed angular correlation of ␥-rays spectroscopy, and a study of metal ion binding, we derived that for Cd(II) 1 -BcII, the thiolate sulfur of D-captopril binds to the metal ion located at the site defined by three histidine ligand residues. This is also the case for the inhibited Co(II) 1 and Co(II) 2 enzymes as observed by UV-visible spectroscopy. Although the single metal ion in Cd(II) 1 -BcII is distributed between both available binding sites in both the uninhibited and the inhibited enzyme, Cd(II) 1 -CphA shows only one defined ligand geometry with the thiolate sulfur coordinating to the metal ion in the site composed of 1 Cys, 1 His, and 1 Asp. CphA shows a strong preference for D-captopril, which is also reflected in a very rigid structure of the complex as determined by perturbed angular correlation spectroscopy. For BcII and CphA, which are representatives of the metallo-␤-lactamase subclasses B1 and B2, we find two different inhibitor binding modes.Metallo-␤-lactamases confer antibiotic resistance to bacteria by catalyzing the hydrolysis of ␤-lactam antibiotics, including carbapenems. This relatively new form of resistance is spreading and thereby escaping the effective inhibitors developed to fight the better known serine-␤-lactamases. For all metallo-␤-lactamases investigated, structurally similar enzyme active sites comprising two zinc binding sites are reported. For Bacillus cereus metallo-␤-lactamase (BcII), 1 one metal-binding site contains three His (H-site); the other one contains 1 Asp, 1 Cys, and 1 His as the metal ligating residues (DCH site) as derived from x-ray crystallography (1). For CphA, 1 His from the H-site (His-116) is supposed to be replaced by an Asn (2). Various thiol-carboxylate compounds were identified as potent inhibitors (3). The active site binding of thiomandelic acid to BcII was studied by NMR spectroscopy (4), whereas the binding of 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4-(phenylbutrylglycine)] to the enzyme from Pseudomonas aeruginosa (IMP-1) was characterized by x-ray crystallography (5). With both approaches, the inhibited binuclear zinc enzymes were studied. Both studies agree in a bridging role of the metal-bound sulfur of the inhibitor, whereas the carboxylate group of the inhibitors binds to an accessible amino acid, thus stabilizing the complex. Other known inhibitory compounds are 2,3-(S,S)-disubstituted succinic acids for IMP-1 (6) or moxalactam and cefoxitin for CphA (7). The latter compounds lead to irreversible inactivation of the enzyme by the hydrolyzed reaction products.The structural investigation of D-and L-captopril binding presented here is based on results obtained from enzyme kinetic and thermodynamic studies. Captopril is known as an ...

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Inactivation of Aeromonas hydrophila metallo‐β‐lactamase by cephamycins and moxalactam

Cited by 31 publications

References 30 publications

Probing the Specificity of the Subclass B3 FEZ-1 Metallo-β-lactamase by Site-directed Mutagenesis

Probing the Specificity of the Subclass B3 FEZ-1 Metallo-β-lactamase by Site-directed Mutagenesis

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

Coordination Geometries of Metal Ions in D- or L-Captopril-inhibited Metallo-β-lactamases

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