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

Abstract

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 ...

Tissue- and Age-Dependent Differences in the Complexation of Cadmium and Zinc in the Cadmium/Zinc Hyperaccumulator Thlaspi caerulescens (Ganges Ecotype) Revealed by X-Ray Absorption Spectroscopy

“…2 and 3, bottom) were attributed to the multiple scattering in the His ring. For Cd, however, this multiple scattering contribution is not that significant (for example, see Heinz et al, 2003). Thus, in the refinement, we did not further discriminate between nitrogen (N) and oxygen (O) donors.…”

“…For Cd, the multiple scattering contributions from imidazoles are not significant. Even in the case of proteins where the coordination of His is known (Heinz et al, 2003), multiple scattering contributions are difficult to refine. S contributions to the first shell at around 2.5 Å are clearly distinct from those of low Z scatterers.…”

New β-Lactamase Inhibitors: a Therapeutic Renaissance in an MDR World

“…Of these, the thiol derivatives have shown the most promise for meeting the need for broad-spectrum activity against each of the MBL subclasses. Thiols, including the clinically available antihypertension agent L-captopril, have shown effective inhibition of NDM-1 and subclass B1, B2, and B3 enzymes (75)(76)(77)(78). For example, synthesis informed by binding mode structural analyses led to a thiol compound with 0.019, 5.7, and 1.8 M affinity values for MBLs from each class, IMP-1, CphA, and L1, respectively (Fig.…”

Mechanistic Studies on the Mononuclear ZnII-Containing Metallo-β-lactamase ImiS from Aeromonas sobria

“…Toney and Moloughney recently reviewed the literature on inhibitors of MβL's (13), and most of these inhibitors are effective against only one or two of the enzymes. This is not surprising since, with few exceptions (14)(15)(16), the design of most of the inhibitors was guided by studies on only one enzyme. To address this problem, we are currently characterizing a metallo-β-lactamase from each of the group B subclasses (CcrA, ImiS, and L1) in an effort to identify common structural/mechanistic properties of the enzymes towards which a single inhibitor can be designed.…”