Comparative genomic analysis revealed a gene for monoglucosyldiacylglycerol synthase, an enzyme for photosynthetic membrane lipid synthesis in cyanobacteria

Abstract

Cyanobacteria have a thylakoid lipid composition very similar to that of plant chloroplasts, yet cyanobacteria are proposed to synthesize monogalactosyldiacylglycerol (MGDG), a major membrane polar lipid in photosynthetic membranes, by a different pathway. In addition, plant MGDG synthase has been cloned, but no ortholog has been reported in cyanobacterial genomes. We report here identification of the gene for monoglucosyldiacylglycerol (MGlcDG) synthase, which catalyzes the first step of galactolipid synthesis in cyanobacteria. Using comparative genomic analysis, candidates for the gene were selected based on the criteria that the enzyme activity is conserved between two species of cyanobacteria (unicellular [Synechocystis sp. PCC 6803] and filamentous [Anabaena sp. PCC 7120]), and we assumed three characteristics of the enzyme; namely, it harbors a glycosyltransferase motif, falls into a category of genes with unknown function, and shares significant similarity in amino acid sequence between these two cyanobacteria. By a motif search of all genes of Synechocystis, BLAST searches, and similarity searches between these two cyanobacteria, we identified four candidates for the enzyme that have all the characteristics we predicted. When expressed in Escherichia coli, one of the Synechocystis candidate proteins showed MGlcDG synthase activity in a UDP-glucose-dependent manner. The ortholog in Anabaena also showed the same activity. The enzyme was predicted to require a divalent cation for its activity, and this was confirmed by biochemical analysis. The MGlcDG synthase and the plant MGDG synthase shared low similarity, supporting the presumption that cyanobacteria and plants utilize different pathways to synthesize MGDG.

Figure 1.

Scheme for MGDG synthesis. A, MGDG synthesis in cyanobacteria. First, MGlcDG is synthesized from DAG and UDP-Glc by MGlcDG synthase. Then, an epimerase converts MGlcDG into MGDG. B, MGDG synthesis in photosynthetic eukaryotes. MGDG is synthesized from UDP-Gal and DAG by the one-step reaction of MGDG synthase. Note that UDP-Gal is also formed by epimerization of UDP-Glc.

Figure 2.

UDP-Glc-dependent glycolipid synthesis activity in both unicellular and filamentous cyanobacteria. Sugar transferase activities were measured using radiolabeled UDP-Glc or UDP-Gal. Lipids were chromatographed by a solvent system (acetone:toluene:water = 90:30:7 [v/v]) and visualized by autoradiography. Crude extract of Arabidopsis leaves was used as a control for UDP-Gal-dependent MGDG synthesis. Lipids of Synechocystis sp. PCC 6803 were visualized by α-naphthol for lipid standard. The identities of the minor radiolabeled bands were not determined.

Figure 3.

Sugar transferase activity of the candidate genes. Open reading frames of the genes were expressed in E. coli and the activity of crude extracts was measured using radiolabeled UDP-Glc (A) or UDP-Gal (B). Cucumber MGDG synthase (csMGD1) was used as a positive control for UDP-Gal-dependent sugar transfer. Small dots at the bottom indicate origin of TLC. TLC was developed with a solvent system (chloroform:methanol:water = 65:20:2). Incubation of the sll1377 enzyme with UDP-Glc resulted in the production of MGlcDG, whereas MGDG was formed in assays of the csMGD1 enzyme with UDP-Gal.

Figure 4.

Glycolipid accumulated in E. coli membrane. Glycolipids were isolated from transformed E. coli and analyzed by TLC. Lipids of Synechocystis sp. PCC 6803 and E. coli-expressing cucumber MGDG synthase were used as a control. TLC was developed by a solvent system (acetone:toluene:water = 90:30:7 [v/v]) and visualized by α-naphthol. pET24a, Vector control; sll1377, E. coli-expressing sll1377 gene; all4933, E. coli-expressing all4933 gene; csMGD1, E. coli-expressing cucumber MGDG synthase gene.

Figure 5.

Anomeric and epimeric portion of cyanobacterial MGDG (A) and glycolipids (B) accumulated in E. coli-expressing sll1377 protein. Glycolipids were isolated from Synechocystis or transformed E. coli and analyzed by ¹H-NMR. Right arrow in the top column (3.48 ppm) points to the two doublet peaks characteristic of Gal, whereas the arrow in the bottom column (3.42 ppm) points to the triplet peak of Glc. Double peaks (around 4.2 ppm) indicated by left arrow are the peaks from a β-glycosidic carbon atom of the hexose moiety in the head group of the glycolipids.

Figure 6.

A, Amino acid alignment of MGlcDG synthase from Synechocystis (sll1377) and Anabaena (all4933). Black boxes, Identical amino acids; gray boxes, similar amino acids. Asterisks on the top of the sequences indicate position of D…DxD and QxxRW motifs. Predicted secondary structure is also shown at the bottom of the alignment. E, β-Strand. H, α-Helix. B, Structural feature of cyanobacterial MGlcDG synthase with other GT2 family transferases. TMs, Transmembrane domains; SpsA, SpsA protein; CesA, cellulose synthase protein.

Figure 7.

Enzymatic property of MGlcDG synthase. Relative activity to control is described. MgCl₂ (control): OPDAG, 25 mm MgCl₂; −MgCl₂: OPDAG, 0 mm MgCl₂; EDTA: OPDAG, 5 mm EDTA; CaCl₂: OPDAG, 25 mm CaCl₂; MnCl₂: OPDAG, 25 mm MnCl₂; DPDAG: DPDAG, 25 mm MgCl₂. sd is based on three independent experiments.