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See detailInsights into the metagenomic approach : identification and characterization of cellulases involved in bacterial cellulose synthesis
Berlemont, Renaud ULg; Delsaute, Maud ULg; Galleni, Moreno ULg

Conference (2010, March 22)

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 ... [more ▼]

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 1C). 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. [less ▲]

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See detailNovel peptide inhibiting both TEM-1 beta-lactamase and penicillin-binding proteins.
Phichith, Denis; Bun, Sylvie; Padiolleau-Lefevre, Severine et al

in The FEBS journal (2010), 277(23), 4965-72

9G4H9, a catalytic antibody displaying beta-lactamase-like activity, has been developed by the anti-idiotypic approach using beta-lactamase as the first antigen. Thus 9G4H9 represents the 'internal image ... [more ▼]

9G4H9, a catalytic antibody displaying beta-lactamase-like activity, has been developed by the anti-idiotypic approach using beta-lactamase as the first antigen. Thus 9G4H9 represents the 'internal image' of beta-lactamase. We selected a cyclic peptide anchored to a bacteriophage M13 library using 9G4H9 as the target. Pep90 is a cyclic heptapeptide enclosed between two cysteine residues. We showed that Pep90 could inhibit both TEM-1 beta-lactamase (K(i) = 333 mum) and several penicillin-binding proteins (IC(5)(0) values ranging from 6-62 mum). We determined that the tryptophan residue of Pep90 is of crucial importance for its inhibitory activity. Using Pep90 as a scaffold, we generated a new class of peptidomimetics that retained inhibitory activity towards TEM-1 beta-lactamase. [less ▲]

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See detailNovel chromosome-encoded CTX-M-78 beta-lactamase from a Kluyvera georgiana clinical isolate as a putative origin of CTX-M-25 subgroup.
Rodriguez, Maria Margarita; Power, Pablo; Sader, Helio et al

in Antimicrobial agents and chemotherapy (2010), 54(7), 3070-1

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See detailcAbVIM4, a nanobody inhibiting the metallo-β-lactamase VIM-4
Sohier, Jean ULg; Laurent, Clémentine ULg; Pardon, Els et al

Poster (2010)

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See detailCA1838, A NANOBODY INHIBITING THE METALLO-β-LACTAMASE VIM-4.
Sohier, Jean ULg; Laurent, Clémentine ULg; Pardon, Els et al

Poster (2010)

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See detailMercaptophosphonate Compounds as Broad-Spectrum Inhibitors of the Metallo-β-lactamases
Lassaux, Patricia ULg; 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 detailThymic self-antigens for the design of a negative/tolerogenic self-vaccination against type 1 diabetes.
Geenen, Vincent ULg; Mottet, Marie ULg; Dardenne, Olivier ULg et al

in Current Opinion in Pharmacology (2010), 10

Before being able to react against infectious non-self antigens, the immune system has to be educated in the recognition and tolerance of neuroendocrine proteins and this critical process takes place only ... [more ▼]

Before being able to react against infectious non-self antigens, the immune system has to be educated in the recognition and tolerance of neuroendocrine proteins and this critical process takes place only in the thymus. The development of the autoimmune diabetogenic response results from a thymus dysfunction in programming central self-tolerance to pancreatic insulin-secreting islet β cells, leading to the breakdown of immune homeostasis with an enrichment of islet β-cell reactive effector T cells and a deficiency of β-cell specific natural regulatory T cells (nTregs) in the peripheral T-lymphocyte repertoire. Insulin-like growth factor 2 (IGF-2) is the dominant member of the insulin family expressed during fetal life by the thymic epithelium under the control of the autoimmune regulator (AIRE) gene/protein. The very low degree of insulin gene transcription in normal murine and human thymus explains why the insulin protein is poorly tolerogenic as evidenced in many studies, including the failure of all clinical trials that have attempted immune tolerance to islet β cells via various methods of insulin administration. Based on the close homology and cross-tolerance between insulin, the primary T1D autoantigen, and IGF-2, the dominant self-antigen of the insulin family, a novel type of vaccination, so-called “negative/tolerogenic self-vaccination”, is currently being developed for prevention and cure of T1D. If this approach were found to be effective for reprogramming immunological tolerance in T1D, it could pave the way for the design of other self-vaccines against autoimmune endocrine diseases, as well as other organ-specific autoimmune diseases. [less ▲]

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See detailCellulases catalysed cellulose polymerisation
Delsaute, Maud ULg; Berlemont, Renaud ULg; Renson, Thomas ULg et al

Poster (2010)

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See detailCharacterisation of B1 metallo-beta-lactamase inhibition by VHHs
Sohier, Jean ULg; Laurent, Clémentine ULg; Chevigné, Andy et al

Poster (2010)

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See detailSecreted subtilisin Sub3 from Microsporum canis is required for adherence to but not for invasion of the epidermis
Baldo, Aline ULg; Mathy, Anne ULg; Tabart, J. et al

in British Journal of Dermatology (2010), 162(5), 990-997

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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 detailIdentification of bla(IMP-22) in Pseudomonas spp. in urban wastewater and nosocomial environments: biochemical characterization of a new IMP metallo-enzyme variant and its genetic location.
Pellegrini, Cristina; Mercuri, Paola ULg; Celenza, Giuseppe et al

in The Journal of antimicrobial chemotherapy (2009), 63(5), 901-8

OBJECTIVES: The aim of the study was the biochemical characterization of a new variant of the metallo-beta-lactamase, IMP-22. Moreover, the genetic environment of the bla(IMP-22) gene was investigated in ... [more ▼]

OBJECTIVES: The aim of the study was the biochemical characterization of a new variant of the metallo-beta-lactamase, IMP-22. Moreover, the genetic environment of the bla(IMP-22) gene was investigated in Pseudomonas fluorescens and Pseudomonas aeruginosa collected from urban wastewater and a teaching hospital in L'Aquila, Italy. METHODS: Molecular characterization of genetic elements was carried out by PCR and DNA sequencing methods. The new enzyme was purified from recombinant Escherichia coli BL21(DE)Rosetta/pBC-SK/IMP-22. Steady-state kinetic parameters (K(m) and V(max)) were determined for a large pattern of substrates. RESULTS: A new IMP metallo-beta-lactamase gene was found in a class 1 integron and in one case, in a plasmid of Pseudomonas spp. The bla(IMP-22) encodes for a pre-protein of 246 amino acids and the N-terminus of the mature beta-lactamase (NH(2)-PDLK) was also determined. The molecular mass and pI were 24 930 Da and 6.2, respectively. On the basis of the kinetic parameters calculated (K(m) and V(max)), IMP-22 was found to hydrolyse narrow- and extended-spectrum beta-lactams. Enzyme activity was found to be inhibited by metal chelators such as EDTA, 1,10-o-phenathroline and dipicolinic acid with an IC(50) of 800, 750 and 300 microM, respectively. CONCLUSIONS: The finding of the bla(IMP-22) gene in P. fluorescens environmental strains and P. aeruginosa clinical isolate suggests the ongoing spread of bla(MBL) genes in several bacterial species and in different environments. [less ▲]

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See detailFirst detection of CTX-M-28 in a Tunisian hospital from a cefotaxime-resistant Klebsiella pneumoniae strain.
Ben Achour, N.; Mercuri, Paola ULg; Power, P. et al

in Pathologie-biologie (2009), 57(5), 343-8

A cefotaxime-resistant Klebsiella pneumoniae ML4313 was obtained from a patient from intensive care unit of Military hospital in Tunisia. This strain was resistant to beta-lactams, aminoglycosides ... [more ▼]

A cefotaxime-resistant Klebsiella pneumoniae ML4313 was obtained from a patient from intensive care unit of Military hospital in Tunisia. This strain was resistant to beta-lactams, aminoglycosides, quinolones and phenicols, and tetracyclines. It was identified as producer of extended-spectrum beta-lactamases (ESBL) by double-disk synergy test between amoxicillin-clavulanate and cefotaxime, ceftriaxone, ceftazidime and aztreonam. The ESBL was identified as CTX-M-28 by sequencing of PCR products and by isoelectric focusing. The ESBL resistance was transferred by a 50kb plasmid. CTX-M-28 is closely related to CTX-M-15. This is the first description of this enzyme in Tunisia. [less ▲]

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See detailCharacterization of a novel extended-spectrum TEM-type beta-lactamase, TEM-164, in a clinical strain of Klebsiella pneumoniae in Tunisia.
Ben Achour, Nahed; Mercuri, Paola ULg; Ben Moussa, Mohamed et al

in Microbial drug resistance (Larchmont, N.Y.) (2009), 15(3), 195-9

Klebsiella pneumoniae ML1708 exhibited a multiresistance phenotype, including resistance to all beta-lactams tested, chloramphenicol, ciprofloxacin, nalidixic acid, tetracycline, and streptomycin. The ... [more ▼]

Klebsiella pneumoniae ML1708 exhibited a multiresistance phenotype, including resistance to all beta-lactams tested, chloramphenicol, ciprofloxacin, nalidixic acid, tetracycline, and streptomycin. The double-disk synergy test was positive. ML1708 harbored a 50 kb conjugative plasmid that encoded a beta-lactamase of pI 5.5. The corresponding bla gene was identified by polymerase chain reaction and sequencing as a bla(TEM) gene. The deduced protein sequence revealed a new variant of TEM-1 beta-lactamase designated TEM-164. TEM-164 contains the unusual following mutations: L40V and I279T. These modifications may result in a change of the pI to 5.5 and hydrolyze cefotaxime and ceftazidime. [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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