Chemistry & Biology
Volume 14, Issue 2, February 2007, Pages 121-129
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Brief Communication
Identification of a Potential General Acid/Base in the Reversible Phosphoryl Transfer Reactions Catalyzed by Tyrosine Recombinases: Flp H305

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Summary

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 ≅ 10) or 3-fluoro-tyrosine (pKa ≅ 8.4). Religation was studied using DNA substrates with 3′-phospho-cresol (pKa ≅ 10) or 3′-para-nitro-phenol (pKa ≅ 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.

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3

These authors contributed equally to this work.

4

Present address: Department of Pharmaceutical Chemistry, University of California, San Francisco, 1700 4th Street, QB3 Building, San Francisco, CA 94158, USA.

5

Present address: Department of Pathology, The University of Chicago, N344, 5841 South Maryland Avenue, Chicago, IL 60637, USA.

6

Present address: Sloan-Kettering Institute, 1275 York Avenue, Box 73, New York, NY 10021, USA.