Mono- and Binuclear Zn2+-β-Lactamase

Self Cite

116

We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-␤-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from Chryseobacterium meningosepticum), B2 (CphA from Aeromonas hydrophila), and B3 (L1 from Stenotrophomonas maltophilia). By competition experiments with metal-free apoenzymes and chromophoric zinc chelators or EDTA, we determined the dissociation constants in the absence and presence of substrates. For the formation of the monozinc enzymes we determined constants of 1.8, 5.1, 0.007, and 2.6 nM in the absence and 13.6, 1.8, 1.2, and 5.7 pM in the presence of substrates for BcII, BlaB, CphA, and L1, respectively. A second zinc ion binds in the absence (presence) of substrates with considerably higher dissociation constants, namely 1.8 (0.8), 0.007 (0.025), 50 (1.9), and 0.006 (0.12) M for BcII, BlaB, CphA, and L1, respectively. We have concluded that the apo form might be the prevailing state of most of the metallo-␤-lactamases under physiological conditions in the absence of substrates. Substrate availability induces a spontaneous self-activation due to a drastic decrease of the dissociation constants, resulting in the formation of active mononuclear enzymes already at picomolar free zinc ion concentrations. In the presence of substrates, the binuclear state of the enzymes only exists at unphysiologic high zinc concentrations and might be of no biological relevance. From the competition experiments with EDTA it is further concluded that the reactivation rate does not depend on the pool of free zinc ions but proceeds via the EDTA-Zn(II)-enzyme ternary complexes.Metallo-␤-lactamases (class B ␤-lactamases) are produced by bacteria as extracellular or periplasmatic enzymes. All known representatives possess two conserved metal binding sites and require zinc ions as enzymatic cofactors. By catalyzing the hydrolysis of ␤-lactams they render the corresponding strains resistant to almost all ␤-lactam antibiotics. Their increasing emergence in pathogenic bacterial strains due to a rapid dissemination by horizontal gene transfer has induced a growing interest in this enzyme family because of the lack of efficient therapies to treat infected patients.The metallo-␤-lactamases constitute a group of heterogeneous proteins that is divided into subclasses B1, B2, and B3 (1). Subclass B1 exhibits a broad substrate profile (2), and its zinc binding sites are composed of His-116, His-118, and His-196 (site 1) and Asp-120, Cys-221, and His-263 (site 2) (numbering according to Ref. 3). In subclass B2 the zinc ligands in site 2 are conserved, whereas His-116 in site 1 is replaced by Asn. Representatives of subclass B2 efficiently hydrolyze only carbapenems (2) and are active as monozinc enzymes, whereas the binding of a second zinc ion causes non-competitive inhibition (4). In subclass B3, Cys-221 is substituted by a Ser and is replaced by His-121 as a zinc ligand in site 2. In the Gob-1 enzyme (Chryseobacterium meningosepticum PINT) an additional H...

“…15,14,16, and 17, respectively. The protein concentrations were determined with the following extinction coefficients:…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Substrate-activated Zinc Binding of Metallo-β-lactamases

Wommer¹,

Rival²,

Heinz³

et al. 2002

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116

“…The metallo-␤-lactamases CphA from Aeromonas hydrophila AE036 and BcII from B. cereus 569/H/9 were purified as described (11,12). The protein concentrations were determined by measuring the absorbance at 280 nm using extinction coefficients of 30,500 M Ϫ1 cm Ϫ1 for BcII 569/H/9 and 38,000 M Ϫ1 cm Ϫ1 for CphA.…”

Section: Production and Characterization Of Enzymes And Apo-enzymesmentioning

confidence: 99%

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

Heinz

Bauer²,

Wommer

et al. 2003

Self Cite

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 ...

“…Bacillus cereus metallo-␤-lactamase (BcII) 1 differs from other homologous subclass B1 lactamases because it exhibits different binding constants for the 2 Zn(II) equivalents, and it is active in both mono-and bi-Zn(II) forms (11)(12)(13). The monoZn(II) forms of the CcrA enzymes from Bacteroides fragilis and IMP-1 have also been characterized as active species (10,14,15) even if results from different groups are contrasting (16).…”

mentioning

confidence: 99%

Structural Determinants of Substrate Binding to Bacillus cereus Metallo-β-lactamase

Rasia¹,

Vila

2004

Binding and hydrolysis of the ␤-lactams cefotaxime, cephapirin, imipenem, and benzylpenicillin by the metallo-␤-lactamase from Bacillus cereus were studied by presteady state kinetic measurements. In all cases, the substrate was unmodified in the most populated reaction intermediate, and no chemically modified substrate species accumulated to a detectable amount. The cephalosporins tested showed similar formation rate constants for this intermediate, and they differed mostly in their decay rates. Formation of a non-productive enzyme⅐substrate complex was detected for imipenem. The substrate binding differences can be accounted for by considering the structural features of each substrate. The apoenzyme could not bind any of the substrates, but binding was restored when the apoenzyme was reconstituted with Zn(II), revealing that the metal ions are the main determinants of substrate binding. This evidence is in line with the lack of an optimized substrate recognition patch in B1 and B3 metallo-␤-lactamases that provides a broad substrate spectrum.The most prevalent mechanism of bacterial resistance to ␤-lactam antibiotics is the production of ␤-lactamases (1, 2), which inactivate these drugs by hydrolyzing the active ␤-lactam bond. Metallo-␤-lactamases (class B ␤-lactamases) constitute a distinct class within this family of enzymes (3-5). In most cases, these metalloenzymes exhibit a broad substrate spectrum, being able to hydrolyze most of the ␤-lactam antibiotics currently available ( Fig. 1) (6 -8). The spread of metallo-␤-lactamase genes among pathogenic bacterial strains is raising an increasing concern in the biomedical community because no clinically useful inhibitors have yet been developed.Metallo-␤-lactamases are classified in three groups according to sequence homology: subclasses B1, B2, and B3 (5). The metal content of the enzymes is variable. Most of them are active as bi-Zn(II) enzymes, while the lactamases from subclass B2 are active as mono-Zn(II) enzymes (9, 10). In particular, Bacillus cereus metallo-␤-lactamase (BcII) 1 differs from other homologous subclass B1 lactamases because it exhibits different binding constants for the 2 Zn(II) equivalents, and it is active in both mono-and bi-Zn(II) forms (11-13). The monoZn(II) forms of the CcrA enzymes from Bacteroides fragilis and IMP-1 have also been characterized as active species (10, 14, 15) even if results from different groups are contrasting (16). Crystal structures have been solved for subclass B1 and B3 enzymes, while no structure of subclass B2 lactamases is yet available (17-21). All class B lactamases share a common fold, and the residues that constitute the metal binding site are mostly conserved, although the overall sequence similarity among them is low. In all structures but one, two Zn(II) ions are found in the active site. One of the metal ions (Zn 1 ) is bound to three histidine residues of the protein and a water molecule (17), which is thought to act as a nucleophile in catalysis. The coordination geometry of the other metal ion ...