| Sin resolvase is a site-specific small serine recombinase from Staphylococcus aureus that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here we present a 1.86A crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported, and the structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites. Since serine recombinases fashion an unusual constellation of conserved amino acids in their active sites compared to other enzymes that perform similar functions, details of this biochemical reaction have remained unclear until now. Here, we have elucidated a mechanism of DNA cleavage where the protein uses a conserved arginine to protonate the 3' oxygen at the scissile position. We used a modified DNA substrate with a 3' phosphorothiolate group instead of the normal oxygen and were able to perform cleavage experiments utilizing a protein lacking a general acid. By observing a significant rescue effect where the modified DNA substrate was able to partially suppress the general acid knockout, we conclude that an arginine residue is responsible for general acid catalysis and protonating the 3' oxygen to activate the scissile phosphate. |
