Identification of the active site tyrosine of Flp recombinase. Possible relevance of its location to the mechanism of recombination.

A combination of site-directed mutagenesis and amino acid sequence analysis identifies Tyr-343 of Flp recombinase as the residue that covalently attaches to DNA during the strand-cleavage step of recombination. This residue is part of the invariant His-Arg-Tyr triad of the Int family of recombinases. Tyr-343 is located in a highly protease-accessible (and hence "open") region of Flp. This placement may provide the conformational flexibility required for the dual role of Tyr-343 in recombination: nicking of the DNA strands to initiate recombination and joining of the nicked strands across partner substrates to complete recombination. In-frame insertion of a few amino acids close to Tyr-343 (and to its amino-terminal side) does not affect substrate recognition by Flp but abolishes its catalytic function.