Inactivation ofAeromonas hydrophilametallo-β-lactamase by cephamycins and moxalactam (original) (raw)
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Competitive Inhibitors of the CphA Metallo- -Lactamase from Aeromonas hydrophila
Antimicrobial Agents and Chemotherapy, 2007
Various inhibitors of metallo--lactamases have been reported; however, none are effective for all subgroups. Those that have been found to inhibit the enzymes of subclass B2 (catalytically active with one zinc) either contain a thiol (and show less inhibition towards this subgroup than towards the dizinc members of B1 and B3) or are inactivators behaving as substrates for the dizinc family members. The present work reveals that certain pyridine carboxylates are competitive inhibitors of CphA, a subclass B2 enzyme. X-ray crystallographic analyses demonstrate that pyridine-2,4-dicarboxylic acid chelates the zinc ion in a bidentate manner within the active site. Salts of these compounds are already available and undergoing biomedical testing for various nonrelated purposes. Pyridine carboxylates appear to be useful templates for the development of morecomplex, selective, nontoxic inhibitors of subclass B2 metallo--lactamases.
Journal of Biological Chemistry, 2005
Among class B β-lactamases, the subclass B2 CphA enzyme is characterized by a unique specificity profile. CphA efficiently hydrolyzes only carbapenems. In this work, we generated site-directed mutants that possess a strongly broadened activity spectrum when compared with the WT CphA. Strikingly, the N116H/N220G double mutant exhibits a substrate profile close to that observed for the broad spectrum subclass B1 enzymes. The double mutant is significantly activated by the binding of a second zinc ion under conditions where the WT enzyme is non-competitively inhibited by the same ion. Abreviations: WT, wild type; CD, circular dichroism In vivo, class B β-lactamases (1) require one or two zinc ions as enzymatic cofactors. By efficiently catalyzing the hydrolysis of the β-lactam amide bond, these enzymes play a key role in bacterial resistance to this group of antibiotics. The metallo-β-lactamase family has been divided into three different subclasses, B1, B2, and B3, on the basis of sequence similarities (2, 3). The CphA metallo-β-lactamase produced by Aeromonas hydrophila belongs to subclass B2. It is characterized by a uniquely narrow specificity profile. CphA efficiently hydrolyzes only carbapenems and shows very poor activity against penicillins and cephalosporins, a behavior in contrast to that of metallo-β-lactamases of subclasses B1and B3, which usually exhibit very broad activity spectra against nearly all β-lactam compounds, with the exception of monobactams (4, 5). Moreover, in contrast to the BcII (Bacillus cereus) and CcrA (Bacteroides fragilis) enzymes belonging to the B1 subclass, and in general to most other metallo-β-lactamases, CphA exhibits a maximum activity as a mono-zinc enzyme. The presence of a Zn 2+ ion in a second low affinity binding site non-competitively inhibits the enzyme with a K i value of 46 µM at pH 6.5 (6). Recently, the structure of the mono-zinc CphA enzyme has been solved by x-ray crystallography (7). Similar to the known structures of metallo-β-lactamases of subclasses B1 (BcII (8), CcrA (9), IMP-I (10), BlaB (11)) and B3 (L1 (12) and FEZ-1 (13)), the x-ray structure of CphA highlights an αββα sandwich with two central β-sheets and α-helices on the external faces. The active site is located at the bottom of the β-sheet core. In agreement with previous spectroscopic results (14, 15) and site-directed mutagenesis studies (16), these structural data show that the sole Zn 2+ ion resides in the Asp 120-Cys 221-His 263 site of the A. hydrophila metallo-βlactamase. In the di-zinc form of subclass B1, the zinc ions occupy both the His 116 , His 118 , and His 196 and the Asp 120 , Cys 221 , and His 263 sites (see Fig. 1). The histidine residue in position 116 in most metallo-β-lactamases is replaced by an asparagine residue in CphA (2, 17). This Asn-116 residue is not responsible for the narrow substrate profile of CphA, because the activity of the N116H mutant (where the three-histidine site found in most metallo-β-lactamases is recreated) against nitrocefin, benzyl-penicillin, and cephaloridine, although increased, remains rather low (16). Moreover, the K D 2 values are similar for the N116S mutant and the wild-type enzyme, indicating that Asn 116 does not participate in the binding of the second metal ion.
Journal of Biological Chemistry, 2005
Among class B -lactamases, the subclass B2 CphA enzyme is characterized by a unique specificity profile. CphA efficiently hydrolyzes only carbapenems. In this work, we generated site-directed mutants that possess a strongly broadened activity spectrum when compared with the WT CphA. Strikingly, the N116H/N220G double mutant exhibits a substrate profile close to that observed for the broad spectrum subclass B1 enzymes. The double mutant is significantly activated by the binding of a second zinc ion under conditions where the WT enzyme is non-competitively inhibited by the same ion.
Antimicrobial agents and chemotherapy, 1997
Ceftriaxone and ceftriaxone S-oxide behaved as inactivators against the metallo-beta-lactamase of Aeromonas hydrophila AE036 and as substrates for the zinc beta-lactamase produced by Bacillus cereus (569/H/9) and Stenotrophomonas maltophilia ULA 511. Moreover, RO 09-1428, a catechol-cephalosporin, was not recognized by the A. hydrophila enzyme. Panipenem, cephalosporin C, cephalosporin C-gamma-lactone, and loracarbef were substrates for the three studied beta-lactamases.
FEBS Letters, 2000
Two metal ion binding sites are conserved in metallo-L L-lactamase from Aeromonas hydrophila. The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS spectroscopy as one Cys, two His and one additional N/O donor. Sulfur-to-metal charge transfer bands are observed for all mono-and di-metal species substituted with Cu(II) or Co(II) due to ligation of the single conserved cysteine residue. Binding of a second metal ion results in non-competitive inhibition which might be explained by an alternative kinetic mechanism. A possible partition of metal ions between the two binding sites is discussed.
Protein Expression and Purification, 2004
Its natural resistance to antiprotozoal chemotherapy characterizes the intestinal protozoan parasite Cryptosporidium parvum and the Pglycoprotein-related multidrug resistance proteins such as CpABC3 could be involved. In order to design and study specific inhibitors of the CpABC3 nucleotide-binding domain, a hexahistidine-tagged recombinant protein encompassing the N-terminal cytosolic NBD1 domain was overexpressed in E. coli and purified. The 45 kDa H6-NBD1 displayed intrinsic fluorescent properties consistent with the presence of two Trp residues in a hydrophobic environment. The binding of ATP and the fluorescent analogue TNP-ATP produced a dose-dependent quenching as well as progesterone and the flavone quercetin. The extrinsic fluorescence of TNP-ATP was enhanced upon binding to H6-NBD1, which was only partially displaced by the natural substrate ATP. The recombinant protein hydrolyzed ATP (K m = 145.4 ± 18.2 M), but ADP (K m = 4.3 ± 0.6 mM) and AMP (K m = 5.4 ± 1.5 M) were also substrates. TNP-ATP is a competitive inhibitor of the catalytic activity (K i = 36.6 ± 4.5 M), but quercetin and progesterone were not inhibitors, evidencing different binding sites. The recombinant C. parvum H6-NBD1 should be a valuable tool for rational drug design and will allow the discrimination between specific inhibitors of the catalytic site and molecules binding to other sites.
Approaches to the simultaneous inactivation of metallo- and serine-β-lactamases
Bioorganic & Medicinal Chemistry Letters, 2009
A series of cephalosporin-derived reverse hydroxamates and oximes were prepared and evaluated as inhibitors of representative metallo-and serine-β-lactamases. The reverse hydroxamates showed submicromolar inhibition of the GIM-1 metallo-β-lactamase. With respect to interactions with the classes A, C, and D serine β-lactamases, as judged by their correspondingly low Km values, the reverse hydroxamates were recognized in a manner similar to the non-hydroxylated N-H amide side chains of the natural substrates of these enzymes. This indicates that, with respect to recognition in the active site of the serine β-lactamases, the O=C-NR-OH functionality can function as a structural isostere of the O=C-NR-H group, with the NO-H group presumably replacing the amide N-H group as a hydrogen bond donor to the appropriate backbone carbonyl oxygen of the protein. The reverse hydroxamates, however, displayed k cat values up to three orders of magnitude lower than the natural substrates, thus indicating substantial slowing of the hydrolytic action of these serine β-lactamases. Although the degree of inactivation is not yet enough to be clinically useful, these initial results are promising. The substitution of the amide N-H bond by N-OH may represent a useful strategy for the inhibition of other serine hydrolases.
Antimicrobial Agents and Chemotherapy, 1993
The Aeromonas hydrophila AE036 chromosome contains a cphA gene encoding a metallo-beta-lactamase highly active against carbapenem antibiotics. This enzyme was induced in strain AE036 to the same extent by both benzylpenicillin and imipenem. When the cphA gene was inserted into plasmid pACYC184, used to transform Escherichia coli DH5 alpha, the MICs of imipenem, meropenem, and penem HRE664 for recombinant clone DH5 alpha(pAA20R), expressing the Aeromonas metallo-beta-lactamase, were significantly increased, but those of penicillins and cephalosporins were not. When the metallo-beta-lactamase purified from E. coli DH5 alpha(pAA20R) was assayed with several beta-lactam substrates, it hydrolyzed carbapenems but not penicillins or cephalosporins efficiently. These results demonstrate that this metallo-beta-lactamase possesses an unusual spectrum of activity compared with all the other class B enzymes identified so far, being active on penems and carbapenems only. This enzyme may thus con...
Biochemical characterization of the metallo-beta-lactamase CcrA from Bacteroides fragilis TAL3636
Antimicrobial Agents and Chemotherapy, 1992
The CcrA beta-lactamase from Bacteroides fragilis TAL3636 was cloned into Escherichia coli and purified from inclusion bodies. This group 3 metalloenzyme hydrolyzed most beta-lactam antibiotics, including cephamycins and carbapenems. Following inhibition by chelators, enzyme activity was recovered with the cations Zn2+ and Co2+. Clavulanate and sulbactam were activators; tazobactam at 10 microM inactivated the enzyme.