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J. Biol. Chem., Vol. 281, Issue 26, 17864-17869, June 30, 2006

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Structural Role for Tyr-104 in Escherichia coli Isopentenyl-diphosphate Isomerase

SITE-DIRECTED MUTAGENESIS, ENZYMOLOGY, AND PROTEIN CRYSTALLOGRAPHY^*

Jérôme de Ruyck¹², Virginie Durisotti¹, Yamina Oudjama, and Johan Wouters

From the Laboratoire de Chimie Biologique Structurale, University of Namur, 61 Rue de Bruxelles, 5000 Namur and Institut de Recherches Microbiologiques J. M. Wiame, 1 Avenue E. Gryzon, 1070 Bruxelles, Belgium

Isopentenyl-diphosphate (IPP):dimethylallyl diphosphate isomerase is a key enzyme in the biosynthesis of isoprenoids. The mechanism of the isomerization reaction involves protonation of the unactivated carbon-carbon double bond in the substrate, but identity of the acidic moiety providing the proton is still not clear. Multiple sequence alignments and geometrical features observed in crystal structures of complexes with IPP isomerase suggest that Tyr-104 could play an important role during catalysis. A series of mutants was constructed by directed mutagenesis and characterized by enzymology. Crystallographic and thermal denaturation data for Y104A and Y104F mutants were obtained. Those data demonstrate the importance of residue Tyr-104 for proper folding of Escherichia coli type I IPP isomerase.

Received for publication, February 27, 2006 , and in revised form, April 10, 2006.

^* This work was supported in part by Fonds National de la Recherche Scientifique Grant IISN 4.4505.00. 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.

The atomic coordinates and structure factors (code 2G74, 1R67, and 2G73) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains Scheme S1 and Figs. S1–S3.

¹ Both authors contributed equally to this work.

² Recipient of support from the Belgian Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture. To whom correspondence should be addressed: Laboratoire de Chimie Biologique Structurale, University of Namur, 61 Rue de Bruxelles 5000 Namur, Belgium. Tel.: 32-81-72-4569; Fax: 32-81-72-4530; E-mail: jerome.deruyck{at}fundp.ac.be.

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