Directed evolution of a DD-peptidase into a β-lactamase

Understanding how proteins or more specifically enzymes evolve is still a challenging scientific issue. This thesis aimed at finding an artificial evolutionary trajectory between phylogenetically related proteins with the objective of uncovering some unknown aspects of the birth of new catalytic properties. It is largely accepted that serine β-lactamases, responsible of β-lactam antibiotics hydrolysis, have evolved from some ancestral DD-peptidases involved in the peptidoglycan metabolism. DD-peptidases are also called Penicillin Binding Proteins because they are covalently inhibited by β-lactams such as penicillins. In this work, a cyanobacterial DD-peptidase (PBP-A), sharing sequence similarities with class A β-lactamases, was used as a starting scaffold to engineer a β-lactamase activity. Our evolutionary scenario hypothetised a loss of the native peptidase activity to become exclusively a Penicillin-Binding Protein as a required step before evolving toward a β-lactamase function. For this purpose, highly diverse libraries of PBP-A mutants were built and drifted without any selective pressure for catalytic activity. Interestingly, libraries were enriched with clones sharing specific mutations in the active site region. The biochemical charaterization of a clone with these mutations showed around 300 fold decrease in the recognition of the native substrate (peptidoglycan precursor) while maintainig the capability to react with penicillin. These two mutations were also responsible of the fitness cost readjustment of the protein. Starting from these neutralized mutants, subsequent rounds of mutagenesis and drifts under sub-lethal then lethal concentrations of β-lactam antibiotic allowed the selection of clones with an ampicillin resistance phenotype. The kinetic parameters estimation of the best mutant have shown an increase in the β-lactamase activity by at least 3 orders of magnitude. This work have shown for the first time, how the neutralization of an enzyme after the gene acquisition promotes its neofunctionalization.