The Role of the Conserved Trp330 in Flp-mediated Recombination
Open Access
- 1 July 2003
- journal article
- Published by Elsevier BV
- Vol. 278 (27), 24800-24807
- https://doi.org/10.1074/jbc.m300853200
Abstract
The active site of Flp contains, in addition to a transdonated nucleophilic tyrosine, five other residues that are highly conserved within the λ-integrase family of site-specific recombinases and the type IB topoisomerases. We have used site-directed mutagenesis and x-ray crystallography to investigate the roles of two such residues, Lys223 and Trp330. Our findings agree with studies on related enzymes showing the importance of Lys223 in catalysis but demonstrate that in Flp-mediated recombination the primary role of Trp330 is architectural rather than catalytic. Eliminating the hydrogen bonding potential of Trp330 by phenylalanine substitution results in surprisingly small changes in reaction rates, compared with dramatic decreases in the activities of W330A, W330H, and W330Q. The structure of a W330F mutant-DNA complex reveals an active site nearly identical to that of the wild type. The phenylalanine side chain preserves most of the van der Waals interactions Trp330 forms with the Tyr343-containing trans helix, which may be particularly important for the docking of this helix. Our studies of Trp330 provide the first detailed examination of this conserved residue in the λ-integrase family, suggesting that the relative importance of active site residues may differ among Flp and related enzymes.Keywords
This publication has 48 references indexed in Scilit:
- Structural Plasticity of the Flp–Holliday Junction ComplexJournal of Molecular Biology, 2003
- Attenuating Functions of the C Terminus of λ IntegraseJournal of Molecular Biology, 2002
- DNA recombination and RNA cleavage activities of the Flp protein: roles of two histidine residues in the orientation and activation of the nucleophile for strand cleavageJournal of Molecular Biology, 2001
- Quasi-equivalence in site-specific recombinase structure and function: crystal structure and activity of trimeric cre recombinase bound to a three-way lox DNA junctionJournal of Molecular Biology, 2001
- A newly identified, essential catalytic residue in a critical secondary structure element in the integrase family of site-specific recombinases is conserved in a similar element in eucaryotic type IB topoisomerasesJournal of Molecular Biology, 1999
- Comparative kinetic analysis of FLP and cre recombinases: mathematical models for DNA binding and recombinationJournal of Molecular Biology, 1998
- Histidine 265 Is Important for Covalent Catalysis by Vaccinia Topoisomerase and Is Conserved in All Eukaryotic Type I EnzymesPublished by Elsevier BV ,1997
- [20] Processing of X-ray diffraction data collected in oscillation modeMethods in Enzymology, 1997
- Half-site strand transfer by step-arrest mutants of yeast site-specific recombinase FlpJournal of Molecular Biology, 1992
- Mutagenesis of a conserved region of the gene encoding the FLP recombinase of Saccharomyces cerevisiae: A role for arginine 191 in binding and ligationJournal of Molecular Biology, 1992