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Patrice Soumillion

Publications and source records attributed to Patrice Soumillion.

7 recordsLinked to original sources
PubMed2006

Active TEM-1 beta-lactamase mutants with random peptides inserted in three contiguous surface loops.

Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non-natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM-1 beta-lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two beta-strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well-conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N-terminal helix and the first beta-strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final beta-strand to the C-terminal helix, particularly if the wild-type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural medium-sized loops. The combination of these activity-selected libraries generated a huge library containing active hybrid enzymes with all three loops modified.

Amino Acid Sequence↗
PubMed2006

Selection of allosteric beta-lactamase mutants featuring an activity regulation by transition metal ions.

Libraries of phage-displayed beta-lactamase mutants in which up to three loops have been engineered by genetic introduction of random peptide sequences or by randomization of the wild-type sequence have been submitted to selection protocols designed to find mutants in which binding of transition metal ions to the engineered secondary binding site leads to significant effects on the enzymatic activity. A double-selection protocol was applied: The phage-displayed libraries were first selected for transition metal ions affinity by panning on IMAC support, then a second selection step was applied to isolate mutants that have retained significant catalytic activity. The analysis of the kinetic properties of mutants in the presence of nickel, copper, or zinc ions allowed isolation of a few mutants whose activity was either enhanced or inhibited by factors up to three and >10, respectively, in a metal-specific manner. A remarkable mutant exhibiting differential allosteric regulation depending on the metal was found. Its activity was activated by nickel ion binding, inhibited by cupric ion binding, and nearly unaffected by zinc ions. These observations point to an interesting potential for up- or down-regulation of activity within a monomeric enzyme by binding to an "allosteric site" relatively remote from the active site.

Allosteric Regulation↗
PubMed2006

Gene reconstitution using high efficiency homologous recombination between a bacteriophage fd and a plasmid.

Highly efficient intermolecular crossing-over was observed occurring between regions of limited homology in a fd filamentous phage and a plasmid. These extraneous regions corresponded to two overlapping fragments of the beta-lactamase gene. Gene reconstitution through homologous recombination of these regions yielded a highly ampicillin-resistant phenotype in Escherichia coli while co-expression of the enzyme fragments afforded low and thermosensitive activity. The recombination rates were between two and three orders of magnitude higher than that reported between plasmids using a similar assay. The fd-plasmid cointegrate was detected in recombined bacteria, as was its encapsidation into phage particles and subsequent transduction. A 100-fold reduction in the recombination rate was observed in a recA mutant strain even though crossing-over was still efficient. This gene reconstitution strategy is generally applicable to phage display technology and provides an easy way for constructing large combinatorial libraries of mutants.

Bacteriophage M13↗
PubMed2004

Crystal structure of a dimeric oxidized form of human peroxiredoxin 5.

Peroxiredoxin 5 is the last discovered mammalian member of an ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Mammalian peroxiredoxin 5 has been recently classified as an atypical 2-Cys peroxiredoxin due to the presence of a conserved peroxidatic N-terminal cysteine (Cys47) and an unconserved resolving C-terminal cysteine residue (Cys151) forming an intramolecular disulfide intermediate in the oxidized enzyme. We have recently reported the crystal structure of human peroxiredoxin 5 in its reduced form. Here, a new crystal form of human peroxiredoxin 5 is described at 2.0 A resolution. The asymmetric unit contains three polypeptide chains. Surprisingly, beside two reduced chains, the third one is oxidized although the enzyme was crystallized under initial reducing conditions in the presence of 1 mM 1,4-dithio-dl-threitol. The oxidized polypeptide chain forms an homodimer with a symmetry-related one through intermolecular disulfide bonds between Cys47 and Cys151. The formation of these disulfide bonds is accompanied by the partial unwinding of the N-terminal parts of the alpha2 helix, which, in the reduced form, contains the peroxidatic Cys47 and the alpha6 helix, which is sequentially close to the resolving residue Cys151. In each monomer of the oxidized chain, the C-terminal part including the alpha6 helix is completely reorganized and is isolated from the rest of the protein on an extended arm. In the oxidized dimer, the arm belonging to the first monomer now appears at the surface of the second subunit and vice versa.

Crystallization↗
PubMed2004

Evidence of an intramolecular interaction between the two domains of the BlaR1 penicillin receptor during the signal transduction.

The BlaR1 protein is a penicillin-sensory transducer involved in the induction of the Bacillus licheniformis beta-lactamase. The amino-terminal domain of the protein exhibits four transmembrane segments (TM1-TM4) that form a four-alpha-helix bundle embedded in the plasma bilayer. The carboxyl-terminal domain of 250 amino acids (BlaR-CTD) fused at the carboxyl end of TM4 possesses the amino acid sequence signature of penicillin-binding proteins. This membrane topology suggests that BlaR-CTD and the BlaR-amino-terminal domain are responsible for signal reception and signal transduction, respectively. With the use of phage display experiments, we highlight herein an interaction between BlaR-CTD and the extracellular, 63-amino acid L2 loop connecting TM2 and TM3. This interaction does not occur in the presence of penicillin. This result suggests that binding of the antibiotic to BlaR1 might entail the release of the interaction between L2 and BlaR-CTD, causing a motion of the alpha-helix bundle and transfer of the information to the cytoplasm of the cell. In addition, fluorescence spectroscopy, CD, and Fourier transform IR spectroscopy experiments indicate that in contrast to the behavior of the corresponding Staphylococcus aureus protein, the beta-lactam antibiotic does not induce a drastic conformational change in B. licheniformis BlaR-CTD.

Acylation↗
PubMed2002

TEM-1 beta-lactamase as a scaffold for protein recognition and assay.

A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules. We have previously shown that the TEM-1 beta-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to beta-lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that beta-lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to beta-lactamases binding to streptavidin, beta-lactamase clones binding to horse spleen ferritin and beta-galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining beta-lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for beta-lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme-conjugated antibodies in enzyme-linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme.

Animals↗