title: The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding.
abstract: Protein MobM, the relaxase involved in conjugative transfer of the streptococcal plasmid pMV158, is the prototype of the MOB(V) superfamily of relaxases. To characterize the DNA-binding and nicking domain of MobM, a truncated version of the protein (MobMN199) encompassing its N-terminal region was designed and the protein was purified. MobMN199 was monomeric in contrast to the dimeric form of the full-length protein, but it kept its nicking activity on pMV158 DNA. The optimal relaxase activity was dependent on Mn(2+) or Mg(2+) cations in a dosage-dependent manner. However, whereas Mn(2+) strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg(2+). Furthermore, MobMN199 exhibited a high affinity binding for Mn(2+) but not for Mg(2+). We also examined the binding-specificity and affinity of MobMN199 for several substrates of single-stranded DNA encompassing the pMV158 origin of transfer (oriT). The minimal oriT was delimited to a stretch of 26 nt which included an inverted repeat located eight bases upstream of the nick site. The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein-DNA complex. We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn(2+) during pMV158 transfer.
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Fabián
authors.initials: F
authors.lastname: Lorenzo-Díaz
authors.forename: Lubomir
authors.initials: L
authors.lastname: Dostál
authors.forename: Miquel
authors.initials: M
authors.lastname: Coll
authors.forename: Joel F
authors.initials: JF
authors.lastname: Schildbach
authors.forename: Margarita
authors.initials: M
authors.lastname: Menéndez
authors.forename: Manuel
authors.initials: M
authors.lastname: Espinosa
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002412:Cations / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D008345:Manganese / Q000737:chemistry* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D002412:Cations; D004277:DNA, Single-Stranded; D008345:Manganese; D004706:Endodeoxyribonucleases; C120220:MobM protein, bacteria