The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions.

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used β-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins.

H, C and N backbone resonance assignments of the β-lactamase BlaP from Bacillus licheniformis 749/C and two mutational variants.

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.

Specificity and reversibility of the transpeptidation reaction catalyzed by the Streptomyces R61 D-Ala-D-Ala peptidase.

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 ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class A penicillin-binding protein.

A 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 14C-labeled lipid II 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 k2/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs.

Catalytic mechanism of the Streptomyces K15 DD-transpeptidase/penicillin-binding protein probed by site-directed mutagenesis and structural analysis.

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.