A metallo-beta-lactamase enzyme in action: Crystal structures of the monozinc carbapenemase CphA and its complex with biapenem

in Journal of Molecular Biology (2005), 345(4), 785-795

One strategy developed by bacteria to resist the action of beta-lactam antibiotics is the expression of metallo-beta-lactamases. CphA from Aeromonas hydrophila is a member of a clinically important ... [more ▼]

One strategy developed by bacteria to resist the action of beta-lactam antibiotics is the expression of metallo-beta-lactamases. CphA from Aeromonas hydrophila is a member of a clinically important subclass of metallo-beta-lactamases that have only one zinc ion in their active site and for which no structure is available. The crystal structures of wild-type CphA and its N220G mutant show the structural features of the active site of this enzyme, which is modeled specifically for carbapenem hydrolysis. The structure of CphA after reaction with a carbapenem substrate, biapenem, reveals that the enzyme traps a reaction intermediate in the active site. These three X-ray structures have allowed us to propose how the enzyme recognizes carbapenems and suggest a mechanistic pathway for hydrolysis of the beta-lactam. This will be relevant for the design of metallo-beta-lactamase inhibitors as well as of antibiotics that escape their hydrolytic activity. (C) 2004 Elsevier Ltd. All rights reserved. [less ▲]

Description of In116, the first blaCTX-M-2-containing complex class 1 integron found in Morganella morganii isolates from Buenos Aires, Argentina

in Journal of Antimicrobial Chemotherapy (2005), 55(4), 461-465

Synthesis of copolymer brushes endowed with adhesion to stainless steel surfaces and antibacterial properties by controlled nitroxide-mediated radical polymerization

in Langmuir (2004), 20(24), 10718-10726

Novel copolymer brushes have been synthesized by a two-step "grafting from" method that consists of the electrografting of poly(2-phenyl-2-(2,2,6,6-tetramethyl-piperidin-1-yloxy)-ethylacrylate) onto ... [more ▼]

Novel copolymer brushes have been synthesized by a two-step "grafting from" method that consists of the electrografting of poly(2-phenyl-2-(2,2,6,6-tetramethyl-piperidin-1-yloxy)-ethylacrylate) onto stainless steel, followed by the nitroxide-mediated radical polymerization of 2-(dimethylamino ethyl)acrylate and styrene or n-butyl acrylate, initiated from the electrografted polyacrylate chains. The grafted copolymers were quaternized in order to endow them with antibacterial properties. Peeling tests have confirmed the strong adhesion of the grafted copolymer onto the stainless steel substrate. Quartz crystal microbalance experiments have proven that fibrinogen adhesion is lower on the hydrophilic quaternized films compared to the nonionic counterpart. Such quaternized copolymers exhibit significant antibacterial activity against the Gram-positive bacteria S. aureus and the Gram-negative bacteria E. coli. [less ▲]

Synthesis and evaluation of N1/C4-substituted beta-lactams as PPE and HLE inhibitors

in Bioorganic & Medicinal Chemistry (2004), 12(1), 129-138

4-(Alkylamino)carbonyl-1-(alkoxy)carbonyl-2-azetidinones (9-11) have been prepared in five steps from 4-(benzyloxy)carbonyl-1-(t-butyidimethyl)silyl-2-azetidinone (1). The P-lactam reactivity of 9 has ... [more ▼]

4-(Alkylamino)carbonyl-1-(alkoxy)carbonyl-2-azetidinones (9-11) have been prepared in five steps from 4-(benzyloxy)carbonyl-1-(t-butyidimethyl)silyl-2-azetidinone (1). The P-lactam reactivity of 9 has been established by H-1 NMR experiment. Compound 11 was a good reversible inhibitor of PPE and HLE. Based on theoretical design, series of 2-azetidinones (12-17) and 4(alkoxy)carbonyl-2-azetidinones (18-21) bearing various carbonyl (ester, thiolester, amide) and thiocarbonyl (thioamide) functionalities at position N1 were similarly prepared. In the absence of C4-substituent, the compounds were inactive against elastases. On the other hand, 4-(benzyloxy)carbonyl-1-(ethylthioxy)carbonyl-2-azetidinone (19) and 4-(benzyloxy)carbonyl-1-(benzylamino)-thiocarbonyl-2-azetidinone (21) were both good reversible inhibitors, but acting most probably via different mechanisms (enzymic processing of the exocyclic ester function or beta-lactam ring opening). (C) 2003 Elsevier Ltd. All rights reserved. [less ▲]

Mutational analysis of the two zinc-binding sites of the Bacillus cereus 569/H/9 metallo-beta-lactamase

in Biochemical Journal (2002), 363(Pt 3), 687-696

The metallo-beta-lactamase BcII from Bacillus cereus 569/H/9 possesses a binuclear zinc centre. The mono-zinc form of the enzyme displays an appreciably high activity. although full efficiency is observed ... [more ▼]

The metallo-beta-lactamase BcII from Bacillus cereus 569/H/9 possesses a binuclear zinc centre. The mono-zinc form of the enzyme displays an appreciably high activity. although full efficiency is observed for the di-zinc enzyme. In an attempt to assign the involvement of the different zinc ligands in the catalytic properties of BcII, individual substitutions of selected amino acids were generated. With the exception of His(116) --> Ser (H116S), C221A and C221S, the mono- and di-zinc forms of all the other mutants were poorly active. The activity of H116S decreases by a factor of 10 when compared with the wild type. The catalytic efficiency of C221A and C221S was zinc-dependent. The monozinc forms of these mutants exhibited a low activity, whereas the catalytic efficiency of their respective di-zinc forms was comparable with that of the wild type. Surprisingly, the zinc contents of the mutants and the wild-type Bell were similar. These data suggest that the affinity of the beta-lactamase for the metal was not affected by the substitution of the ligand. The pH-dependence of the H196S catalytic efficiency indicates that the zinc ions participate in the hydrolysis of the beta-lactam ring by acting as a Lewis acid. The zinc ions activate the catalytic water molecule, but also polarize the carbonyl bond of the beta-lactam ring and stabilize the development of a negative charge on the carbonyl oxygen of the tetrahedral reaction intermediate. Our studies also demonstrate that Asn(233) is not directly involved in the interaction with the substrates. [less ▲]

Inactivation of Aeromonas Hydrophila Metallo-Beta-Lactamase by Cephamycins and Moxalactam

in European Journal of Biochemistry (2001), 268(13), 3840-50

Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by ... [more ▼]

Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3' leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate. [less ▲]

Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae.

in Antimicrobial Agents and Chemotherapy (2001), 45(10), 2807-12

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries ... [more ▼]

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII beta-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those beta-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 beta-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of beta-lactamases. [less ▲]

Peptidase Activity of Beta-Lactamases

in Biochemical Journal (1999), 341((Pt 2)), 409-13

Although beta-lactamases have generally been considered as being devoid of peptidase activity, a low but significant hydrolysis of various N-acylated dipeptides was observed with representatives of each ... [more ▼]

Although beta-lactamases have generally been considered as being devoid of peptidase activity, a low but significant hydrolysis of various N-acylated dipeptides was observed with representatives of each class of beta-lactamases. The kcat/Km values were below 0.1 M(-1). s(-1), but the enzyme rate enhancement factors were in the range 5000-20000 for the best substrates. Not unexpectedly, the best 'peptidase' was the class C beta-lactamase of Enterobacter cloacae P99, but, more surprisingly, the activity was always higher with the phenylacetyl- and benzoyl-d-Ala-d-Ala dipeptides than with the diacetyl- and alpha-acetyl-l-Lys-d-Ala-d-Ala tripeptides, which are the preferred substrates of the low-molecular-mass, soluble dd-peptidases. A comparison between the beta-lactamases and dd-peptidases showed that it might be as difficult for a dd-peptidase to open the beta-lactam ring as it is for the beta-lactamases to hydrolyse the peptides, an observation which can be explained by geometric and stereoelectronic considerations. [less ▲]

Mechanistic Diversity of Beta-Lactamases

in Biochemical Society Transactions (1999), 27(2), 58-63

Résistance bactérienne aux beta-lactamines

in Medecine Sciences : M/S (1998), 14(5), 544-555