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See detail1H, 13C and 15N backbone resonance assignments of the β-lactamase BlaP from Bacillus licheniformis 749/C and two mutational variants
Kay, Jennifer ULiege; Thorn, David; Rhazi, Noureddine ULiege et al

in Biomolecular NMR Assignments (2017)

Class A β-lactamases have been widely used as versatile scaffolds to create hybrid (or chimeric) proteins for a series of applications ranging from basic research to medicine. We have, in particular, used ... [more ▼]

Class A β-lactamases have been widely used as versatile scaffolds to create hybrid (or chimeric) proteins for a series of applications ranging from basic research to medicine. We have, in particular, used the β-lactamase BlaP from Bacillus licheniformis 749/C (BlaP) as a protein scaffold to create model polyglutamine (polyQ) proteins in order to better understand the mechanism(s) by which an expanded polyQ sequence triggers the formation of amyloid fibrils. The model chimeras were designed by inserting a polyQ sequence of various lengths at two different locations within BlaP (i.e. position 197 or position 216) allowing a detailed comparison of the effects of subtle differences in the environment of the polyQ sequence on its ability to trigger protein aggregation. In order to investigate the effects of the polyQ insertion at both positions on the structure, stability and dynamics of BlaP, a series of NMR experiments including H/D exchange are foreseen. Accordingly, as necessitated by these studies, here we report the NMR assignment of the wild-type BlaP (BlaP-WT) and of the two reference proteins BlaP197Q0 and BlaP216Q0, wherein a dipeptide Pro-Gly has been introduced at position 197 and 216, respectively; this dipeptide originates from the addition of the Sma1 restriction site at the genetic level to allow further polyQ sequence insertion. [less ▲]

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See detailSpecificity and reversibility of the transpeptidation reaction catalyzed by the Streptomyces R61 D-Ala-D-Ala peptidase
Rhazi, Noureddine ULiege; Delmarcelle, Michaël ULiege; Sauvage, Eric ULiege et al

in Protein Science (2005), 14(11), 2922-2928

The specificity of the Streptomyces R61 penicillin-sensitive D-Ala-D-Ala peptidase has been re-examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with ... [more ▼]

The specificity of the Streptomyces R61 penicillin-sensitive D-Ala-D-Ala peptidase has been re-examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with Gly-L-Xaa dipeptides as acceptor substrates are themselves poor substrates of the enzyme. This is in apparent contradiction with the classically accepted specificity rules for D-Ala-D-Ala peptidases. The Gly-L-Xaa dipeptide is regenerated by both the hydrolysis and transpeptidation reactions. The latter reaction is observed when another Gly-L-Xaa peptide or D-Alanine are supplied as acceptors. Utilization of substrates in which the terminal -COO(-) group has been esterified or amidated shows that a free carboxylate is not an absolute prerequisite for activity. The results are discussed in the context of the expected reversibility of the transpeptidation reaction. [less ▲]

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See detailThe ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class a penicillin-binding protein
Duez, Colette ULiege; Hallut, Séverine; Rhazi, Noureddine ULiege et al

in Journal of Bacteriology (2004), 186(13), 4412-4416

soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli (14)C(-)labeled lipid 11 ... [more ▼]

soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli (14)C(-)labeled lipid 11 precursor. As a DD-peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k(2)/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs. [less ▲]

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See detailCatalytic mechanism of the Streptomyces K15 DD-transpeptidase/penicillin-binding protein probed by site-directed mutagenesis and structural analysis
Rhazi, Noureddine ULiege; Charlier, Paulette ULiege; Dehareng, Dominique ULiege et al

in Biochemistry (2003), 42(10), 2895-2906

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic ... [more ▼]

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic mechanism, the roles of residues Lys38, Ser96, and Cys98, belonging to the structural elements defining the active site cleft, have been investigated by site-directed mutagenesis, biochemical studies, and X-ray diffraction analysis. The Lys38His and Ser96Ala mutations almost completely abolished the penicillin binding and severely impaired the transpeptidase activities while the geometry of the active site was essentially the same as in the wild-type enzyme. It is proposed that Lys38 acts as the catalytic base that abstracts a proton from the active serine Ser35 during nucleophilic attack and that Ser96 is a key intermediate in the proton transfer from the Ogamma of Ser35 to the substrate leaving group nitrogen. The role of these two residues should be conserved among penicillin-binding proteins containing the Ser-Xaa-Asn/Cys sequence in motif 2. Conversion of Cys98 into Asn decreased the transpeptidase activity and increased hydrolysis of the thiolester substrate and the acylation rate with most beta-lactam antibiotics. Cys98 is proposed to play the same role as Asn in motif 2 of other penicilloyl serine transferases in properly positioning the substrate for the catalytic process. [less ▲]

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See detailPeptidase Activity of Beta-Lactamases
Rhazi, Noureddine ULiege; Galleni, Moreno ULiege; Page, Michael I. et al

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 ▲]

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See detailSite-Directed Mutagenesis of the Actinomadura R39 DD-Peptidase
Zhao, GuoHua; Duez, Colette ULiege; Forceille, Christine et al

in Biochemical Journal (1997), 327(2), 377-381

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive dd-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the ... [more ▼]

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive dd-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the 'SDN loop' by Ala or Gly significantly decreased the kcat/Km value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate and various cephalosporins. Similar fusion proteins obtained with the N300A, K410H and K410N mutants were unstable and their catalytic and penicillin-binding properties were very strongly affected. In transpeptidation reactions, the presence of the acceptor influenced the kcat/Km values, which suggested a catalytic pathway more complex than a simple partition of the acyl-enzyme between hydrolysis and aminolysis. These results are compared with those obtained with two other penicillin-sensitive enzymes, the Streptomyces R61 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5. [less ▲]

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See detailThe Bimodular G57-V577 Polypeptide Chain of the Class B Penicillin-Binding Protein 3 of Escherichia Coli Catalyzes Peptide Bond Formation from Thiolesters and Does Not Catalyze Glycan Chain Polymerization from the Lipid II Intermediate
Adam, Maggy; Fraipont, Claudine ULiege; Rhazi, Noureddine ULiege et al

in Journal of Bacteriology (1997), 179(19), 6005-6009

Because the specificity profile of the membrane anchor-free G57-V577 penicillin-binding protein 3 (PBP3) of Escherichia coli for a large series of beta-lactam antibiotics is similar to that of the full ... [more ▼]

Because the specificity profile of the membrane anchor-free G57-V577 penicillin-binding protein 3 (PBP3) of Escherichia coli for a large series of beta-lactam antibiotics is similar to that of the full-size membrane-bound PBP, the truncated PBP is expected to adopt the native folded conformation. The truncated PBP3 functions as a thiolesterase. In aqueous media and in the presence of millimolar concentrations of a properly structured amino compound, it catalyzes the aminolysis of the thiolester until completion, suggesting that the penicillin-binding module of PBP3 is designed to catalyze transpeptidation reactions. In contrast, the truncated PBP3 is devoid of glycan polymerization activity on the E. coli lipid II intermediate, suggesting that the non-penicillin-binding module of PBP3 is not a transglycosylase. [less ▲]

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