title: Genetic and biochemical characterization of MbeA, the relaxase involved in plasmid ColE1 conjugative mobilization.
abstract: MbeA is a 60 kDa protein encoded by plasmid ColE1. It plays a key role in conjugative mobilization. MbeA*, a slightly truncated version of MbeA, was purified for in vitro analysis. MbeA* catalysed DNA cleavage and strand-transfer reactions using oligonucleotides embracing the ColE1 nic site, which was mapped to 5'-(1469)CTGG/CTTA(1462)-3'. Thus MbeA is the relaxase for ColE1 conjugal mobilization, in spite of the fact that it lacks a three histidine motif considered the invariant signature of conjugative relaxases. Amino acid sequence comparisons suggest MbeA is nevertheless related to the common relaxase protein family. For instance, MbeA residue Y19 could correspond to the invariant tyrosine in Motif I, whereas H97, E104 and N106 may constitute the equivalent residues to the histidine triad in Motif III. This hypothesis was tested by site-directed mutagenesis. MbeA amino acid residues Y19, H97, E104 and N106 were changed to alanine. MbeA mutant N106A showed reduced oligonucleotide cleavage and strand-transfer activities, whereas mutation in the other three residues resulted in proteins without detectable activity, suggesting they are directly implicated in catalysis of DNA-cleavage and strand-transfer reactions. A double substitution of E104 and N106 by histidines, therefore reconstituting the canonical histidine triad, restored relaxase activities to 1% of wild type. Thus, MbeA is a variant of the common relaxase theme with a HEN signature motif, which has to be added to the canonical three histidine motif of previously reported relaxases.
authors.affiliation: Sector of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, 451 10 Ioannina, Greece.
authors.forename: Athanasia
authors.initials: A
authors.lastname: Varsaki
authors.forename: María
authors.initials: M
authors.lastname: Lucas
authors.forename: Amalia S
authors.initials: AS
authors.lastname: Afendra
authors.forename: Constantin
authors.initials: C
authors.lastname: Drainas
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cruz
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004706:Endodeoxyribonucleases