JBC Transcription and Nuclear Factor Monoclonals

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Originally published In Press as doi:10.1074/jbc.M300853200 on April 27, 2003

J. Biol. Chem., Vol. 278, Issue 27, 24800-24807, July 4, 2003
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The Role of the Conserved Trp330 in Flp-mediated Recombination

FUNCTIONAL AND STRUCTURAL ANALYSIS*

Yu Chen and Phoebe A. Rice {ddagger}

From the Department of Biochemistry and Molecular Biology, the University of Chicago, Chicago, Illinois 60637

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.

Received for publication, January 27, 2003 , and in revised form, April 24, 2003.

The atomic coordinates and structure factors (code 1P4E) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by National Institutes of Health (NIH) Grant GM58827. Use of the Advanced Photon Source was supported by the United States Department of Energy, Basic Energy Sciences, Office of Science, under Contract W-31-109-Eng-38. Use of the BioCARS Sector 14 was supported by the NIH, National Center for Research Resources, under Grant RR07707. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

{ddagger} To whom correspondence should be addressed. Tel.: 773-834-1723; Fax: 773-702-0439; E-mail: price{at}midway.uchicago.edu.


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