title: Engineering allosteric regulation into the hinge region of a circularly permuted TEM-1 beta-lactamase.
abstract: In nature, the activity of many enzymes involved in important biochemical pathways is controlled by binding a ligand in a site remote from the active site. The allosteric sites are frequently located in hinge regulatory subunits, in which a conformational change can occur and propagate to the active site. The enzymatic activity is then enhanced or decreased depending on the type of effectors. Many artificial binding sites have been created to engineer an allosteric regulation. Generally, these sites were engineered near the active site in loops or at the surface of contiguous helices or strands but rarely in hinge regions. This work aims 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. Here, we describe the design of two mutants in a circularly permuted TEM-1 (cpTEM-1) beta-lactamase. The first one, cpTEM-1-His(3) was created by a rational design. It shows little regulation upon metal ion binding except for a weak activation with Zn(2+). The second one, cpTEM-1-3M-His(2), was selected by a directed evolution strategy. It is allosterically down-regulated by Zn(2+), Ni(2+) and Co(2+) with binding affinities around 300 microM.
authors.affiliation: Biochimie et Génétique Moléculaire Bactérienne, Institut des Sciences de la Vie, Université catholique de Louvain, Place Croix du Sud, 4-5 boîte 3, 1348 Louvain-la-Neuve, Belgium.
authors.forename: Valéry
authors.initials: V
authors.lastname: Mathieu
authors.forename: Jacques
authors.initials: J
authors.lastname: Fastrez
authors.forename: Patrice
authors.initials: P
authors.lastname: Soumillion
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D019216:Metals, Heavy / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D019216:Metals, Heavy; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1