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J. Biol. Chem., Vol. 280, Issue 41, 34691-34701, October 14, 2005

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Molecular Modeling and Site-directed Mutagenesis of Plant Chloroplast Monogalactosyldiacylglycerol Synthase Reveal Critical Residues for Activity^*

Cyrille Botté1, Charlotte Jeanneau1, Lenka Snajdrova, Olivier Bastien¶, Anne Imberty, Christelle Breton, and Eric Maréchal2

From the UMR 5168 CNRS-Commissariat à l'Energie Atomique-Institut National de la Recherche Agronomique, Université Joseph Fourier, Département Réponse et Dynamique Cellulaires, 17 rue des Martyrs, Commissariat à l'Energie Atomique, 38054 Grenoble cedex 9, the Centre de Recherche sur les Macromolécules Végétales-CNRS, 38041 Grenoble, and the ^¶Gene-IT, 92500 Rueil-Malmaison, France

Monogalactosyldiacylglycerol (MGDG), the major lipid of plant and algal plastids, is synthesized by MGD (or MGDG synthase), a dimeric and membrane-bound glycosyltransferase of the plastid envelope that catalyzes the transfer of a galactosyl group from a UDP-galactose donor onto a diacylglycerol acceptor. Although this enzyme is essential for biogenesis, and therefore an interesting target for herbicide design, no structural information is available. MGD monomers share sequence similarity with MURG, a bacterial glycosyltransferase catalyzing the transfer of N-acetyl-glucosamine on Lipid 1. Using the x-ray structure of Escherichia coli MURG as a template, we computed a model for the fold of Spinacia oleracea MGD. This structural prediction was supported by site-directed mutagenesis analyses. The predicted monomer architecture is a double Rossmann fold. The binding site for UDP-galactose was predicted in the cleft separating the two Rossmann folds. Two short segments of MGD (2–2 and 6–7 loops) have no counterparts in MURG, and their structure could not be determined. Combining the obtained model with phylogenetic and biochemical information, we collected evidence supporting the 2–2 loop in the N-domain as likely to be involved in diacylglycerol binding. Additionally, the monotopic insertion of MGD in one membrane leaflet of the plastid envelope occurs very likely at the level of hydrophobic amino acids of the N-terminal domain.

Received for publication, May 23, 2005 , and in revised form, June 29, 2005.

^* This work was supported by Ministère de la Recherche and by Oseo Agence Nationale de la Valorisation de la Recherche Rhône-Alpes. 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 on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1S.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 33-4-38-78-49-85; Fax: 33-4-38-78-50-91; E-mail: emarechal{at}cea.fr.

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