Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases

Plasmid pAD1 is a 60-kb conjugative element commonly found in clinical isolates of Enterococcus faecalis. The relaxase TraX and the primary origin of transfer oriT2 are located close to each other and have been shown to be essential for conjugation. The oriT2 site contains a large inverted repeat (where the nic site is located) adjacent to a series of short direct repeats. TraX does not show any of the typical relaxase sequence motifs but is the prototype of a unique family of relaxases (MOBC). The present study focuses on the genetic, biochemical, and structural analysis of TraX, whose 3D structure could be predicted by protein threading. The structure consists of two domains: (i) an N-terminal domain sharing the topology of the DNA binding domain of the MarR family of transcriptional regulators and (ii) a C-terminal catalytic domain related to the PD-(D/E)XK family of restriction endonucleases. Alignment of MOBC relaxase amino acid sequences pointed to several conserved polar amino acid residues (E28, D152, E170, E172, K176, R180, Y181, and Y203) that were mutated to alanine. Functional analysis of these mutants (in vivo DNA transfer and cleavage assays) revealed the importance of these residues for relaxase activity and suggests Y181 as a potential catalytic residue similarly to His-hydrophobe-His relaxases. We also show that TraX binds specifically to dsDNA containing the oriT2 direct repeat sequences, confirming their role in transfer specificity. The results provide insights into the catalytic mechanism of MOBC relaxases, which differs radically from that of His-hydrophobe-His relaxases.This work was supported by Spanish Fondo de Investigación Sanitaria (FIS) and Instituto de Salud Carlos III Grant FIS PI10/01081 (to M.V.F.); Spanish FIS, Instituto de Salud Carlos III and Fundación Marqués de Valdecilla–Instituto de Formación e Investigación Marqués de Valdecilla Grant CES08/008 (to M.V.F.); Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Grant Red Española de Investigación en Patología Infecciosa RD06/0008/0031 (to M.V.F.); European Union Sixth Framework Programme Grant LSHE-CT-2007-037410 (to M.F.V.); Ministerio de Ciencia e Innovación (Spain) Grants BIO2010-14809 (to G.M.) and BFU2011-26608 (to F.d.l.C.); and European Union Seventh Framework Programme Grants 248919/FP7-ICT-2009-4 and 282004/FP7-HEALTH.2011.2.3.1-2 (to F.d.l.C.).Peer Reviewe