Dynamics of Mononuclear Cadmium β-Lactamase Revealed by the Combination of NMR and PAC Spectroscopy

Hemmingsen, Lars; Damblon, Christian; Antony, Jens; Jensen, Mikael; Adolph, Hans-Werner; Wommer, Sandra; Roberts, G. C. K.; Bauer, Rogert

doi:10.1021/ja0112240

Cited by 53 publications

(84 citation statements)

References 34 publications

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“…The main difference in the active site relative to BcII is a substitution of a His residue with an Asn residue in the H-site. The PAC results for Cd(II) 1 -CphA clearly demonstrate a distribution of the single cadmium ion bound between both binding sites, which are different from the one found for Cd(II) 1 -BcII (18). However, the DCH site is strongly preferred.…”

Section: Discussionmentioning

confidence: 67%

“…Both the cadmium and zinc enzymes of BcII exhibit negative cooperativity with respect to metal binding to the two conserved sites (17,18). This, however, does not mean that there is a high and a low affinity binding site.…”

Section: Discussionmentioning

confidence: 93%

“…In the presence of 25 M D-captopril and 5 M BcII, a first Cd(II) ion binds with a K D of 1.6 nM as compared with 8.3 nM in absence of D-captopril (18). A second cadmium ion is bound with a K D of 50 M as compared with 5.9 M in the absence of the inhibitor (18). These K D values are macroscopic constants, and in case of the mononuclear enzymes, they do not reflect any binding site assignment (see below).…”

Section: Resultsmentioning

confidence: 99%

“…This, however, does not mean that there is a high and a low affinity binding site. We have shown earlier that a single metal ion, when bound to the binuclear site, is distributed between both binding sites (10, 12) and that rapid exchange among binding sites occurs (18). A recent work (28) shows that only mononuclear metallo-␤-lactamases might be physiologically important.…”

Section: Discussionmentioning

confidence: 99%

“…The dissociation constants for a first (K mono ) and second (K bi ) cadmium ion bound to BcII in presence of 25 M D-captopril and 0.1 M NaCl were obtained from competition experiments with the chromophoric chelator Mag-fura-2 (Molecular Probes, Eugene, OR) in 15 mM HEPES, pH 7.0, as described previously (17,18).…”

Section: Determination Of Metal Ion Dissociation Constantsmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Determination Of Metal Ion Dissociation Constantsmentioning

confidence: 99%

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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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Metallo β‐Lactamases

Herzberg

Fitzgerald

2004

Handbook of Metalloproteins

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The metallo‐β‐lactamases (class B β‐lactamases) confer bacterial resistance to β‐lactam antibiotics and are therefore targets for the development of clinical agents against resistant strains of bacteria. They are found in a wide variety of bacteria and act on a broad spectrum of substrates including penicillins, cephalosporins, and carbapenems, as well as on some inhibitors of serine‐β‐lactamases. These ∼25 000 Da zinc‐dependent enzymes have been studied extensively in recent years, and crystal structures have been determined for the enzymes from five different bacteria representing two different sequence subclasses (B1 and B3). The active site is located at the interface between two α/β domains where two zinc atoms bind. This review surveys the current understanding of these enzymes, with emphasis on structural studies, spectroscopy, kinetics, and proposals for the catalytic mechanism.

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Perturbed Angular Correlations of γ‐rays (PAC) Spectroscopy

Hemmingsen¹,

Butz²

2005

Encyclopedia of Inorganic Chemistry

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In this article, we give an elementary introduction to the theory and applications of perturbed angular correlation of γ‐rays (PAC) spectroscopy. In most applications in bioinorganic and inorganic chemistry the nuclear quadrupole interaction (NQI) is measured, providing a spectroscopic fingerprint of the local electronic and molecular structure and dynamics at the PAC probe site. The focus is on selected illustrative applications of PAC spectroscopy in bioinorganic chemistry: (i) local structure at metal ion binding sites in the bacterial mercury sensor MerR; de novo designed proteins; alcohol dehydrogenase–pH dependence; carboxypeptidase A—crystalline state versus solution; and carboxypeptidase A–during the hydrolysis of substrate; (ii) trapping intermediates in reactions using rapid freeze quenching; (iii) in vivo experiments on Cd(II) binding to biomolecules in cyanobacteria; (iv) cooperativity of metal ion binding in multinuclear sites; (v) dynamics of protein folding; (vi) rotational diffusion of biomolecules as a tool to monitor protein–protein interactions; (vii) exchange dynamics; and (viii) internal dynamics in proteins. Examples from inorganic chemistry and solid state physics are included for cases where the information derived is different from that already presented in the examples from bioinorganic chemistry: (i) magnetic properties of metals; (ii) defects trapped by the impurity PAC probe atom; (iii) temperature and pressure dependence of the EFG in metals; (iv) amorphous metals, alloys and internal oxidation; (v) metal surfaces and interfaces; (vi) semiconductors; (vii) phase transitions; (viii) charge density waves in transition metal dichalcogenides; (ix) intercalation reactions in 2H‐TaS 2 ; (x) structure and bonding; (xi) single crystal experiments.

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Dynamics of Mononuclear Cadmium β-Lactamase Revealed by the Combination of NMR and PAC Spectroscopy

Cited by 53 publications

References 34 publications

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

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

Metallo β‐Lactamases

Perturbed Angular Correlations of γ‐rays (PAC) Spectroscopy

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