Identification of residues indirectly involved in cation coordination by HUH endonucleases

Abstract

Metal ions are necessary in about one third of all proteins, playing both functional and structural roles. HUH endonucleases are metalloproteins containing conserved His-hydrophobic-His residues at the active site. However, the nature of the metallic cofactor is not identical in all members of this family. TrwC, a protein involved in the single strand DNA (ssDNA) processing during bacterial conjugation, is one of the most studied HUH endonucleases at a biochemical and structural level. The physiological cofactors of TrwC relaxase (TrwCR) are Mg+2 and Mn+2 while other relaxases only use Mn+2 as a cofactor. In vitro assays demonstrated that cleavage ability of TrwCRT87I is notably reduced in the presence of Mg+2. Moreover, structural analysis showed that in TrwCT87I the orientation of H163 has changed. In order to evaluate the role of the catalytic ion in vivo, conjugation assays were performed. Strikingly, no differences in conjugation frequencies were observed when using TrwC T87I. Thus, TrwC probably has evolved to be active using Mg+2 or Mn+2 as cofactors to increase the probability to find an appropriate metal in the conjugative environment. These findings open a new path for modification of cofactor specificity and de novo protein design.