HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 8, 7162-7169, February 25, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/8/7162    most recent M412028200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lindner, H. A. Articles by Cygler, M. Search for Related Content PubMed PubMed Citation Articles by Lindner, H. A. Articles by Cygler, M. Social Bookmarking                      What's this?

Site-directed Mutagenesis of the Active Site Region in the Quinate/Shikimate 5-Dehydrogenase YdiB of Escherichia coli^*

Holger A. Lindner, Guy Nadeau¶, Allan Matte¶, Gurvan Michel||, Robert Ménard, and Miroslaw Cygler¶

From the Biotechnology Research Institute, National Research Council of Canada (NRCC), Montréal, Québec H4P 2R2, Canada, the ^¶Montréal Joint Center for Structural Biology, Montréal, Québec H3G 1Y6, Canada, and the ^||Végétaux Marins et Biomolécules, CNRS/Université Pierre-et-Marie-Curie/Laboratories Goëmar, Station Biologique de Roscoff BP 74, 29682 Roscoff Cedex, Brittany, France

YdiB and its paralog AroE are members of the quinate/shikimate 5-dehdrogenase family. Enzymes from this family function in the shikimate pathway that is essential for survival of microorganisms and plants and represent potential drug targets. Recent YdiB and AroE crystal structures revealed the presence of a NAD(P)-binding and a catalytic domain. We carried out site-directed mutagenesis of 8 putative active site residues in YdiB from Escherichia coli and analyzed structural and kinetic properties of the mutant enzymes. Our data indicate critical roles for an invariant lysine and aspartate residue in substrate binding and allowed us to differentiate between two previously proposed models for the binding of the substrate in the active site. Comparison of several YdiB and AroE structures led us to conclude that, upon cofactor binding and domain closure, the 2 identified binding residues are repositioned to bind to the substrate. Although the lysine residue contributes to some extent to the stabilization of the transition state, we did not identify any residue as catalytically essential. This indicates that catalysis does not operate through a general acid-base mechanism, as thought originally. Our improved understanding of the medically and agriculturally important quinate/shikimate 5-dehydrogenase family at the molecular level may prove useful in the development of novel herbicides and antimicrobial agents.

Received for publication, October 25, 2004 , and in revised form, November 26, 2004.

^* This research was supported by the Canadian Institutes of Health Research through Grant 200103GSP-90094-GMX-CFAA-19924 (to M. C.). This is NRCC publication number 46231. 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.

^* To whom correspondence should be addressed: Biotechnology Research Institute, NRCC, 6100 Ave. Royalmount, Montréal, Québec H4P 2R2, Canada. Tel.: 514-496-1887; Fax: 514-496-5143; E-mail: Holger.Lindner{at}cnrc-nrc.gc.ca.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?