Molecular modelling and site directed mutagenesis of plant chloroplast monogalactosyldiacylglycerol synthase reveal critical residues for activity
Botté , Cyrille (INRA (France). UMR 1200 Laboratoire de Physiologie Cellulaire Végétale) | Jeanneau , Charlotte (Centre National de la Recherche Scientifique, Grenoble(France). Centre de Recherche sur les Macromolécules Végétales) | Snajdrova , Lenka (Centre National de la Recherche Scientifique, Grenoble(France). Centre de Recherche sur les Macromolécules Végétales) | Bastien , Olivier (INRA (France). UMR 1200 Laboratoire de Physiologie Cellulaire Végétale) | Imberty , Anne (Centre National de la Recherche Scientifique, Grenoble(France). Centre de Recherche sur les Macromolécules Végétales) | Breton , Christelle (Centre National de la Recherche Scientifique, Grenoble(France). Centre de Recherche sur les Macromolécules Végétales) | Maréchal , Éric (INRA (France). UMR 1200 Laboratoire de Physiologie Cellulaire Végétale)
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 (〈β2–α2〉 and 〈β6–β7〉 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 〈β2–α2〉 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.
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