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See detailZinc Beta-lactamase superfamily
Bebrone, Carine ULg

in Kretsinger, Robert; Uversky, Vladimir; Permyakov, Eugene (Eds.) Encyclopedia of Metalloproteins (2013)

The metallo-beta-lactamase superfamily was first defined in 1997 on the basis of a sequence alignment. The members of this superfamily are characterized by the presence of a common alpha-beta-beta-alpha ... [more ▼]

The metallo-beta-lactamase superfamily was first defined in 1997 on the basis of a sequence alignment. The members of this superfamily are characterized by the presence of a common alpha-beta-beta-alpha fold and share five conserved motifs: Asp84, His116-Xaa-His118-Xaa-Asp120-His121, His196, Asp221 and His263, which are (with the exception of Asp84) involved in the binding of two metal ions. This superfamily contains now more than 23,900 members divided into 17 biological groups. Besides metallo-beta-lactamases which cleave the amide bond of the β-lactam ring of penicillins, cephalosporins or carbapenems (thus inactivating the antibiotic), the metallo-beta-lactamase superfamily includes enzymes which hydrolyze thiol-ester, phosphodiester and sulfuric ester bonds as well as oxydoreductases. [less ▲]

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See detailGES-18, a new carbapenem-hydrolyzing GES-Type β-lactamase from pseudomonas aeruginosa that contains Ile80 and Ser170 residues.
Bebrone, Carine ULg; Bogaerts, Pierre; Delbrück, Heinrich et al

in Antimicrobial Agents and Chemotherapy (2013), 57(1)

A clinical isolate of Pseudomonas aeruginosa recovered from the lower respiratory tract of an 81-year-old patient hospitalized in Belgium was sent to the national reference center to determine its ... [more ▼]

A clinical isolate of Pseudomonas aeruginosa recovered from the lower respiratory tract of an 81-year-old patient hospitalized in Belgium was sent to the national reference center to determine its resistance mechanism. PCR sequencing identified a new GES variant, GES-18, which differs from the carbapenem-hydrolyzing enzyme GES-5 by a single amino acid substitution (Val80Ile, in the numbering according to Ambler) and from GES-1 by two substitutions (Val80Ile and Gly170Ser). Detailed kinetic characterization showed that GES-18 and GES-5 hydrolyze imipenem and cefoxitin with similar kinetic parameters and that GES-18 was less susceptible than GES-1 to classical β-lactamase inhibitors such as clavulanate and tazobactam. The overall structure of GES-18 is similar to the solved structures of GES-1 and GES-2, the Val80Ile and Gly170Ser substitutions causing only subtle local rearrangements. Notably, the hydrolytic water molecule and the Glu166 residue were slightly displaced compared to their counterparts in GES-1. Our kinetic and crystallographic data for GES-18 highlight the pivotal role of the Gly170Ser substitution which distinguishes GES-5 and GES-18 from GES-1. [less ▲]

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See detailKinetic and crystallographic studies of extended-spectrum GES-11, GES-12, and GES-14 β-lactamases.
Delbrück, Heinrich; Bogaerts, Pierre; Kupper, Michaël et al

in Antimicrobial Agents and Chemotherapy (2012), 56(11)

GES-1 is a class A extended-spectrum β-lactamase conferring resistance to penicillins, narrow- and expanded-spectrum cephalosporins, and ceftazidime. However, GES-1 poorly hydrolyzes aztreonam and ... [more ▼]

GES-1 is a class A extended-spectrum β-lactamase conferring resistance to penicillins, narrow- and expanded-spectrum cephalosporins, and ceftazidime. However, GES-1 poorly hydrolyzes aztreonam and cephamycins and exhibits very low k(cat) values for carbapenems. Twenty-two GES variants have been discovered thus far, differing from each other by 1 to 3 amino acid substitutions that affect substrate specificity. GES-11 possesses a Gly243Ala substitution which seems to confer to this variant an increased activity against aztreonam and ceftazidime. GES-12 differs from GES-11 by a single Thr237Ala substitution, while GES-14 differs from GES-11 by the Gly170Ser mutation, which is known to confer increased carbapenemase activity. GES-11 and GES-12 were kinetically characterized and compared to GES-1 and GES-14. Purified GES-11 and GES-12 showed strong activities against most tested β-lactams, with the exception of temocillin, cefoxitin, and carbapenems. Both variants showed a significantly increased rate of hydrolysis of cefotaxime, ceftazidime, and aztreonam. On the other hand, GES-11 and GES-12 (and GES-14) variants all containing Ala243 exhibited increased susceptibility to classical inhibitors. The crystallographic structures of the GES-11 and GES-14 β-lactamases were solved. The overall structures of GES-11 and GES-14 are similar to that of GES-1. The Gly243Ala substitution caused only subtle local rearrangements, notably in the typical carbapenemase disulfide bond. The active sites of GES-14 and GES-11 are very similar, with the Gly170Ser substitution leading only to the formation of additional hydrogen bonds of the Ser residue with hydrolytic water and the Glu166 residue. [less ▲]

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See detailX-RAY STRUCTURES AND MECHANISMS OF METALLO-beta-LACTAMASES
Bebrone, Carine ULg; Garau, Gianpiero; Garcia-Saez, Isabel et al

in Frère, Jean-Marie (Ed.) Beta-Lactamases (Molecular Anatomy and Physiology of Proteins) (2012)

The metallo--lactamase superfamily comprises a remarkable set of enzymes that catalyse the hydrolysis of a wide range of substrates such as peptides, nucleic acids, antibiotics of the penicillin family ... [more ▼]

The metallo--lactamase superfamily comprises a remarkable set of enzymes that catalyse the hydrolysis of a wide range of substrates such as peptides, nucleic acids, antibiotics of the penicillin family and organophosphorus derivatives. In the past ten years, X-Ray structures of representative enzymes from different families have been determined, with the metallo--lactamases being the most represented. The salient common structural feature is the presence of a catalytic metal centre embedded within a  fold. The wealth of sequence and structural information on metallo--lactamases has allowed their classification into three subclasses. Structural information is now available for members of each subclass. Interestingly, these structures show the presence of either a mono or a di-nuclear metal centre in the active sites raising questions on the metal to protein stoichiometry under physiological conditions. In addition, the structures reveal a wide variability in the shape of the active site, which involves three variable loops lining the metal centre. For each enzyme a clear correlation is found between active site shape and substrate specificity. [less ▲]

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See detailDetection and characterization of VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in a clinical isolate of Enterobacter cloacae.
Bogaerts, Pierre; Bebrone, Carine ULg; Huang, Te-Ding et al

in Antimicrobial Agents and Chemotherapy (2012), 56(6)

We report the first description of the metallo-β-lactamase VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in Enterobacter cloacae 11236, which was isolated from blood specimens of ... [more ▼]

We report the first description of the metallo-β-lactamase VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in Enterobacter cloacae 11236, which was isolated from blood specimens of a patient with colonic adenocarcinoma in Belgium. bla(VIM-31) was found on a class 1 integron located on a self-transferable but not typeable 42-kb plasmid. Compared to values published elsewhere for VIM-2, the purified VIM-31 enzyme showed weaker catalytic efficiency against all the tested beta-lactam agents (except for ertapenem), resulting from lower k(cat) (except for ertapenem) and higher K(m) values for VIM-31. [less ▲]

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See detailThe CphAII protein from Aquifex aeolicus exhibits a metal-dependent phosphodiesterase activity
Kupper, Michaël; Bauvois, Cédric; Frère, Jean-Marie ULg et al

in Extremophiles : Life Under Extreme Conditions (2012), 16(1)

The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-β-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass ... [more ▼]

The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-β-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass B2 CphA MBL. The gene encoding CphAII was amplified by PCR from the A. aeolicus genomic DNA and overexpressed in Escherichia coli using a pLex-based expression system. The recombinant CphAII protein was purified by a combination of heating (to denature E. coli proteins) and two steps of immobilized metal affinity chromatography. The purified enzyme preparation did not exhibit a β-lactamase activity but showed a metal-dependent phosphodiesterase activity versus bis-p-nitrophenyl phosphate and thymidine 5'-monophosphate p-nitrophenyl ester, with an optimum at 85°C. The circular dichroism spectrum was in agreement with the percentage of secondary structures characteristic of the MBL αββα fold. [less ▲]

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See detailOXA-198, an acquired carbapenem-hydrolyzing class D beta-lactamase from Pseudomonas aeruginosa.
El Garch, Farid; Bogaerts, Pierre; Bebrone, Carine ULg et al

in Antimicrobial Agents and Chemotherapy (2011), 55(10)

A carbapenem-resistant Pseudomonas aeruginosa strain (PA41437) susceptible to expanded-spectrum cephalosporins was recovered from several consecutive lower-respiratory-tract specimens of a patient who ... [more ▼]

A carbapenem-resistant Pseudomonas aeruginosa strain (PA41437) susceptible to expanded-spectrum cephalosporins was recovered from several consecutive lower-respiratory-tract specimens of a patient who developed a ventilator-associated pneumonia while hospitalized in an intensive care unit. Cloning experiments identified OXA-198, a new class D β-lactamase which was weakly related (less than 45% amino acid identity) to other class D β-lactamases. Expression in Escherichia coli TOP10 and in P. aeruginosa PAO1 led to transformants that were resistant to ticarcillin and showed reduced susceptibility to carbapenems and cefepime. The bla(OXA-198) gene was harbored by a class 1 integron carried by a ca. 46-kb nontypeable plasmid. This study describes a novel class D β-lactamase involved in carbapenem resistance in P. aeruginosa. [less ▲]

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See detailBroad antibiotic resistance profile of the subclass B3 metallo-β-lactamase GOB-1, a di-zinc enzyme.
Horsfall, Louise; Izougarhane, Youssef; Lassaux, Patricia et al

in FEBS Journal (2011), 278(8)

The metallo-β-lactamase (MBL) GOB-1 was expressed via a T7 expression system in Escherichia coli BL21(DE3). The MBL was purified to homogeneity and shown to exhibit a broad substrate profile, hydrolyzing ... [more ▼]

The metallo-β-lactamase (MBL) GOB-1 was expressed via a T7 expression system in Escherichia coli BL21(DE3). The MBL was purified to homogeneity and shown to exhibit a broad substrate profile, hydrolyzing all the tested β-lactam compounds efficiently. The GOB enzymes are unique among MBLs due to the presence of a glutamine residue at position 116, a zinc-binding residue in all known class B1 and B3 MBL structures. Here we produced and studied the Q116A, Q116N and Q116H mutants. The substrate profiles were similar for each mutant, but with significantly reduced activity compared with that of the wild-type. In contrast to the Q116H enzyme, which bound two zinc ions just like the wild-type, only one zinc ion is present in Q116A and Q116N. These results suggest that the Q116 residue plays a role in the binding of the zinc ion in the QHH site. [less ▲]

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See detailZinc complexes with 1,2,4-triazole functionalized amino acid derivatives: Synthesis, structure and b-lactamase assay
Naik, Anil; Beck, Joséphine; Dîrtu, Marinela et al

in Inorganica Chimica Acta (2011), 368

Coordinating abilities of 4R-1,2,4-triazole derivatives (R = glycine ethyl ester (L1), glycine (L2), diethylamino malonate (L3), methionine (L4) and diethyl aminomethylphosphonate (L5)) towards ZnII ions ... [more ▼]

Coordinating abilities of 4R-1,2,4-triazole derivatives (R = glycine ethyl ester (L1), glycine (L2), diethylamino malonate (L3), methionine (L4) and diethyl aminomethylphosphonate (L5)) towards ZnII ions have been studied in solution, in solid state and versus three zinc-b-lactamases. The crystal structure of [Zn3(L4)6(H2O)6] (6) is described; it is the first crystal structure involving a 1,2,4-triazole functionalized methionine. It forms a trinuclear complex with central zinc octahedrally coordinated by only L4, whereas terminal zinc ions coordination sphere is completed by three water molecules. L4 exhibits a dual functionality of a bridging bidentate ligand as well as an anion. A dense hydrogen bonding network connects these trinuclear entity into a 3D supramolecular network. The ZnII ions in 6 are held at equidistance (3.848 Å) which coincidently matches with the corresponding Zn Zn distance in the binuclear zinc enzyme from Bacillus cereus (3.848 and 4.365 Å). Among L1–L5 screened for b-lactamase assay, L4 shows modest inhibition for BcII enzyme. [less ▲]

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See detailNH-1,2,3-Triazole-based Inhibitors of the VIM-2 Metallo-β- Lactamase: Synthesis and Structure-Activity Studies
Weide, Timo; Saldanha, S. Adrian; Minond, Dmitriy et al

in ACS Medicinal Chemistry Letters (2010), 1(4)

Metallo-ß-lactamases (MBL) are an emerging cause of bacterial resistance to antibiotic treatment. The VIM-2 ß-lactamase is the most commonly encountered MBL in clinical isolates worldwide. Described here ... [more ▼]

Metallo-ß-lactamases (MBL) are an emerging cause of bacterial resistance to antibiotic treatment. The VIM-2 ß-lactamase is the most commonly encountered MBL in clinical isolates worldwide. Described here are potent and selective small molecule inhibitors of VIM-2 containing the arylsulfonyl-NH-1,2,3-triazole chemotype that potentiate the efficacy of the ß-lactam, imipenem, in E. coli. [less ▲]

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See detailCurrent challenges in antimicrobial chemotherapy: focus on beta-lactamase inhibition.
Bebrone, Carine ULg; Lassaux, Patricia ULg; Vercheval, Lionel ULg et al

in Drugs (2010), 70(6)

The use of the three classical beta-lactamase inhibitors (clavulanic acid, tazobactam, sulbactam) in combination with beta-lactam antibiotics is currently the most successful strategy to combat the beta ... [more ▼]

The use of the three classical beta-lactamase inhibitors (clavulanic acid, tazobactam, sulbactam) in combination with beta-lactam antibiotics is currently the most successful strategy to combat the beta-lactamase mediated resistance. However, these inhibitors are efficient in inactivating class A beta-lactamases only and the efficiency of the inhibitor/antibiotic combination can be compromised by several mechanisms among which the production of naturally resistant class B or class D enzymes, the hyperproduction of AmpC or even the production of evolved inhibitor-resistant class A enzymes. There is thus an urgent need in the development of novel inhibitors. For serine active enzymes (classes A, C and D), derivatives of the beta-lactam ring such as 6-beta-halogenopenicillanates, beta-lactam sulfones, penems and oxapenems, monobactams or trinems seem to be potential starting points to design efficient molecules (among which AM-112 and LK-157). Moreover, a promising non-beta-lactam molecule, NXL-104 is now under clinical trial. In contrast, an ideal inhibitor of metallo-beta-lactamases (class B) remains to be found, despite the huge number of potential molecules already described (biphenyl tetrazoles, cysteinyl peptides, mercaptocarboxylates, succinic acid derivatives, etc). The search for such an inhibitor is complicated by the absence of a covalent intermediate in their catalytic mechanisms and the fact that beta-lactam derivatives often behave as substrates rather than as inhibitors. Currently, the most promising broad spectrum inhibitors of class B enzymes are molecules presenting chelating groups (thiols, carboxylates, etc) combined with an aromatic group. This review describes all the types of molecules already tested as potential beta-lactamase inhibitors and thus constitutes an update of the current status in beta-lactamase inhibitor discovery. [less ▲]

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See detailMercaptophosphonate Compounds as Broad-Spectrum Inhibitors of the Metallo-β-lactamases
Lassaux, Patricia; Hamel, Matthieu; Gulea, Mihaela et al

in Journal of Medicinal Chemistry (2010), 53

In this paper, we investigated the inhibitory effect of mercaptophosphonate derivatives against the three subclasses of MBLs (B1, B2, and B3). All 14 tested mercaptophosphonates, with the exception of one ... [more ▼]

In this paper, we investigated the inhibitory effect of mercaptophosphonate derivatives against the three subclasses of MBLs (B1, B2, and B3). All 14 tested mercaptophosphonates, with the exception of one, behaved as competitive inhibitors for the three subclasses. <br />Apart from two compounds, all the mercaptophosphonates tested exhibit a good inhibitory effect on the subclass B2 MBL CphA with low inhibition constants (Ki<15 μM). Interestingly, compound 18 turned out to be a potent broad spectrum MBL inhibitor. <br />The crystallographic structures of the CphA-10a and CphA-18 complexes indicated that the sulfur atom of 10a and the phosphonato group of 18 interact with the Zn2þ ion, respectively. Molecular modeling studies of the interactions between two compounds and the VIM-4 (B1), CphA (B2), and FEZ-1 (B3) enzymes brought to light different binding modes depending on the enzyme and the inhibitor, consistent with the crystallographic structures. [less ▲]

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See detailInhibitors of VIM-2 by screening pharmacologically active and click-chemistry compound libraries
Minond, D.; Saldanha, S. A.; Spaargaren, M. et al

in Bioorganic & Medicinal Chemistry (2009), 17

VIM-2 is an Ambler class B metallo-beta-lactamase (MBL) capable of hydrolyzing a broad-spectrum of beta-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area ... [more ▼]

VIM-2 is an Ambler class B metallo-beta-lactamase (MBL) capable of hydrolyzing a broad-spectrum of beta-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active research, an array of potent, small molecule inhibitors is yet to be fully characterized for VIM-2. In the presented research, a compound library screening approach was used to identify and characterize VIM-2 inhibitors from a library of pharmacologically active compounds as well as a focused 'click' chemistry library. The four most potent VIM-2 inhibitors resulting from a VIM-2 screen were characterized by kinetic studies in order to determine K(i) and mechanism of enzyme inhibition. As a result, two previously described pharmacologic agents, mitoxantrone (1,4-dihydroxy-5,8-bis([2-([2-hydroxyethyl]amino)ethyl]amino)-9,10-anthracenedione) and 4-chloromercuribenzoic acid (pCMB) were found to be active, the former as a non-competitive inhibitor (K(i)=K(i)(')=1.5+/-0.2microM) and the latter as a slowly reversible or irreversible inhibitor. Additionally, two novel sulfonyl-triazole analogs from the click library were identified as potent, competitive VIM-2 inhibitors: N-((4-((but-3-ynyloxy)methyl)-1H-1,2,3-triazol-5-yl)methyl)-4-iodobenzenesulfonamide (1, K(i)=0.41+/-0.03microM) and 4-iodo-N-((4-(methoxymethyl)-1H-1,2,3-triazol-5-yl)methyl)benzenesulfonamide (2, K(i)=1.4+/-0.10microM). Mitoxantrone and pCMB were also found to potentiate imipenem efficacy in MIC and synergy assays employing Escherichia coli. Taken together, all four compounds represent useful chemical probes to further investigate mechanisms of VIM-2 inhibition in biochemical and microbiology-based assays. [less ▲]

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See detailDiscovery of novel lipophilic inhibitors of OXA-10 enzyme (class D beta-lactamase) by screening amino analogs and homologs of citrate and isocitrate.
Beck, Joséphine; Vercheval, Lionel ULg; Bebrone, Carine ULg et al

in Bioorganic & Medicinal Chemistry Letters (2009), 19

Aminocitrate (and homolog) derivatives have been prepared by bis-alkylation of glycinate Schiff bases with bromoacetates (and ethyl acrylate), followed by N-acylation and esters (partial or complete ... [more ▼]

Aminocitrate (and homolog) derivatives have been prepared by bis-alkylation of glycinate Schiff bases with bromoacetates (and ethyl acrylate), followed by N-acylation and esters (partial or complete) deprotection. Aminoisocitrate was similarly obtained by mono-alkylation with diethyl fumarate. Evaluation against representative beta-lactamases revealed that the free acid derivatives are modest inhibitors of class A enzymes, whilst their benzyl esters showed a good inhibition of OXA-10 (class D enzyme). A docking experiment featured hydrophobic interactions in the active site. [less ▲]

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See detailAminophosphonic Acids and Aminobis(phosphonic acids) as Potential Inhibitors of Penicillin-Binding Proteins
Beck, Josephine; Gharbi, Sonia; Herteg-Fernea, Adriana et al

in European Journal of Organic Chemistry (2009), (1), 85-97

Abstract Aminophosphonic acids and aminobis(phosphonic acids) have been prepared by the alkylation of Schiff bases with methyl bromoacetate or ethyl acrylate. Other pathways, like the modified Pudovik ... [more ▼]

Abstract Aminophosphonic acids and aminobis(phosphonic acids) have been prepared by the alkylation of Schiff bases with methyl bromoacetate or ethyl acrylate. Other pathways, like the modified Pudovik reaction and Kabachnik-Fields reaction, have been considered for the synthesis of the -phosphonic bioisoster of aminocitrate. Partial or complete deprotection of the phosphonate ester have been realised by either acidic hydrolysis or by treatment with trimethylsilyl bromide. Evaluation against penicillin-binding proteins has shown that our compounds are modest inhibitors of class A -lactamases, but have an interesting activity against R39 (D,D-peptidase/carboxypeptidase). [less ▲]

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See detailThe structure of the di-zinc subclass B2 metallo-beta-lactamase CphA reveals that the second inhibitory zinc ion binds in the "histidine" site.
Bebrone, Carine ULg; Delbrück, Heinrich; Kupper, Michaël et al

in Antimicrobial Agents and Chemotherapy (2009)

Bacteria can defend themselves against beta-lactam antibiotics through the expression of class B beta-lactamases, which cleave the beta-lactam amide bond and render the molecule harmless. There are three ... [more ▼]

Bacteria can defend themselves against beta-lactam antibiotics through the expression of class B beta-lactamases, which cleave the beta-lactam amide bond and render the molecule harmless. There are three subclasses of class B beta-lactamases (B1, B2 and B3), all of which require Zn(2+) for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-beta-lactamases are most active as di-zinc enzymes, subclass B2 enzymes such as Aeromonas hydrophila CphA are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the di-zinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the "cysteine" site, as previously determined for the mono-zinc form of the enzyme. The second zinc ion occupies a slightly modified "histidine" site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed in enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically-important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site. [less ▲]

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See detailMutational analysis of the zinc- and substrate-binding sites in the CphA metallo-beta-lactamase from Aeromonas hydrophila.
Bebrone, Carine ULg; Anne, Christine; Kerff, Frédéric ULg et al

in Biochemical Journal (2008), 414(1), 151-9

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate ... [more ▼]

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His(118), Asp(120), His(196) and His(263)) and in substrate specificity (Val(67), Thr(157), Lys(224) and Lys(226)), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp(120) and His(263), and thus is located in the 'cysteine' zinc-binding site. His(118) and His(196) residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val(67) plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val(67) also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys(224) in the binding of substrate. Lys(226) is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding. [less ▲]

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See detailMetallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily
Bebrone, Carine ULg

in Biochemical Pharmacology (2007), 74(12), 1686-1701

One strategy employed by bacterial strains to resist beta-lactam antibiotics is the expression of metallo-beta-lactamases requiring Zn+2 for activity. In the last few years, many new zinc beta-lactamases ... [more ▼]

One strategy employed by bacterial strains to resist beta-lactam antibiotics is the expression of metallo-beta-lactamases requiring Zn+2 for activity. In the last few years, many new zinc beta-lactamases have been described and several pathogens are now known to synthesize members of this class. Metallo-beta-lactamases are especially worrisome due to: (1) their broad activity profiles that encompass most beta-lactam antibiotics, including the carbapenems; (2) potential for horizontal transference; and (3) the absence of clinically useful inhibitors. on the basis of the known sequences, three different lineages, identified as subclasses B1, B2, and B3 have been characterized. The three-dimensional structure of at least one metallo-p-lactamase of each subclass has been solved. These very similar 3D structures are characterized by the presence of an alpha beta beta alpha-fold. In addition to metallo-beta-lactamases which cleave the amide bond of the beta-lactam ring, the metallo-beta-lactamase superfamily includes enzymes which hydrolyze thiol-ester, phosphodiester and sulfuric ester bonds as well as oxydoreductases. Most of the 6000 members of this superfamily share five conserved motifs, the most characteristic being the His116-X-His118-X-Asp120-His121 signature. They all exhibit an alpha beta beta alpha-fold, similar to that found in the structure of zinc beta-lactamases. Many members of this superfamily are involved in mRNA maturation and DNA reparation. This fact suggests the hypothesis that metallo-beta-lactamases may be the result of divergent evolution starting from an ancestral protein which did not have a beta-lactamase activity. (c) 2007 Elsevier Inc. All rights reserved. [less ▲]

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See detailMonitoring the zinc affinity of the metallo-beta-lactamase CphA by automated nanoESI-MS
De Vriendt, K.; Van Driessche, G.; Devreese, B. et al

in Journal of the American Society for Mass Spectrometry (2006), 17(2), 180-188

Metallo-beta-lactamases are zinc containing enzymes that are able to hydrolyze and inactivate beta-lactam antibiotics. The subclass B2 enzyme CphA of Aeromonas hydrophila is a unique metallo-p-lactamase ... [more ▼]

Metallo-beta-lactamases are zinc containing enzymes that are able to hydrolyze and inactivate beta-lactam antibiotics. The subclass B2 enzyme CphA of Aeromonas hydrophila is a unique metallo-p-lactamase because it degrades only carbapenems efficiently and is only active when it has one zinc ion bound. A zinc titration experiment was used to study the zinc affinity of the wild-type and of several mutant CphA enzymes. It shows that a second Zn2+ is also bound at high ion concentrations. All samples were analyzed using mass spectrometry in combination with an automated nanoESI source. The metal-free enzyme has a bimodal charge distribution indicative of two conformational states. A completely folded enzyme is detected when the apo-enzyme has bound the first zinc. Intensity ratios of the different enzyme forms were used to deduce the zinc affinities. CphA enzymes mutated in metal ligands show decreased zinc affinity compared to wild-type, especially D120 mutants. [less ▲]

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See detailDramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-beta-lactamase by site-directed mutagenesis
Bebrone, Carine ULg; Anne, C.; De Vriendt, K. et al

in Journal of Biological Chemistry (2005), 280(31), 28195-28202

Among class B beta-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 ... [more ▼]

Among class B beta-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. [less ▲]

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