photosystems in relation to chlorophyll a and removal of phycocyanin. Biochim. Biophys. Acta 357:231-245. 4. Bradford, M. M. 1976. A rapid and sensitive ...
JOURNAL OF BACTERIOLOGY, Jan. 1993, p. 544-547 0021-9193/93/020544-04$02.00/0 Copyright X 1993, American Society for Microbiology
Vol. 175, No. 2
In Vitro Ferredoxin-Dependent Desaturation of Fatty Acids in Cyanobacterial Thylakoid Membranes HAJIME WADA,1* HERMANN SCHMIDT,2 ERNST HEINZ,2
National Institute for Basic Biology,
NORIO MURATA1 Myodaiji Okazaki 444, Japan,1 and Institut fir Allgemeine Botanik, AND
Universitat Hamburg, 2000 Hamburg 52, Germany2 Received 19 August 1992/Accepted 12 November 1992
Thylakoid membranes isolated from the cyanobacterium Synechocystis sp. strain PCC6803 were capable of desaturating the acyl groups in monogalactosyl diacylglycerol. This desaturation reaction required the reduced form of ferredoxin.
In eukaryotic plants, desaturation of fatty acids occurs in two different compartments (8), namely the plastids and the endoplasmic reticulum. Desaturation of fatty acids is catalyzed by several desaturases, each of which introduces a double bond at a specific position in fatty acids (7, 8). Desaturases require electrons, which are donated by an electron-donating system, for the desaturation of fatty acids. The stearoyl-acyl carrier protein (stearoyl-ACP) desaturase, which is the only soluble desaturase known to date, requires ferredoxin (Fd) as its electron donor (10). Although other membrane-bound desaturases in plastids and the endoplasmic reticulum also require electron donors, their electron donors have not been identified because of the difficulties associated with making measurements in vitro. Recently, Schmidt and Heinz (16, 17) demonstrated that the desaturation of fatty acids by membrane-bound desaturases in chloroplasts involves Fd, Fd-NADP+ oxidoreductase, and NADPH, and they suggested that Fd is a potential candidate for the donor of electrons to chloroplast desaturases. By contrast, Kearns et al. (9) and Smith et al. (18) suggested that cytochrome b5 is involved in the desaturation of fatty acids in the endoplasmic reticulum as an electron donor. Cyanobacteria are photosynthetic prokaryotic algae that use a desaturation system to synthesize unsaturated fatty acids, as do eukaryotic plants (11). Although a gene for cyanobacterial desaturation has been identified (20), the characteristics of the electron-donating system that is required for cyanobacterial desaturation have not yet been clarified. Since the structure and the lipid composition of cyanobacterial and chloroplast thylakoids are similar (11, 19), one might assume that Fd, Fd-NADP+ oxidoreductase, and NADPH constitute the electron-donating system for the desaturation of fatty acids in cyanobacteria. This system in cyanobacteria is clearly also of particular interest from an
during the exponential phase of growth. After incubation overnight at 22°C, the cells were used for isolation of thylakoid membranes. Isolation of membranes. A total of 750 ml of cultures at a chlorophyll concentration of approximately 3 jig ml-' was centrifuged at 8,000 x g for 10 min. The sedimented cells were resuspended in 80 ml of a medium that contained 50 mM 3-(N-morpholino)propanesulfonic acid-KOH (MOPSKOH; pH 7.5) and 10 mM MgCl2, and the suspension was centrifuged at 8,000 x g for 10 min. The collected cells were finally resuspended in a 10-ml volume of 50 mM MOPSKOH (pH 7.5), 300 mM sorbitol, and 10 mM MgCl2 that also contained 50,000 U of catalase (C 3155; Sigma, St. Louis, Mo.). All of the above steps were carried out at room temperature, but the subsequent preparation of membranes was performed at 0 to 4°C. The suspension was gassed with argon and mixed with an equal volume of glass beads (G 8893; Sigma), and then the cells were disrupted by agitation five times (1 min each with intervals of 1 min) on a vortex mixer (Vortex-Genie; Scientific Industries, Biochemia, Bohemia, N.Y.) operated at maximum speed. Glass beads were removed by centrifugation of the suspension at 2,000 x g for 1 min. The supernatant was then centrifuged at 8,000 x g for 10 min to remove unbroken cells. The resultant supernatant, containing membranes, was placed on a discontinuous sucrose gradient which was made in a 14-ml tube by layering the following solutions of sucrose: 2 ml of 55% sucrose, 2 ml of 40% sucrose, and 1 ml of 15% sucrose in 50 mM MOPS-KOH (pH 7.5)-10 mM MgCl2. After centrifugation for 20 min at 200,000 x g, the top 8 ml of the gradient, corresponding to the loading zone, was removed by aspiration. A green fraction containing thylakoid membranes was apparent at the interface between the layers of 40 and 55% sucrose. This fraction was withdrawn with a Pasteur pipette and was immediately used for the assay of desaturation activity. Concentrations of protein and chlorophyll were determined by the methods of Bradford (4) and Arnon et al. (3), respectively. Fatty acid nomenclature. The fatty acids are represented by numbers of carbon atoms and numbers of double bonds before and after a colon, respectively. Desaturation of fatty acids. Amounts of thylakoid membranes corresponding to 60 ,g of protein were mixed with various components to give the following final concentrations or quantities in a total volume of 100 ,ul: 40 mM
evolutionary standpoint. In this communication, we report the characteristics of the desaturation in vitro of fatty acids in thylakoid membranes isolated from the cyanobacterium Synechocystis sp. strain PCC6803, from which the gene for desaturation of fatty acids has been isolated as mentioned above. We demonstrate that the desaturation in this system depends on Fd. Growth conditions. Synechocystis sp. strain PCC6803 was grown photoautotrophically at 34°C in BG-11 medium as described previously (21). In order to accelerate the desaturation activity, cultures were transferred from 340C to 220C *
N-tris[hydroxymethyl]methylglycine-KOH (Tricine-KOH; pH 8.0), 10 mM MgCI2, 20 ,g of Fd from spinach (F 3013; Sigma) or from a Spirulina sp. (F 2513; Sigma), 5 mM
Corresponding author. 544
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VOL. 175, 1993
NADPH (N 1630; Sigma), 5,000 U of catalase (C 3155; Sigma), 20 mU of Fd-NADP+ oxidoreductase (F 0628; Sigma), 55 puM lyso-mono alactosyl diacylglycerol (lysoMGDG), and 0.7 kBq of [1-' C]acyl-ACP (generous gift from M. Frentzen, Universitat Hamburg) or 3.7 kBq of [1-14CJ acyl-coenzyme A ([1-14C]acyl-CoA; DuPont, NEN Research Products, Boston, Mass.). The specific radioactivities of acyl-ACP and acyl-CoA were 2.0 to 2.1 MBq ,umol-1. The reaction mixture was incubated for 4 h at approximately 250C, and then the lipids were extracted from the mixture and analyzed by thin-layer chromatography as described previously (1, 16, 17). Methyl esters prepared from MGDG and the free fatty acid (FFA) fractions were analyzed isocratically on a radio-high performance liquid chromatography (radio-HPLC) system as described previously (1, 16, 17). Hydrogenation of fatty acid methyl esters was carried out by the method of Appelqvist (2). Lyso-MGDG was prepared according to the method of Fischer et al. (6) from MGDG that had been isolated from the cells of Synechococcus sp. strain PCC7942 by digestion with a lipase from Rhizopus delemar (Boehringer Mannheim, Mannheim, Germany). For examination of the desaturation in vitro of fatty acids in thylakoid membranes, the thylakoid membrane lipids that are good substrates for desaturases must be labeled in situ (14, 15). Chen et al. (5) reported that the membrane fraction of Anabaena variabilis has an acyl-ACP:lyso-MGDG acyltransferase activity that catalyzes the transfer of the acyl moiety from acyl-ACP to lyso-MGDG. If the same enzyme is present in the thylakoid membranes of Synechocystis sp. strain PCC6803, it should be possible to label MGDG with [1-14C]acyl-ACP and lyso-MGDG. Figure 1 shows the results of attempts at the incorporation of acyl groups from either acyl-ACP or acyl-CoA into lipid classes. When either 18:0-ACP or 18:1-CoA was used as a substrate (Fig. 1A and C), most of the radioactivity was recovered in the MGDG fraction, and only a small proportion of the radioactivity was detected in the FFA fraction. The extent of incorporation of 18:0 into the MGDG fraction was much reduced in the absence of lyso-MGDG (Fig. 1D). When 18:0-CoA was used as a substrate, only a small proportion of radioactivity was detected in MGDG and FFA fractions even in the presence of lyso-MGDG (data not shown). These results indicate that the thylakoid membranes of Synechocystis sp. strain PCC6803 are capable of transferring acyl groups from both 18:0-ACP and 18:1-CoA to lyso-MGDG. It is likely that this acyl-transfer reaction is catalyzed by an acyl-ACP:lysoMGDG acyltransferase that is present in the thylakoid membrane fraction (5). It is worth noting that acyl groups were transferred from both 18:0-ACP and 18:1-CoA to lyso-MGDG and not only from acyl-ACP as was the case for A. variabilis (5). In addition, when 18:1-ACP was used as a substrate, most of the radioactivity was recovered in the FFA fraction and not in MGDG (Fig. 1B). In correlation with the possible occurrence of acyl-ACP hydrolase activity in cyanobacterial thylakoid membranes, this observation requires further investigation. Figure 2 shows the results of analysis of the desaturation of 18:0 and 18:1 in the MGDG of the thylakoid membranes. When MGDG was labeled with [1-14CJ18:0-ACP and methyl esters were analyzed after the incubation, 18:0 and 18:1 were detected on the chromatograms of fatty acid methyl esters (Fig. 2A and B). When the purified radioactive 18:1 methyl ester was subjected to hydrogenation and rechromatographed, only one radioactive peak, with the retention time of 18:0 methyl ester, was observed. These results suggest
A
A
~~B
545
~~FFA
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e
MGDG
C1
MGD.
0
CA
FFA
D
MGDG
AA Origin Front FIG. 1. Hydrolysis to FFAs and transfer to lyso-MGDG of acyl groups from acyl-ACP and acyl-CoA by thylakoid membranes from Synechocystis sp. strain PCC6803. The thylakoid membranes were incubated with various cofactors (see text) at room temperature for 4 h in the presence of [1-14C]18:0-ACP and exogenous lyso-MGDG (A), [1-14C]18:1-ACP and exogenous lyso-MGDG (B), [1-14C]18:0CoA and exogenous lyso-MGDG (C), or only [1-14C]18:0-ACP (D). Lipids were extracted and then separated by thin-layer chromatography, and the radioactive components were detected with a thinlayer chromatography radioactivity scanner. Each bar corresponds to 2 cm.
that 18:0 was desaturated to 18:1 in MGDG of the thylakoid membranes. Seventeen percent of 18:0 was desaturated to 18:1 in the presence of Fd from spinach (Fig. 2A), but only 6% of 18:0 was desaturated in its absence (Fig. 2B). Fd from a Spirulina sp. was as effective as Fd from spinach for the desaturation of 18:0 (data not shown), indicating the equivalence of Fd from different sources in this assay. When
546
NOTES
J. BAcTERIOL.
18:0
A
18:1
18:0
B I
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0
0
:6~
c
~~
1
C
0
0
0A
O
~~~~1~~18:2 8:2
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Elution time FIG. 2. Effects of Fd on the desaturation of stearate and oleate.
The thylakoid membranes from Synecho ytis sp. strain PCC6803 (see text) at room tempera-
were incubated with various cofactors,
911C18:0-ACP and Fd from spinach (A), [1_14C]18:0-ACP alone (B), [1-1 C]l8:1-CoA and Fd from spinach (C), or [1_l44]l8:1-CoA alone (D)). After the incubation, total lipids were extracted and MGDG was purified. Fatty acid methyl esters prepared from MGDG were analyzed isocratically by radioHPLC. The bar corresponds to 5 nun. ture for 4 h in the presence of
MGDG labeled with [1_14C]18:1-CoA was incubated in the presence of Fd from spinach, both 18:1 and 18:2 were detected in the subsequent analysis (Fig. 2C). After the radioactive 18:2 methyl ester had been hydrogenated, only one radioactive peak was observed, with a retention time of the 18:0 methyl ester. These results suggest that 18:1 was desaturated to 18:2 in the MGDG of the thylakoid membranes. About 7% of 18:1 in MGDG was desaturated to 18:2 in the presence of Fd from spinach (Fig. 2C), but the extent of desaturation was insignificant in the absence of Fd (Fig. 2D). The results of the present study demonstrate that the thylakoid membranes of Synechocystis sp. strain PCC6803
contain 18:0 and 18:1 desaturases and that the reduced form of Fd is required as an electron donor for the desaturation of 18:0 and 18:1. Omata and Murata (12) compared the fatty acid composition of lipids in thylakoid membranes with that in plasma membranes from Anacystis nidulans and found that they were very similar. Omata and Murata also demonstrated that the synthesis of monoglucosyl diacylglycerol takes place in both types of membrane from A. nidulans (13). At present, we do not know whether desaturases are located only in thylakoid membranes or in both thylakoid and plasma membranes. Our newly developed system for assays of desaturases in vitro should help us answer this question. We thank M. Frentzen of the University of Hamburg for the generous gift of 14C-labeled acyl-ACP and for helpful suggestions. REFERENCES 1. Andrews, J., and E. Heinz. 1987. Desaturation of newly synthesized monogalactosyldiacylglycerol in spinach chloroplasts. J. Plant Physiol. 131:75-90. 2. Appelqvist, L. A. 1972. A simple and convenient procedure for the hydrogenation of lipids on the micro- and nanomole scale. J. Lipid Res. 13:146-148. 3. Arnon, D. I., B. D. McSwain, H. Y. Tsujimoto, and K. Wada. 1974. Photochemical activity and components of membrane preparations from blue-green algae. I. Coexistence of two photosystems in relation to chlorophyll a and removal of phycocyanin. Biochim. Biophys. Acta 357:231-245. 4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 5. Chen, H.-H., A. Wickrema, and J. G. Jaworski. 1988. Acyl-acylcarrier protein: lysomonogalactosyldiacylglycerol acyltransferase from the cyanobacterium Anabaena variabilis. Biochim. Biophys. Acta 963:493-500. 6. Fischer, W., E. Heinz, and M. Zeus. 1973. The suitability of lipase from Rhizopus arrhizus delemar for analysis of fatty acid distribution in dihexosyl diglycerides, phospholipids and plant sulfolipids. Hoppe-Seyler's Z. Physiol. Chem. 354:1115-1123. 7. Harwood, J. L. 1988. Fatty acid metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:101-138. 8. Jaworskd, J. G. 1987. Biosynthesis of monoenoic and polyenoic fatty acids, p. 159-174. In P. K. Stumpf (ed.), The biochemistry of plants, vol. 9. Academic Press, Orlando, Fla. 9. Kearns, E. V., S. Hugly, and C. R. Somerville. 1991. The role of cytochrome b5 in A12 desaturation of oleic acid by microsomes of safflower (Carthamus tinctorius L.). Arch. Biochem. Biophys. 284:431-436. 10. McKeon, T. A., and P. K. Stumpf. 1982. Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J. Biol. Chem. 257:12141-12147. 11. Murata, N., and I. Nishida. 1987. Lipids of blue-green algae (cyanobacteria), p. 315-347. In P. K. Stumpf (ed.), The biochemistry of plants, vol. 9. Academic Press, Orlando, Fla. 12. Omata, T., and N. Murata. 1983. Isolation and characterization of the cytoplasmic membranes from the blue-green alga (cyanobacterium) Anacystis nidulans. Plant Cell Physiol. 24:11011112. 13. Omata, T., and N. Murata. 1986. Glucolipid synthesis activities in cytoplasmic and thylakoid membranes from the cyanobacterium Anacystis nidulans. Plant Cell Physiol. 27:485-490. 14. Sato, N., and N. Murata. 1982. Lipid biosynthesis in the blue-green alga, Anabaena vanabilis. II. Fatty acids and lipid molecular species. Biochim. Biophys. Acta 710:279-289. 15. Sato, N., Y. Seyama, and N. Murata. 1986. Lipid-linked desaturation of palmitic acid in monogalactosyl diacylglycerol in the blue-green alga (cyanobacterium) Anabaena variabilis studied in vivo. Plant Cell Physiol. 27:819-835. 16. Schmidt, H., and E. Heinz. 1990. Desaturation of oleoyl groups in envelope membranes from spinach chloroplasts. Proc. Natl.
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Acad. Sci. USA 87:9477-9480. 17. Schmidt, H., and E. Heinz. 1990. Involvement of ferredoxin in desaturation of lipid-bound oleate in chloroplasts. Plant Physiol. 94:214-220. 18. Smith, M. A., A. R. Cross, T. G. Jones, W. T. Griffiths, S. Stymne, and K. Stobart. 1990. Electron-transport components of the 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine A12-desaturase (A12-desaturase) in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons. Biochem. J. 272:23-29.
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19. Stanier, R. Y., and G. Cohen-Bazire. 1977. Phototrophic prokaryotes: the cyanobacteria. Annu. Rev. Microbiol. 31:225274. 20. Wada, H., Z. Gombos, and N. Murata. 1990. Enhancement of chilling tolerance of a cyanobacterium by genetic manipulation of fatty acid desaturation. Nature (London) 347:200-203. 21. Wada, H., and N. Murata. 1989. Synechocystis PCC6803 mutants defective in desaturation of fatty acids. Plant Cell Physiol. 30:971-978.
