Flp provides a unique opportunity to apply the tools of chemical biology to phosphoryl transfer reactions. Flp and other tyrosine recombinases catalyze site-specific DNA rearrangements via a phosphotyrosine intermediate. Unlike most related enzymes, Flp's nucleophilic tyrosine derives from a different protomer than the remainder of its active site. Because the tyrosine can be supplied exogenously, nonnatural synthetic analogs can be used. Here we examine the catalytic role of Flp's conserved H305. DNA cleavage was studied using a peptide containing either tyrosine (pKa congruent with 10) or 3-fluoro-tyrosine (pKa congruent with 8.4). Religation was studied using DNA substrates with 3'-phospho-cresol (pKa congruent with 10) or 3'-para-nitro-phenol (pKa congruent with 7.1). In both cases, the tyrosine analog with the lower pKa specifically restored the activity of an H305 mutant. These results provide experimental evidence that this conserved histidine functions as a general acid/base catalyst in tyrosine recombinases.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|