I. Development of a general method for assaying protein activity in vivo. II. Efforts toward the conversion of a penicillin-binding protein into a beta-lactamase by rational design and directed evolution

The penicillin-binding proteins (PBPs) and the beta-lactamases are structurally homologous families of bacterial enzymes. Although their overall three-dimensional fold and active-site residues are nearly identical, differences both in sequence and structural topology render these enzymes different in their biological responses to antibiotics. The beta-lactamases efficiently catalyze hydrolysis of beta-lactams, while the PBPs do not. It has been difficult to propose a unique mechanism for the hydrolysis of beta-lactams. The first part of the thesis describes developing a general assay for enzyme catalysis, which uses the beta-lactamase as a model system. The second is aimed at trying to understand the difference in chemical reactivity between the PBPs and beta-lactamases by creating chimeras of the two, either through rational design or directed evolution.; Chapters 2 and 3 describe chemical complementation, an in vivo assay which links enzyme catalysis to reporter gene transcription. After optimization of the assay, the reaction mechanism for the cleavage of the novel beta-lactam substrate was first determined. Then a series of beta-lactamase variants was designed to span several orders of magnitude in kcat/K m. beta-lactamase variants spanning three-orders of magnitude in k cat/Km could be distinguished in the assay, and the catalytic efficiency of each variant correlated with its level of transcription activation in vivo.; Chapter 4 and 5 describe studies toward the directed evolution of a PBP into a beta-lactamase. Rationally designed hybrid enzymes of R61 DD-peptidase and P99 class C beta-lactamase were generated via either single or double crossovers did not result in any properly folded proteins due to disruptions in their tertiary structures. Increasing the beta-lactamase activity of an evolved PBP5 mutant was to be achieved by shuffling the PBP5 gene with several homologous PBPs from a variety of species. DNA shuffling was first optimized using the Escherichia coli PBP6 and Salmonella typhimurium PBP6a genes that share 72% nucleotide identity. Shuffling between the two genes was successful, with no bias in crossover locations.