title: Two distinct regions of the large serine recombinase TnpX are required for DNA binding and biological function.
abstract: The large serine recombinase, TnpX, from the Clostridium perfringens integrative mobilizable element Tn4451, consists of three domains and has two known DNA binding regions. In this study random and site-directed mutagenesis was used to identify other regions of TnpX that were required for biological activity. Genetic and biochemical analysis of these mutants led to the identification of important TnpX residues in the N-terminal catalytic pocket. In addition, another region of TnpX (aa 243-261), which is conserved within large serine recombinases, was shown to be essential for both excision and insertion. Mutation of charged residues within this region led to a loss of biological activity and aberrant DNA binding. This phenotype was mediated by interaction with the distal DNA binding region (aa 598-707). In these mutants, removal of residues 598-707 resulted in loss of DNA binding, despite the presence of the primary DNA binding region (aa 533-583). Analysis of mutations within the aa 243-261 region indicated that different protein conformations were involved in the insertion and the excision reactions. In summary, we have shown that TnpX is a complex protein that has multiple intra- and intermolecular interaction sites, providing insight into the structural and functional complexity of this important enzyme family.
authors.affiliation: Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Vic. 3800, Australia. vicki.adams@med.monash.edu.au
authors.forename: Vicki
authors.initials: V
authors.lastname: Adams
authors.forename: Isabelle S
authors.initials: IS
authors.lastname: Lucet
authors.forename: Fleur E
authors.initials: FE
authors.lastname: Tynan
authors.forename: Martina
authors.initials: M
authors.lastname: Chiarezza
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Howarth
authors.forename: Jonathan
authors.initials: J
authors.lastname: Kim
authors.forename: Jamie
authors.initials: J
authors.lastname: Rossjohn
authors.forename: Dena
authors.initials: D
authors.lastname: Lyras
authors.forename: Julian I
authors.initials: JI
authors.lastname: Rood
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D045522:Recombinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D045522:Recombinases; C497158:TnpX protein, Clostridium perfringens