Molecular Modeling and Site-directed Mutagenesis of Plant Chloroplast Monogalactosyldiacylglycerol Synthase Reveal Critical Residues for Activity
2005; Elsevier BV; Volume: 280; Issue: 41 Linguagem: Inglês
10.1074/jbc.m505622200
ISSN1083-351X
AutoresCyrille Y. Botté, Charlotte Jeanneau, Lenka Šnajdrová, Olivier Bastien, Anne Imberty, Christelle Breton, Éric Maréchal,
Tópico(s)Plant biochemistry and biosynthesis
ResumoMonogalactosyldiacylglycerol (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. 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. Membranes of plant plastids have a unique lipid composition. Whereas phospholipids are the major polar lipids of cellular membranes, plastids contain up to 80% galactosylglycerolipids, i.e. 1,2-diacyl-3-O-(β-d-galactopyranosyl)-sn-glycerol (called monogalactosyldiacylglycerol or MGDG) 3The abbreviations used are: MGDG, monogalactosyldiacylglycerol; DAG, 1,2-diacylglycerol-sn-glycerol; DGDG, digalactosyldiacylglycerol; MGD, MGDG synthase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; WT, wild-type; UDP-gal, UDP-galactose; MGlcDG, 1,2-diacyl-3-O-(β-d-glucopyranosyl)-sn-glycerol; MGDG, 1,2-diacyl-3-O-(β-d-galactopyranosyl)-sn-glycerol; MURG, UDP-α-d-(N-acetyl)-glucosamine:N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol-4β-d-(N-acetyl)-glucosaminyl transferase. and 1,2-diacyl-3-O-(α-d-galactopyranosyl-(1→6)-β-d-galactopyranosyl)-sn-glycerol (called digalactosyldiacylglycerol or DGDG) (1Douce R. Holtz R.B. Benson A.A. J. Biol. Chem. 1973; 248: 7215-7222Abstract Full Text PDF PubMed Google Scholar, 2Joyard J. Block M.A. Malherbe A. Maréchal E. Douce R. Moore T.S. Lipid Metabolism in Plants. CRC Press, Boca Raton, FL1993: 231-257Google Scholar). Because chloroplast thylakoids constitute the largest membrane surface in photosynthetic plants and algae, galactosylglycerides are considered as the most abundant polar lipids in the biosphere (3Gounaris K. Barber J. Trends Biochem. Sci. 1983; 8: 378-381Abstract Full Text PDF Scopus (114) Google Scholar). MGDG is generated by transfer of a β-galactosyl moiety from a water-soluble UDP-α-d-galactose (UDP-Gal) donor onto the sn-3 position of the hydrophobic 1,2-diacyl-sn-glycerol (DAG) acceptor (4Ferrari R.A. Benson A.A. Arch. Biochem. Biophys. 1961; 93: 185-192Crossref PubMed Scopus (56) Google Scholar, 5Neufeld E.F. Hall C.W. Biochem. Biophys. Res. Commun. 1964; 14: 503-508Crossref PubMed Scopus (30) Google Scholar). This reaction is catalyzed by a UDP-α-d-galactose:1,2-diacyl-sn-glycerol 3-β-d-galactosyltransferase or MGDG synthase (TABLE ONE). Plastids derive from a single primary endosymbiosis between a cyanobacterium and a eukaryotic host (6Archibald J.M. Keeling P.J. Trends Genet. 2002; 18: 577-584Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 7Keeling P.J. Archibald J.M. Fast N.M. Palmer J.D. Science. 2004; 306: 2191Crossref PubMed Scopus (29) Google Scholar). Cyanobacteria share with plastids this unique galactolipid composition (8Joyard J. Douce R. Stumpf P.K. The Biochemistry of Plant Lipids: Structure and Function. 9. Academic Press, New York1987: 215-274Google Scholar). However, synthesis of MGDG in cyanobacteria is a two-step process (9Feige G.B. Heinz E. Wrage K. Cochems N. Ponzelar E. Mazliak P. Benveniste P. Costes C. Douce R. Biogenesis and Function of Plant Lipids. Elsevier-North-Holland Biomedical Press, Amsterdam1980: 135-140Google Scholar, 10Murata N. Sato N. Wintermanns J.F.G.M. Kuiper P.J.C. Biochemistry and Metabolism of Plant Lipids. Elsevier Biomedical Press, Amsterdam1982: 165-168Google Scholar). A first transfer of a β-glucosyl moiety from a UDP-α-d-glucose (UDP-Glc) donor onto DAG generates monoglucosyldiacylgycerol. Then an epimerase converts the β-glucosyl polar head into β-galactosyl, producing MGDG (TABLE ONE).TABLE ONESummary of plant plastid MGDG synthases and related enzymes involed in glycolipid syntheses in algae, cyanobacteria, and bacteria Function Activity experimentally detected EC number Molecular characterization Name CAZy family Three-dimensional structure Sugar donor Acceptor Product Synthesis of MGDG in cyanobacteria (i.e. Synechocystis) Synthesis of MGlcDG UDP-α-d-glucose: 1,2-diacyl-sn-glycerol 3β-d-glucosyltransferase 2.4.1.157 No gene candidate Other than GT-28aIn the Synechocystis PCC 6803 complete genome, the only recorded amino acid sequence of the CAZy GT-28 family is the cyanobacterial MURG homologue UDP-α-d-Glc DAG MGlcDG Synthesis of MGDG 1,2-Diacyl-3-O-(β-d-glucopyranosyl)-sn-glycerol glucosyl isomerase 5.3.1.x No gene candidate dnbdn, does not apply dn MGDG Synthesis of chloroplast MGDG in glaucophytes (i.e. Cyanophora) Synthesis of MGDG ? MGDG Synthesis of chloroplast MGDG in plants (i.e. Physcomitrella, Spinacia, and Arabidopsis), green algae (i.e. Chlamydomonas and Prototheca), and red algae (Cyanidioschyzon) Synthesis of MGDG UDP-α-d-galactose: 1,2-diacyl-sn-glycerol 3β-d-galactosyltransferase 2.4.1.46 exp. and sequence similaritycExp., experimental evidence MGD GT-28 This work UDP-α-d-Gal DAG MGDG Synthesis of chloroplast MGDG in Heterokonts (i.e. Thalassiosira) Synthesis of MGDG UDP-α-d-galactose: 1,2-diacyl-sn-glycerol 3β-d-galactosyltransferase 2.4.1.46 Sequence similarity MGD GT-28 UDP-α-d-Gal DAG MGDG Synthesis of MGDG in Euglenids (i.e. Euglena) Synthesis of MGDG UDP-α-d-galactose: 1,2-diacyl-sn-glycerol 3β-d-galactosyltransferase 2.4.1.46 No gene candidate UDP-α-d-Gal DAG MGDG Synthesis of MGDG in Apicomplexa (i.e. Plasmodium and Toxoplasma) Synthesis of MGDG UDP-α-d-galactose: 1,2-diacyl-sn-glycerol 3β-d-galactosyltransferase 2.4.1.46 No gene candidate Other than GT-28dNo GT-28 candidate in the Plasmodium or Toxoplasma predicted open reading frames UDP-α-d-Gal DAG MGDG Synthesis of MGlcDG in bacteria Synthesis of MGlcDG UDP-α-d-glucose: 1,2-diacyl-sn-glycerol 3α-d-glucosyltransferase 2.4.1.157 Exp. and sequence similarity MGS GT-4eModel from Ref. 60 Model UDP-α-d-Glc DAG MGlcDG Synthesis of Lipid II peptidoglycan in bacteria and cyanobacteria Synthesis of lipid II UDP-α-d-(N-acetyl)-glucosamine: N-acetylmuramyl(pentapeptide) pyrophosphoryl undecaprenol 4β-d-(N-acetyl)-glucosaminyl transferase 2.4.1.227 Exp. and sequence similarity MURG GT-28 X-ray UDP-α-d-GlcNAc (N-acetyl)muramyl (pentapeptide)-pyrophosphoryl-undecaprenol (Lipid I) β-(N-acetyl)glucosaminyl (N-acetyl)muramyl (pentapeptide) pyrophosphoryl-undecaprenol (Lipid II)a In the Synechocystis PCC 6803 complete genome, the only recorded amino acid sequence of the CAZy GT-28 family is the cyanobacterial MURG homologueb dn, does not applyc Exp., experimental evidenced No GT-28 candidate in the Plasmodium or Toxoplasma predicted open reading framese Model from Ref. 60Edman M. Berg S. Storm P. Wikstrom M. Vikstrom S. Ohman A. Wieslander A. J. Biol. Chem. 2003; 278: 8420-8428Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Open table in a new tab Evolution from a two-enzyme MGDG synthesis in cyanobacteria to the one-enzyme synthesis in algae and plants is one of the major puzzling questions to understand the history of plastids. As soon as the first MGDG synthase (csMGD1) was molecularly characterized in cucumber (Cucumis sativa), the search for potential cyanobacterial homologues was conducted (11Shimojima M. Morikawa S. Maeda K. Tohya Y. Miyazawa T. Mikami T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 333-337Crossref PubMed Scopus (123) Google Scholar). However, based on the primary sequences, no gene candidate for cyanobacterial galactolipid synthesis could be identified (TABLE ONE). It is possible that the MGDG synthetic machinery is phylogenetically unrelated between cyanobacteria and eukaryotes. The picture is likely more complicated, because the MGDG synthase evolution cannot be fully traced in eukaryotes either. A collection of MGDG synthase (MGD) genes is now well established in Angiosperms, molecularly characterized in spinach (Spinacia oleracea) (12Miège C. Maréchal E. Shimojima M. Awai K. Block M.A. Ohta H. Takamiya K. Douce R. Joyard J. Eur. J. Biochem. 1999; 265: 990-1001Crossref PubMed Scopus (94) Google Scholar), thale cress (Arabidopsis thaliana) (13Awai K. Maréchal E. Block M.A. Brun D. Masuda T. Shimada H. Takamiya K. Ohta H. Joyard J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10960-10965Crossref PubMed Scopus (236) Google Scholar), and rice (Oriza sativa) (14Qi Y.H. Yamauchi Y. Ling J.Q. Kawano N. Li D.B. Tanaka K. Planta. 2004; 219: 450-458PubMed Google Scholar). TABLE ONE gives a summary of what is currently known about MGDG synthases and related enzymes involved in glycolipid syntheses. MGD orthologues have been identified in the moss Physcomitrella patens, the green algae Chlamydomonas reinhardtii and Prototheca wickerhamii, and the red algae Cyanidioschyzon merolae. In the case of glaucophytes like Cyanophora paradoxa, whose plastids preserved a cyanobacterial peptidoglycan wall, it is not clear yet if MGDG is synthesized owing to a one-enzyme or a two-enzyme process. In eukaryotes that contain complex plastids inherited from a secondary endosymbiosis (Euglenids, Chlorarachniophytes, Cryptomonads, Haptophytes, Heterokonts, Dinoflagellates, and Apicomplexa) (7Keeling P.J. Archibald J.M. Fast N.M. Palmer J.D. Science. 2004; 306: 2191Crossref PubMed Scopus (29) Google Scholar), MGD orthologues could only be found in a Heterokont, the diatom Thalassiosira pseudonana. In Euglenids (Euglena gracilis) or Apicomplexans for which we have abundant genomic information (Plasmodium falciparum and Toxoplasma gondii) no MGD candidate gene could be identified using classic bioinformatic tools. Lack of MGD orthologue in these organisms is surprising, because chloroplastic galactolipid syntheses could be assessed in Euglena (15Matson R.S. Fei M. Chang S.B. Plant. Physiol. 1970; 45: 531-532Crossref PubMed Scopus (11) Google Scholar) or in Toxoplasma and Plasmodium (16Maréchal E. Azzouz N. De Macedo C.S. Block M.A. Feagin J.E. Schwarz R.T. Joyard J. Eukaryot. Cell. 2002; 1: 653-656Crossref PubMed Scopus (47) Google Scholar). Because fold is more conserved than sequence by evolution, three-dimensional structure comparison is a powerful means to establish relatedness of proteins (17Jones D. Thornton J J. Comput. Aided Mol. Des. 1993; 7: 439-456Crossref PubMed Scopus (81) Google Scholar), even in the absence of sequence similarity. Some clues to comprehend galactolipid synthesis in cyanobacteria, glaucophytes, euglenids, or apicomplexans might therefore benefit from the knowledge of the molecular structure of Angiosperm MGD. Synthesis of MGDG is a key process for the biogenesis of plastid membranes, particularly for thylakoid expansion (12Miège C. Maréchal E. Shimojima M. Awai K. Block M.A. Ohta H. Takamiya K. Douce R. Joyard J. Eur. J. Biochem. 1999; 265: 990-1001Crossref PubMed Scopus (94) Google Scholar, 13Awai K. Maréchal E. Block M.A. Brun D. Masuda T. Shimada H. Takamiya K. Ohta H. Joyard J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10960-10965Crossref PubMed Scopus (236) Google Scholar, 18Maréchal E. Awai K. Block M.A. Brun D. Masuda T. Shimada H. Takamiya K. Ohta H. Joyard J. Biochem. Soc. Trans. 2000; 28: 732-738Crossref PubMed Scopus (11) Google Scholar). MGDG is also involved in the functional integrity of the photosynthetic machinery (19Pali T. Garab G. Horvath L.I. Kota Z. Cell. Mol. Life. Sci. 2003; 60: 1591-1606Crossref PubMed Scopus (55) Google Scholar). Arabidopsis mutants containing half the normal MGDG amount are consistently severely affected, with defects in chloroplast development, impairment of photosynthesis, and an overall chlorotic phenotype. MGDG is the substrate for another essential lipid, DGDG (20Dörmann P. Balbo I. Benning C. Science. 1999; 284: 2181-2184Crossref PubMed Scopus (165) Google Scholar, 21Benning C. Ohta H. J. Biol. Chem. 2005; 280: 2397-2400Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), which is exported to plasma membrane (22Andersson M.X. Stridh M.H. Larsson K.E. Liljenberg C. Sandelius A.S. FEBS Lett. 2003; 537: 128-132Crossref PubMed Scopus (216) Google Scholar) and mitochondria (23Jouhet J. Maréchal E. Bligny R. Baldan B. Joyard J. Block M.A. J. Cell Biol. 2004; 167: 863-874Crossref PubMed Scopus (187) Google Scholar) under phosphate deprivation, likely to replace missing phosphatidylcholine (24Härtel H. Benning C. Biochem. Soc. Trans. 2000; 28: 729-732Crossref PubMed Google Scholar, 25Härtel H. Dörmann P. Benning C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10649-10654Crossref PubMed Scopus (294) Google Scholar). In addition to plastid membranes, MGDG synthesis is therefore essential for the biogenesis of most cell membranes. Taken together, the roles played by MGDG are vital and imply that MGD enzymes are potent targets for herbicide screening (26Nishiyama Y. Hardré-Liénard H. Miras S. Miège C. Block M.A. Revah F. Joyard J. Maréchal E. Protein. Expr. Purif. 2003; 31: 79-87Crossref PubMed Scopus (18) Google Scholar). Therefore, the MGD three-dimensional structure would also be an important starting point to dissect the MGD molecular mechanism and orientate the rational design of herbicide candidates. In Angiosperms, MGDG production is restricted to the two membranes of the envelope that surrounds plastids (27Douce R. Science. 1974; 183: 852-853Crossref PubMed Scopus (127) Google Scholar, 28Block M.A. Dorne A.J. Joyard J. Douce R. J. Biol. Chem. 1983; 258: 13281-13286Abstract Full Text PDF PubMed Google Scholar, 29Cline K. Keegstra K. Plant Physiol. 1983; 85: 780-785Google Scholar). Our current knowledge about MGDG synthase function, structure, and membrane topology is established from the enzymatic activity of purified chloroplast envelope from spinach (30Maréchal E. Block M.A. Joyard J. Douce R. J. Biol. Chem. 1994; 289: 5788-5798Abstract Full Text PDF Google Scholar, 31Maréchal E. Miège C. Block M.A. Joyard J. Douce R. J. Biol. Chem. 1995; 270: 5714-5722Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), latter attributed to soMGD1 (12Miège C. Maréchal E. Shimojima M. Awai K. Block M.A. Ohta H. Takamiya K. Douce R. Joyard J. Eur. J. Biochem. 1999; 265: 990-1001Crossref PubMed Scopus (94) Google Scholar). In A. thaliana, synthesis of MGDG is catalyzed by a family of three proteins (atMGD1, atMGD2, and atMGD3) of which activity and subcellular targeting to plastid were analyzed in depth (13Awai K. Maréchal E. Block M.A. Brun D. Masuda T. Shimada H. Takamiya K. Ohta H. Joyard J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10960-10965Crossref PubMed Scopus (236) Google Scholar, 18Maréchal E. Awai K. Block M.A. Brun D. Masuda T. Shimada H. Takamiya K. Ohta H. Joyard J. Biochem. Soc. Trans. 2000; 28: 732-738Crossref PubMed Scopus (11) Google Scholar). Unfortunately, our attempts to crystallize MGD proteins from either of these models, spinach or Arabidopsis, after functional expression in Escherichia coli (26Nishiyama Y. Hardré-Liénard H. Miras S. Miège C. Block M.A. Revah F. Joyard J. Maréchal E. Protein. Expr. Purif. 2003; 31: 79-87Crossref PubMed Scopus (18) Google Scholar), were unsuccessful. Glycosyltransferases have been hierarchically classified on the basis of sequence similarities and stereochemistry of the reactions (32Coutinho P.M. Deleury E. Davies G.J. Henrissat B. J. Mol. Biol. 2003; 328: 307-317Crossref PubMed Scopus (931) Google Scholar). Despite their number and functional diversity, glycosyltransferases fall into two major protein fold superfamilies named GT-A and GT-B, respectively (32Coutinho P.M. Deleury E. Davies G.J. Henrissat B. J. Mol. Biol. 2003; 328: 307-317Crossref PubMed Scopus (931) Google Scholar, 33Breton C. Imberty A. Curr. Opin. Struct. Biol. 1999; 9: 563-571Crossref PubMed Scopus (171) Google Scholar). This classification is constantly evolving and updated (CAZy classification available at afmb.cnrs-mrs.fr/CAZY/) (34Coutinho P.M. Henrissat B. J. Mol. Microbiol. Biotechnol. 1999; 1: 307-308PubMed Google Scholar). From sequence alignments and fold recognition, MGD enzymes are members of the GT-B superfamily. The closest homologues of MGD proteins were found to be MURG glycosyltransferases (11Shimojima M. Morikawa S. Maeda K. Tohya Y. Miyazawa T. Mikami T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 333-337Crossref PubMed Scopus (123) Google Scholar), with whom they are classified in the GT-28 family of the CAZy systematics. To date the GT-28 family does only contain one three-dimensional structure, i.e. that of E. coli MURG (ecMURG) (35Ha S. Walker D. Shi Y. Walker S. Protein Sci. 2000; 9: 1045-1052Crossref PubMed Scopus (230) Google Scholar, 36Hu Y. Chen L. Ha S. Gross B. Falcone B. Walker D. Mokhtarzadeh M. Walker S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 845-849Crossref PubMed Scopus (186) Google Scholar). MURG catalyzes the last intracellular step in bacterial and cyanobacterial peptidoglycan biosynthesis, i.e. transfer of an N-acetyl-β-d-glucosaminyl from a UDP-α-d-N-acetyl-glucosamine (UDP-GlcNAc) donor onto lipid 1 (TABLE I). For both MGD and MURG activities, a β-glycosyl moiety is transferred from a UDP-α-sugar donor to a hydrophobic acceptor, after an (α→β) inversion of the anomeric configuration of the sugar (TABLE I). It is therefore tempting to suppose that MGD from algae and plants derived from a MURG sequence of the ancestral symbiotic cyanobacteria. Here, we used the ecMURG structure as a template for secondary structure comparison and fold prediction. This approach was combined with enzymological data and phylogenetical comparisons to deduce molecular models for spinach soMGD1 and Arabidopsis atMGD1, atMGD2, and atMGD3, focusing particularly on the active site. The soMGD1 model was then challenged and sustained by site-directed mutagenesis analyses. Materials—Unlabeled and 14C-labeled (11.0 GBq.mmol-1) UDP-Gal were obtained from Sigma and New England Nuclear, respectively. 1,2-Dioleoyl-sn-glycerol (DAG), isopropyl-β-d-thiogalactopyranoside, phosphatidylglycerol, and CHAPS were purchased from Sigma. Wild-type soMGD1 Expression Vector and Site-directed Mutagenesis via PCR—The soMGD1 sequence used in this study was inserted in NdeI-BamHI cloning site of the pET-y3a vector (12Miège C. Maréchal E. Shimojima M. Awai K. Block M.A. Ohta H. Takamiya K. Douce R. Joyard J. Eur. J. Biochem. 1999; 265: 990-1001Crossref PubMed Scopus (94) Google Scholar). The full-length wild-type (WT) soMGD1 refers to the coding sequence truncated of its predicted chloroplastic transit peptides (12Miège C. Maréchal E. Shimojima M. Awai K. Block M.A. Ohta H. Takamiya K. Douce R. Joyard J. Eur. J. Biochem. 1999; 265: 990-1001Crossref PubMed Scopus (94) Google Scholar), i.e. from leucine 99 to alanine 522. Mutations were introduced into the cloned soMGD1 by using the QuikChange site-directed mutagenesis kit (Stratagene). Mutations were confirmed by sequencing (Genome Express, Grenoble). Recombinant Wild-type and Mutated soMGD1 Functional Expression in E. coli at 28 °C —Isolated colonies of transfected E. coli (BL21-DE3) were inoculated in LB medium (2.5 ml, 100 μg/ml carbenicillin) and grown at 37 °C. When A600 reached 0.5, the cell suspension was transferred to 15 ml of LB medium (100 μg/ml carbenicillin) and grown at 37 °C until A600 reached 0.5. Cells were then transferred to 400 ml of LB medium (100 μg/ml carbenicillin) and grown until A600 reached 0.5. Isopropylthio-β-d-galactopyranoside (0.4 mm) was subsequently added to induce soMGD1 expression, and the suspension was incubated at 28 °C for 4 h. Cells were harvested by centrifugation and stored at -20 °C. Detergent Solubilization of Wild-type and Mutated soMGD1 and Purification by Hydroxyapatite Chromatography —All the operations were carried out at 4 °C. After expression of soMGD1, bacteria pellets (1 mg of proteins) were incubated for 30 min in 1 ml of medium A (6 mm CHAPS, 1 mm dithiothreitol, 10 mm MOPS-KOH, pH 7.8) containing 50 mm KH2PO4/K2HPO4. The mixture was centrifuged for 30 min at 7,000 × g. The pellets containing inclusion bodies of improperly folded and inactive soMGD1 polypeptides (26Nishiyama Y. Hardré-Liénard H. Miras S. Miège C. Block M.A. Revah F. Joyard J. Maréchal E. Protein. Expr. Purif. 2003; 31: 79-87Crossref PubMed Scopus (18) Google Scholar) were discarded. The supernatants, containing soMGD1 enzymes extracted from bacterial membranes by CHAPS and solubilized in mixed micelles (12Miège C. Maréchal E. Shimojima M. Awai K. Block M.A. Ohta H. Takamiya K. Douce R. Joyard J. Eur. J. Biochem. 1999; 265: 990-1001Crossref PubMed Scopus (94) Google Scholar, 13Awai K. Maréchal E. Block M.A. Brun D. Masuda T. Shimada H. Takamiya K. Ohta H. Joyard J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10960-10965Crossref PubMed Scopus (236) Google Scholar, 26Nishiyama Y. Hardré-Liénard H. Miras S. Miège C. Block M.A. Revah F. Joyard J. Maréchal E. Protein. Expr. Purif. 2003; 31: 79-87Crossref PubMed Scopus (18) Google Scholar), were loaded onto the top of a 5- × 15-mm column containing 500 μl of a Hydroxyapatite-Ultrogel (LKD) matrix equilibrated with buffer A, at a 1 ml/min flow rate (37Maréchal E. Block M.A. Joyard J. Douce R. C. R. Acad. Sci. Paris. 1991; 313: 521-528Google Scholar). The column was washed with 2 ml of buffer A. The matrix-bound soMGD1 wild-type and mutated proteins were eluted with 2 ml of 500 mm KH2PO4/K2HPO4 in buffer A. Fractions were collected and used for enzymatic assays and protein determination. MGDG Synthase Enzymatic Assay—Enzyme activity was assayed in mixed micelles at 25 °C (30Maréchal E. Block M.A. Joyard J. Douce R. J. Biol. Chem. 1994; 289: 5788-5798Abstract Full Text PDF Google Scholar). Phosphatidylglycerol (1.3 mm) and DAG (160 μm) dissolved in chloroform were first introduced into glass tubes. After evaporation of the solvent under a stream of argon, 300 μl of incubation medium containing 4.5 mm CHAPS, 1 mm dithiothreitol, 250 mm KCl, 250 mm KH2PO4/K2HPO4, and 10 mm MOPS-KOH, pH 7.8, and purified soMGD1 were added. The mixture was mixed vigorously and kept 30 min at 25 °C for equilibration of mixed micelles. The reaction was then started by addition of 1 mm UDP-[14C]galactose (37 Bq.μmol-1) and stopped by addition of chloroform/methanol (1:2, v/v). The lipids were subsequently extracted (38Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar), and the radioactivity of the 14C-labeled MGDG ultimately produced was determined by liquid scintillation counting. Activity is expressed in micromoles of incorporated galactose.h-1.mg protein-1. MGD and MURG Sequences—In this study, twelve MGD and eight MURG sequences were used as reference sets for sequence analyses and structure model predictions. Each sequence is given with its accession reference in the source data base. Annotated MGD sequences from representative Angiosperms, i.e. eight in dicots, S. oleracea, soMGD1 (CAB56218); A. thaliana, atMGD1 (BAB12042), atMGD2 (T52269), and atMGD3 (BAB12041); C. sativa, csMGD1 (AAC49624.1), Nicotiana tabacum, ntMGD1 (BAB11980), and Glycine max, gmMGD1 (BAB11979); and two in the monocot O. sativa, osMGD1 (BAD33425) and osMGD2 (XM_481404), were obtained via the National Center for Biotechnology Information (NCBI) web site (www.ncbi.nlm.nih.gov/, February of 2005). Full-length sequence from the moss P. patens, ppMGD was deduced from clustered expressed sequence tag (EST) sequences (Php_AX155049, Php_dbEST_Id: 10946475_Frame-2 and Php_AX150691_Frame-3) obtained via the Moss Genome Initiative web site (www.leeds.moss.ac.uk, August of 2004). Partial sequence from the green alga C. reinhardtii MGD, crMGD (partial, C_21260001) was obtained via the ChlamyDB website (www.chlamy.org/chlamydb.html). Partial sequence from the non-photosynthetic green alga P. wickerhamii, pwMGD (partial, AAV65358) was obtained via the NCBI website. Annotated MGD sequence from the red alga C. merolae, cmMGD (#3974) was obtained via the Cyanidioschyzon Genome Project web site (merolae.biol.s.u-tokyo.ac.jp/). The three MGD sequences from the diatom T. pseudonana, tpMGD1 (full-length; Thp_grail.23.172.1 and Thp_newV2.0.genewise.23.85.1), tpMGD2 (partial, Thp_grail. 120.10.1), and tpMGD3 (partial, genewise.89.116.1) were obtained via the DOE Joint Genome Institute (genome.jgi-psf.org/thaps1/thaps1.home.html). The MURG sequences of seven bacteria, i.e. E. coli, ecMURG (CAA38867), Bacillus subtilis, bsMURG (P37585), Mycobacterium avium, maMURG (NP_960831), Vibrio vulnificus, vvMURG (Q7MNV1), Listeria innocua, liMURG (NP_471475), Lactococcus lactis, llMURG (NP_267745), and Bartonella bacilliformis, bbMURG (AAT38530), and one cyanobacteria, Synechocystis sp. PCC 6803, syMURG (NP_442963), were down-loaded from the NCBI web site. These sequences were obtained by BLASTP similarity searches and selected for their representation of prokaryotic phylogenetic diversity. Phylogenetic Reconstruction—MGD and MURG phylogeny was inferred from the 22 sequences described under “Materials and Methods.” Distance between sequences was computed using the TULIP methods (39Bastien O. Aude J.C. Roy S. Maréchal E. Bioinformatics. 2004; 20: 534-537Crossref PubMed Scopus (29) Google Scholar, 40Bastien O. Aude J.C. Roy S. Maréchal E. BMC Bioinformatics. 2005; 6: 49Crossref PubMed Scopus (21) Google Scholar). Alignment was achieved with the Smith-Waterman method and the BLOSUM 62 scoring matrix, using the Biofacet package from Gene-IT, France (41Codani J.J. Comet J.P. Aude J.C. Glémet E. Wozniak A. Risler J.L. Hénaut A. Slonimski P.P. Methods Microbiol. 1999; 28: 229-244Crossref Scopus (19) Google Scholar). We computed estimated z-scores with 2000 sequence shuffling (39Bastien O. Aude J.C. Roy S. Maréchal E. Bioinformatics. 2004; 20: 534-537Crossref PubMed Scopus (29) Google Scholar, 42Comet J.P. Aude J.C. Glemet E. Risler J.L. Henaut A. Slonimski P.P. Codani J.J. Comput. Chem. 1999; 23: 317-331Crossref PubMed Scopus (61) Google Scholar). Distance between sequences was then calculated using the z-score matrix (40Bastien O. Aude J.C. Roy S. Maréchal E. BMC Bioinformatics. 2005; 6: 49Crossref PubMed Scopus (21) Google Scholar). Tree topology was generated using the Neighbor-Joining algorithm (43Felsenstein J. Cladistics. 1989; 5: 164-166Google Scholar). Homology Modeling—Homology modeling of soMGD1 was based on the crystal structure of ecMURG (36Hu Y. Chen L. Ha S. Gross B. Falcone B. Walker D. Mokhtarzadeh M. Walker S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 845-849Crossref PubMed Scopus (186) Google Scholar) using sequence alignment described in this report. α-Helix and β-strand distributions in the MGD sequences were predicted with several secondary structure prediction servers (www.sbg.bio.ic.ac.uk/~3dpssm/ and www.npsa-pbil.ibcp.fr/cgi-bin) (44Kelley L.A. MacCallum R.M. Sternberg M.J. J. Mol. Biol. 2000; 299: 499-520Crossref PubMed Scopus (1121) Google Scholar, 45Combet C. Blanchet C. Geourjon C. Deleage G. Trends Biochem. Sci. 2000; 25: 147-150Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar). Hydrophobic cluster analysis method was used to refine the sequence alignment (46Callebaut I. Labesse G. Durand P. Poupon A. Canard L. Chomilier J. Henrissat B. Mornon J.P. Cell Mol. Life Sci. 1997; 53: 621-645Crossref PubMed Scopus (436) Google Scholar). Hydrophobic cluster analysis is a graphical method based on the detection and comparison of hydropho
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