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Genetic and biochemical characterization of MbeA, the relaxase involved in plasmid ColE1 conjugative mobilization
Author(s) -
Varsaki Athanasia,
Lucas María,
Afendra Amalia S.,
Drainas Constantin,
De La Cruz Fernando
Publication year - 2003
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2003.03441.x
Subject(s) - cole1 , biology , histidine , mutant , tyrosine , alanine scanning , site directed mutagenesis , alanine , microbiology and biotechnology , peptide sequence , oligonucleotide , plasmid , genetics , amino acid , biochemistry , dna , gene , mutagenesis
Summary 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.