Engineering allosteric regulation into a circularly permuted TEM-1 beta-lactamase

The control of enzyme-catalysed reactions involved in natural biochemical pathways is essential for all organisms. The activity of many enzymes is controlled by allosteric regulation. This proceeds by the binding of a ligand in a site distinct from the active site and so called allosteric site. When the ligand binds, a conformational change in the three-dimensional structure occurs and is transmitted to the active site. The resulting perturbations in the catalytic site lead to an increased or decreased activity depending on the effectors, called respectively activators or inhibitors. Based on these observations, many artificial binding sites have been created to engineer allosteric regulation. In most cases, the sites were engineered near the active site in loops or at the surface of contiguous helices or strands but rarely in hinge regions. This thesis aimed at exploring the possibility of regulating a monomeric enzyme whose active site is located at the interface between two domains. We anticipated that binding of a ligand in the hinge region linking the domains would modify their positioning and, consequently, modulate the activity. The bacterial enzyme chosen for this work was the circularly permuted TEM-1 beta-lactamase, whose structure is made of two domains, alpha and alpha/beta connected by one hinge. By rational design or directed evolution, mutants endowed with potential binding sites were engineered in this region. The phage-display technique was used to select for variants having affinity for nickel ions. The activity of one particular clone was modulated upon metal ions binding. This demonstrates the successful engineering of an allosteric site remote from the active site and between structural domains of an enzyme.(CHIM 3) -- UCL, 201