Importance des résidus lysine 70 et tryptophane 154 sur la structure, la stabilité, la carbonatation et l'activité de la béta-lactamase OXA-10 de Pseudomonas aeruginosa

SUMMARY :
This thesis was conducted to highlight the biochemical and structures impact of conserved residues of the beta-lactamase OXA-10 isolated from P. aeruginosa. The beta-lactamase OXA-10 production is one of the main responsible to the beta-lactam antibiotics resistance of Pseudomonas strains.
This work is subdivided in five chapters.
The first is focused on the organization of class D beta-lactamase in sub classes. We have created by identity analysis, 21 sub classes that reflect an easy discrimination between proteins variability and mutants. This methodology is able to organize the enzymes as function of a parental enzyme but also based on the substrate profiles.
The second chapter regroups the studies that target the importance of the residue tryptophan 154 carried by the Omega loop. The substitution of the residue tryptophan 154 by an alanine, a glycine or an phenylalanine leads to a large decrease of i) the catalytic activity, ii) of the affinity toward to CO2 and iii) on the enzyme stability. The structural data show an absence of lysine 70 modification and a large modification of the hydrogen network throughout the mutants structures. The various complexes resolved by R-x diffraction of the W154A mutant indicate that the deacylation rate is the most impacted.
The third chapter makes an analogy between the available structural informations of others proteins characterized by a carboxylation modification. Others serine enzymes known to be not modified were also integrated in the analysis. The data reveal that the presence of a tryptophan residue seems to be necessary. We notice the presence of a large hydrophobic area with various shape near of the modified lysines. The calculation of the theoretical pKa values of the lysines seems to indicate a large decrease of the values that lead to an unprotonated form at physiological pH.
The fourth chapter describes, the role of the lysine 70. The substitution of the lysine by a cysteine or a glutamate leads to a large decrease of the catalytic activity and an absence of carboxylation. At the opposite, the protein stability is not impacted, and an increase of stability is observed for the K70C mutant. The structural data show that the overall fold and structures localization are conserved. The complex obtained with the nitrocefin proves that the mutant is always able to react with the beta-lactams compounds and show that the deacylation step is rate limiting.
Finally, the last chapter regroups the effect of pH on the structure, the dimerization, the stability and the catalytic activity. The data show that in the tested conditions the OXA-10 beta-lactamase is in a dimeric form at physiological pH. The addition of CO2, leads the formation of a more compact enzyme form. At high pH (10), the monomer is observed, while a fast equilibrium between the monomer and the dimer is observed between the pH 7.5 and 10. The enzyme is characterized with similar stability as function of pH. The addition of CO2 leads an increase of the stabilities. In tested conditions, the OXA-10 beta-lactamase is characterized by two optimum pH in presence or not of CO2. The CO2 addition leads to a large increase of the catalytic constant. The biophysics' studies reveal that in absence of CO2, subtle modifications are observed as function of pH, but that the addition of CO2 leads theirs disappearance.