1p4e
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Flpe W330F mutant-DNA Holliday Junction Complex
OverviewOverview
The active site of Flp contains, in addition to a transdonated, nucleophilic tyrosine, five other residues that are highly conserved, within the lambda-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 lambda-integrase family, suggesting that the relative importance of, active site residues may differ among Flp and related enzymes.
About this StructureAbout this Structure
1P4E is a Protein complex structure of sequences from Saccharomyces cerevisiae with 2PO as ligand. Full crystallographic information is available from OCA.
ReferenceReference
The role of the conserved Trp330 in Flp-mediated recombination. Functional and structural analysis., Chen Y, Rice PA, J Biol Chem. 2003 Jul 4;278(27):24800-7. Epub 2003 Apr 27. PMID:12716882
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