References of "Galleni, Moreno"
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See detailCarbohydrate actives enzymes derived from metagenomes: from microbial ecology to enzymology
Berlemont, Renaud ULg; Galleni, Moreno ULg

in Li, Robert W. (Ed.) Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies (2010)

In the last ten years, the intensive mining of various environmental metagenomes has led to the discovery of numerous new genes and corresponding putative enzymes. Some enzymes were isolated for their ... [more ▼]

In the last ten years, the intensive mining of various environmental metagenomes has led to the discovery of numerous new genes and corresponding putative enzymes. Some enzymes were isolated for their ability to hydrolyze carbohydrates, including starch, xylan, chitin and cellulose derivatives. The accurate characterization of these proteins highlights their variability and their biophysical adaptation in order to cope with specific environmental conditions. In this perspective, the sampling of extreme environments for metagenomic library construction resulted in the isolation of enzymes harbouring tailor made properties aimed at their implementation in various industrial processes. Although these new catalysts appear to be of particular interest for biotechnological applications, little is known about their physiological functions in their natural host. In the field of glycosides hydrolases, different functions have been suggested including both hydrolysis and synthesis of polymers. On the one hand, indeed in the environment microorganisms compete for ecological niches by producing enzymes active against vast ranges of substrates which allow them to thrive on various carbon sources. On the other hand, production of structural (cellulose) or reserve (glycogen) polymers by bacteria such as Gluconacetobacter sp. was reported. Polysaccharides can be associated with bacterial biofilm and feed stock, compounds that are required for bacteria to live in various environments. Interestingly, the synthesis of these polymers requires enzymes which act on carbohydrate including enzymes referred to as glycoside hydrolases acting as transglycosylases. In this chapter, a review of the representative glycoside hydrolases isolated by metagenomic and their possible physiological functions are presented. [less ▲]

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See detailComparative study of mature and zymogen mite cysteine protease stability and pH unfolding.
Chevigne, A.; Dumez, Marie-Eve ULg; Dumoulin, Mireille ULg et al

in Biochimica et Biophysica Acta (2010), 1800(9), 937-945

BACKGROUND: Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes. METHODS: To ... [more ▼]

BACKGROUND: Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes. METHODS: To have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1. RESULTS: We showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen. GENERAL SIGNIFICANCE: Finally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis. [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 detailInsights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples.
Berlemont, Renaud ULg; Delsaute, Maud ULg; Pipers, Delphine ULg et al

in ISME Journal (The) (2009), 3(9), 1070-1081

In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is ... [more ▼]

In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained significantly active when temperature decreased (18% of activity at 10 degrees C). It is interesting that RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover, by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1 protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.The ISME Journal advance online publication, 21 May 2009; doi:10.1038/ismej.2009.48. [less ▲]

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See detailCritical role of tryptophan 154 for the activity and stability of class D beta-lactamases.
Baurin, Stephane; Vercheval, Lionel ULg; Bouillenne, Fabrice ULg et al

in Biochemistry (2009), 48(47), 11252-63

The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of ... [more ▼]

The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of catalysis. Evidence is presented that the interaction between the indole group of Trp154 and the carboxylated lysine is essential for the stability of the posttranslationally modified Lys70. Substitution of Trp154 by Gly, Ala, or Phe yielded noncarboxylated enzymes which displayed poor catalytic efficiencies and reduced stability when compared to the wild-type OXA-10. The W154H mutant was partially carboxylated. In addition, the maximum values of k(cat) and k(cat)/K(M) were shifted toward pH 7, indicating that the carboxylation state of Lys70 is dependent on the protonation level of the histidine. A comparison of the three-dimensional structures of the different proteins also indicated that the Trp154 mutations did not modify the overall structures of OXA-10 but induced an increased flexibility of the Omega-loop in the active site. Finally, the deacylation-impaired W154A mutant was used to determine the structure of the acyl-enzyme complex with benzylpenicillin. These results indicate a role of the Lys70 carboxylation during the deacylation step and emphasize the importance of Trp154 for the ideal positioning of active site residues leading to an optimum activity. [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 detailThe in-vitro antimicrobial activity of some medicinal plants against beta-lactam-resistant bacteria
Gangoue Pieboji, Joseph ULg; Eze, N.; Ngongang Djintchui, A. et al

in J Infect Dev Ctries (2009), 3(9), 671-80

BACKGROUND: In effort to identify novel bacterial agents, this study was initiated to evaluate the antimicrobial properties of 17 crude extracts from 12 medicinal plants against beta-lactam-resistant ... [more ▼]

BACKGROUND: In effort to identify novel bacterial agents, this study was initiated to evaluate the antimicrobial properties of 17 crude extracts from 12 medicinal plants against beta-lactam-resistant bacteria. METHODOLOGY: The antimicrobial activities of plant extracts were evaluated against clinically proved beta-lactam-resistant bacteria (Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Serratia marcescens, Acinetobacter baumannii, Staphylococcus aureus and Enterococcus sp.) and reference strains of bacteria (Escherichia coli ATCC 35218, Enterobacter aerogenes ATCC 29751, E. aerogenes ATCC 13048, Pseudomonas aeruginosa ATCC 27853 and Enterococcus hirae ATCC 9790) by using disc-diffusion and agar-dilution assays. RESULTS: The crude plant extracts demonstrated broad spectrum activity against all bacteria tested with inhibition zones in the range of 8-30 mm. The minimal inhibitory concentration (MIC) values of different plant extracts against the tested bacteria were found to range from <or= 0.3 to >or= 10 mg ml(-1). The most active plant extracts were from Dortenia picta and Bridelia micrantha (MIC: 1.25-10 mg ml(-1)) on beta-lactam-resistant Gram-negative bacilli and the extracts from B. micrantha, Mallotus oppositifolius, Garcinia lucida, Garcinia. kola, Campylospermum densiflorum (leaves) and C. zenkeri (root) on beta-lactam-resistant Gram-positive cocci (MIC: <or= 0.3-5 mg ml(-1)). CONCLUSION: Of the 17 plant extracts studied, seven showed good antimicrobial activity against the tested bacteria. The stem bark of B. micrantha and the leaves of D. picta were most active towards beta-lactamase producing Gram-negative bacilli. This study shows that medicinal plants could be sources of compounds which can be used to fight against beta-lactam resistant bacteria. [less ▲]

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See detailPolyolefin matrixes with permanent antibacterial activity: preparation, antibacterial activity, and action mode of the active species
Lenoir, Sandrine; Pagnoulle, Christophe; Galleni, Moreno ULg et al

Poster (2008, May 22)

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See detailDynamic combinatorial mass spectrometry leads to metallo-beta-lactamase inhibitors.
Lienard, Benoit M. R.; Huting, Rebekka; Lassaux, Patricia ULg et al

in Journal of medicinal chemistry (2008), 51(3), 684-8

The use of protein ESI mass spectrometry under non-denaturing conditions to analyze a dynamic combinatorial library of thiols/disulfides with the BcII metallo-beta-lactamase enabled the rapid ... [more ▼]

The use of protein ESI mass spectrometry under non-denaturing conditions to analyze a dynamic combinatorial library of thiols/disulfides with the BcII metallo-beta-lactamase enabled the rapid identification of an inhibitor with a K(i) of < 1 microM. The study exemplifies the utility of protein-MS for screening dynamic mixtures of potential enzyme-inhibitors. [less ▲]

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See detailIsolation of novel hydrolytic genes from an Antarctic metagenomic library
Pipers, D.; Berlemont, R.; Power, P. et al

Poster (2008)

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See detailCharacterization of the cattle serum antibody responses against TEM beta-lactamase and the nonimmunogenic Escherichia coli heat-stable enterotoxin (STaI)
Zervosen, Astrid ULg; Saegerman, Claude ULg; Antoniotti, Ingrid et al

in FEMS Immunology & Medical Microbiology (2008), 54(3), 319-329

In order to test the use of a subunit recombinant vaccine for its capacity to induce antibodies against the nonimmunogenic heat-stable enterotoxin STa from Escherichia coli and the TEM-1 beta-lactamase ... [more ▼]

In order to test the use of a subunit recombinant vaccine for its capacity to induce antibodies against the nonimmunogenic heat-stable enterotoxin STa from Escherichia coli and the TEM-1 beta-lactamase, cattle were immunized with a hybrid protein created by insertion of the STa sequence at position 197 of the TEM-1 beta-lactamase. Specific anti-STa IgG and IgG1 antibodies were detected at low levels, while no IgG2 antibodies were detected. In contrast, high levels of the different anti-TEM IgG subtypes were detected in cattle sera. In addition, beta-lactamase activity was inhibited by the sera. The presence of antibodies against STa and TEM-1 beta-lactamase was assessed in sera from 366 cattle taken from the field. No significant level of IgGs against the toxin or the TEM-1 was detected. A comparison of the antibody level between the immunized and the nonimmunized animals clearly demonstrated that STa was not able to induce a significant level of antibodies in the vaccinated animals. In contrast, a strong antibody response against TEM-1 beta-lactamase was demonstrated. [less ▲]

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See detailActivation mechanism of recombinant Der p 3 allergen zymogen - Contribution of cysteine protease Der p 1 and effect of propeptide glycosylation
Dumez, Marie-Eve ULg; Teller, Nathalie; Mercier, Frédéric ULg et al

in Journal of Biological Chemistry (2008), 283(45), 30606-30617

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been ... [more ▼]

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence TP1R. [less ▲]

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See detailCreating hybrid proteins by insertion of exogenous peptides into permissive sites of a class A beta-lactamase
Ruth, Nadia ULg; Quinting, Brigitte; Mainil, Jacques ULg et al

in FEBS Journal (2008), 275

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See detailAstrobiologie
Javaux, Emmanuelle ULg; Claeys, Philippe; Dehant, Véronique et al

Learning material (2008)

powerpoint sur MyULg, notes partielles imprimées

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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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