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

Botté, Cyrille Y.; Jeanneau, Charlotte; Šnajdrová, Lenka; Bastien, Olivier; Imberty, Anne; Breton, C.; Maréchal, Éric

doi:10.1074/jbc.m505622200

Cited by 38 publications

(36 citation statements)

References 60 publications

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“…Upon phosphate deprivation, DGDG is exported outside plastids to the plasma membrane (60) and the mitochondria (31). No MGDG synthase or DGDG synthase homologous sequences could be detected in the genome of some plastid-containing organisms, such as Euglena, in which MGDG and DGDG are well-established constituents (61), suggesting that their synthesizing enzymes might have strongly diverged during evolution. Searching both Toxoplasma and Plasmodium databases did not reveal any gene candidate for MGDG synthesis, leaving the question of the monogalactolipid-synthesizing enzymes unresolved.…”

Section: Discussionmentioning

confidence: 99%

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

Botté

Saïdani

Mondragón

et al. 2008

Journal of Lipid Research

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Toxoplasma gondii is a unicellular parasite characterized by unique extracellular and intracellular membrane compartments. The lipid composition of subcellular membranes has not been determined, limiting our understanding of lipid homeostasis, control, and trafficking, a series of processes involved in pathogenesis. In addition to a mitochondrion, Toxoplasma contains a plastid called the apicoplast. The occurrence of a plastid raised the question of the presence of chloroplast galactolipids. Using three independent rabbit and rat antibodies against digalactosyldiacylglycerol (DGDG) from plant chloroplasts, we detected a class of Toxoplasma lipids harboring a digalactolipid-like epitope (DGLE). Immunolabeling characterization supports the notion that the DGLE polar head is similar to that of DGDG. Mass spectrometry analyses indicated that dihexosyl lipids having various hydrophobic moieties (ceramide, diacylglycerol, and acylalkylglycerol) might react with anti-DGDG, but we cannot exclude the possibility that more complex dihexosyl-terminated lipids might also be immunolabeled. DGLE localization was analyzed by immunofluorescence and immunoelectron microscopy and confirmed by subcellular fractionation. No immunolabeling of the apicoplast could be observed. DGLE was scattered in pellicle membrane domains in extracellular tachyzoites and was relocalized to the anterior tip of the cell upon invasion in an actin-dependent manner, providing insights on a possible role in pathogenetic processes. DGLE was detected in other Apicomplexa (i.e

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Section: Discussionmentioning

confidence: 99%

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

Botté

Saïdani

Mondragón

et al. 2008

Journal of Lipid Research

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“…The MGD enzymes synthesizing the lipid substrate GalDAG in the preceding pathway are of a similar GT-B fold (22) but are grouped in another CAZy family (GT-28). Several sequences features are conserved between the three isoenzymes, including a number of Trp residues on the potential interface-binding surfaces (72).…”

Section: Discussionmentioning

confidence: 99%

“…The MPEx profile for atC-DGD1 was very similar to the profile of atDGD2, including the existence of all Trp residues, which are totally conserved (data not shown). This feature can vary among membrane-bound GTs; the strongly binding alMGS (54) has only one Trp in the full sequence, whereas high Trp numbers were found in the sequences of atMGD2 and atMGD3 and a lower number in atMGD1, soMGD1 (the spinach (Spinacia oleracea) homologue) (22), and MurG (55). Given that Trp has the strongest bilayer interface affinity of all amino acid residues (56), the numbers and localization of Trp residues in these monotopic proteins must clearly be important.…”

Section: Journal Of Biological Chemistry 6675mentioning

confidence: 99%

“…The majority of these adopt either a single (GT-A) or a double (GT-B) Rossmann-like fold structure, the latter organized in two domains (the N and C domains) (21). It seems that most membrane GTs synthesizing major glycolipids are of the larger GT-B organization type (22)(23)(24). Many of these lack transmembrane segments and are anchored in the membrane interface by charge and hydrophobic interactions, qualifying as monotopic membrane proteins (25).…”

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Tryptophan Residues Promote Membrane Association for a Plant Lipid Glycosyltransferase Involved in Phosphate Stress

Georgiev

Öhman

et al. 2011

Journal of Biological Chemistry

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Chloroplast membranes contain a substantial excess of the nonbilayer-prone monogalactosyldiacylglycerol (GalDAG) over the biosynthetically consecutive, bilayer-forming digalactosyldiacylglycerol (GalGalDAG), yielding a high membrane curvature stress. During phosphate shortage, plants replace phospholipids with GalGalDAG to rescue phosphate while maintaining membrane homeostasis. Here we investigate how the activity of the corresponding glycosyltransferase (GT) in Arabidopsis thaliana (atDGD2) depends on local bilayer properties by analyzing structural and activity features of recombinant protein. Fold recognition and sequence analyses revealed a two-domain GT-B monotopic structure, present in other plant and bacterial glycolipid GTs, such as the major chloroplast GalGalDAG GT atDGD1. Modeling led to the identification of catalytically important residues in the active site of at-DGD2 by site-directed mutagenesis. The DGD synthases share unique bilayer interface segments containing conserved tryptophan residues that are crucial for activity and for membrane association. More detailed localization studies and liposome binding analyses indicate differentiated anchor and substratebinding functions for these separated enzyme interface regions. Anionic phospholipids, but not curvature-increasing nonbilayer lipids, strongly stimulate enzyme activity. From our studies, we propose a model for bilayer "control" of enzyme activity, where two tryptophan segments act as interface anchor points to keep the substrate region close to the membrane surface. Binding of the acceptor substrate is achieved by interaction of positive charges in a surface cluster of lysines, arginines, and histidines with the surrounding anionic phospholipids. The diminishing phospholipid fraction during phosphate shortage stress will then set the new GalGalDAG/ phospholipid balance by decreasing stimulation of atDGD2.At least 30% of the genes in most cells encode proteins that are associated with the surface or embedded in the lipid matrix of the membranes, either as peripheral or as integral membrane proteins (1). Lipids form a bulk bilayer with certain properties and can interact specifically with sites in certain membrane proteins or complexes, potentially as structural cofactors. Hence, lipids exert a substantial control over the activity of many proteins or assist protein-protein interactions. A key characteristic of plants and photosynthetic bacteria is the high abundance of galactolipids in their membranes harboring the photosynthetic complexes. Analogous glycolipids are major components in many Gram-positive bacteria. Most are uncharged, consisting of a glycerol backbone esterified with two acyl chains and carrying one (monoglycosyldiacylglycerol) or two (diglycosyldiacylglycerol) sugar moieties in the headgroup. They are prone to interact by hydrogen bonding, and pure monoglycosyl-DAGs 3 tend to form nonbilayer aggregates due to their small headgroups, whereas diglycosylDAGs are always bilayer-forming. Their relative amounts will affect sponta...

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“…Native and recombinant MGD1 are known to be active as homodimers (5). Each MGD1 monomer is likely to be organized in two Rossmann folds (N-and C-domains) (7). Visualization of surface hydrophobic regions suggested that MGD1 interacts with the membrane surface by its N-domain, whereas the C-domain protrudes above the membrane.…”

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Activation of the Chloroplast Monogalactosyldiacylglycerol Synthase MGD1 by Phosphatidic Acid and Phosphatidylglycerol

Dubots

Audry

Yamaryo

et al. 2010

Journal of Biological Chemistry

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101

106

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One of the major characteristics of chloroplast membranes is their enrichment in galactoglycerolipids, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG), whereas phospholipids are poorly represented, mainly as phosphatidylglycerol (PG). All these lipids are synthesized in the chloroplast envelope, but galactolipid synthesis is also partially dependent on phospholipid synthesis localized in non-plastidial membranes. MGDG synthesis was previously shown essential for chloroplast development. In this report, we analyze the regulation of MGDG synthesis by phosphatidic acid (PA), which is a general precursor in the synthesis of all glycerolipids and is also a signaling molecule in plants. We demonstrate that under physiological conditions, MGDG synthesis is not active when the MGDG synthase enzyme is supplied with its substrates only, i.e. diacylglycerol and UDP-gal. In contrast, PA activates the enzyme when supplied. This is shown in leaf homogenates, in the chloroplast envelope, as well as on the recombinant MGDG synthase, MGD1. PG can also activate the enzyme, but comparison of PA and PG effects on MGD1 activity indicates that PA and PG proceed through different mechanisms, which are further differentiated by enzymatic analysis of point-mutated recombinant MGD1s. Activation of MGD1 by PA and PG is proposed as an important mechanism coupling phospholipid and galactolipid syntheses in plants.Chloroplast membrane lipids are mostly composed of nonphosphorous galactoglycerolipids i.e. monogalactosyldiacylglycerol (MGDG) 2 and digalactosyldiacylglycerol (DGDG) that represent more than 50 and 30% of thylakoid lipids, respectively. Phosphatidylglycerol is the main phospholipid in plastids, representing about 10% of thylakoid lipids. Each of those glycerolipid classes is represented by a range of molecular species differing in the acyl composition at sn-1 and sn-2 positions of the glycerol backbone. Based on the model of cyanobacterial lipids, the prokaryotic type of glycerolipids contains a 16-carbon fatty acid at the sn-2 position of glycerol. The eukaryotic type contains an 18-carbon fatty acid at the sn-2 position. Some plants, such as Arabidopsis or spinach, have both prokaryoticand eukaryotic-type MGDG, whereas other plants, such as pea or cucumber, have only eukaryotic-type MGDG. DGDG is mostly of eukaryotic type in all plants. Chloroplast PG contains exclusively a prokaryotic-type DAG moiety in contrast to nonplastidial PG. These different chloroplast lipids are assembled in the chloroplast envelope (1). Most enzymes have now been identified, but their functioning and regulation remain largely unknown.MGDG is synthesized by MGDG synthase (MGD), which transfers galactose from UDP-gal to DAG. In Arabidopsis, there are three MGDG synthases, and among them, MGD1, is necessary for synthesis of galactolipids and for development of photosynthetic membranes (2, 3). MGD1 utilizes DAG derived from two main sources, either from a DAG de novo synthesized in plastid envelope by double acylation of glycer...

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

Cited by 38 publications

References 60 publications

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

Tryptophan Residues Promote Membrane Association for a Plant Lipid Glycosyltransferase Involved in Phosphate Stress

Activation of the Chloroplast Monogalactosyldiacylglycerol Synthase MGD1 by Phosphatidic Acid and Phosphatidylglycerol

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