title: Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach.
abstract: Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre's enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine.
authors.affiliation: Structural Bioinformatics, BIOTEC TU Dresden, Tatzberg 47-51, 01037 Dresden, Germany.
authors.forename: Josephine
authors.initials: J
authors.lastname: Abi-Ghanem
authors.forename: Janet
authors.initials: J
authors.lastname: Chusainow
authors.forename: Madina
authors.initials: M
authors.lastname: Karimova
authors.forename: Christopher
authors.initials: C
authors.lastname: Spiegel
authors.forename: Helga
authors.initials: H
authors.lastname: Hofmann-Sieber
authors.forename: Joachim
authors.initials: J
authors.lastname: Hauber
authors.forename: Frank
authors.initials: F
authors.lastname: Buchholz
authors.forename: M Teresa
authors.initials: MT
authors.lastname: Pisabarro
mesh_terms: D001287:Attachment Sites, Microbiological; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D008958:Models, Molecular; D011485:Protein Binding; D045522:Recombinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D045522:Recombinases; D004247:DNA