Identification of a potential general acid/base in the reversible phosphoryl transfer reactions catalyzed by tyrosine recombinases: Flp H305.
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.
Whiteson
Katrine L
KL
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
Chen
Yu
Y
Chopra
Neeraj
N
Raymond
Amy C
AC
Rice
Phoebe A
PA
D002384:Catalysis; D053837:DNA Cleavage; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D004271:DNA, Fungal / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase