The Tyrosine Recombinase Like Component of the Active Site

For both the tyrosine recombinases and the type IB topoisomerases the attacking nucleophile for DNA cleavage is an active site tyrosine. In each enzyme family there is also a constellation of active site residues involved in activating cleavage of the phosphodiester bond in the DNA and of the phosphotyrosyl bond in the enzyme-DNA complex, through transition state stabilization and facilitation of leaving group expulsion. For the tyrosine re-combinases it consists of a conserved pentad RKHR(H/W), while for the type IB topoisomerases it is a conserved tetrad of RKR(H/N) (Perry et al. 2006; Shuman 1998; Tian et al. 2004; Van Duyne 2001).

Changing the putative active site tyrosine (Y335) of ResT to phenylalan-ine produced a catalytically dead protein (Kobryn and Chaconas 2002). The assignment of Y335 as the active site nucleophile was confirmed by trapping the phosphotyrosyl ResT-DNA intermediate with a biotinylated rTel suicide substrate. The resulting complex was subjected to tryptic digestion, the phosphopeptide-DNA fragment purified with streptavidin-conjugated magnetic beads, the DNA component of the complex P1 nuclease-digested and the resulting purified phosphopeptide sequenced by tandem mass spectroscopy (Deneke et al. 2004).

Systematic mutagenesis of putative active site residues based on alignment of the telomere resolvases with the tyrosine recombinases, type IB topoiso-merases and themselves was performed by alanine scanning, except in the case of tyrosines which were changed to phenylalanines. As expected from the alignment mutation of R199, K224, Y293 and R296, it produces a resolvase still proficient for DNA binding but defective for telomere resolution. The last amino acid of the pentad (H/W) is H324 of ResT which appears to be dispensable for the reaction. This side chain is generally thought to play a catalytic role in the tyrosine recombinases but there is one example, the yeast Flp re-combinase, in which the corresponding W330 plays a purely structural role (Chen and Rice 2003).

ResT R199 and K224 are side chains that correspond to the general acid catalyst (R199 donates a proton to K224 which donates the proton to the leaving group) involved in leaving group expulsion for the tyrosine recombi-nases and type IB topoisomerases, for which this contention has been tested (Ghosh et al. 2005; Krogh and Shuman 2000, 2002). While these residues do appear to be important for catalysis, they fail to show convincing evi dence (at least the mutations to alanine tested) of being directly involved in proton donation to the DNA leaving group during cleavage. None of the other mutants tested in this study met the criteria of a general acid catalyst. Mutagenesis of two additional residues conserved amongst the telomere resolvases, but with no counterpart in the recombinase or topoisomerase families (R198 and Y299), suggest their possible involvement in catalysis (Deneke et al. 2004).

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