Báo cáo khoa học: Geranylgeranyl reductase involved in the biosynthesis of archaeal membrane lipids in the hyperthermophilic archaeon Archaeoglobus fulgidus potx

Geranylgeranyl reductase involved in the biosynthesis of archaeal membrane lipids in the hyperthermophilic archaeon Archaeoglobus fulgidus Motomichi Murakami 1 , Kyohei Shibuya 2 , Toru Nakayama 2 , Tokuzo Nishino 2 , Tohru Yoshimura 1 and Hisashi Hemmi 1 1 Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Aichi, Japan 2 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Miyagi, Japan The structure of membrane lipids is the most striking characteristic of the Archaea (one of the three domains of life), which includes many extremophiles, such as thermophiles, halophiles and methanogens [1–3]. The archaeal membrane lipids are different from the typ- ical glycerolipids in organisms of the other domains – Bacteria and Eucarya – in the following respects. First, Archaeal lipids have fully reduced prenyl chains, whereas glycerolipids typically have fatty acyl chains. Almost all archaea produce membrane lipids that contain phytanyl groups (i.e. fully saturated C 20 prenyl groups). Second, the connection of the hydrocarbon chains with the glycerol moiety occurs via an ether bond in archeal lipids, not via the ester bond generally Keywords archaea; geranylgeranyl reductase; isoprenoid; lipid; oxidoreductase Correspondence H. Hemmi, Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464–8601, Japan Fax: +81 52 7894120 Tel: +81 52 7894134 E-mail: hhemmi@agr.nagoya-u.ac.jp (Received 27 September 2006, revised 29 November 2006, accepted 5 December 2006) doi:10.1111/j.1742-4658.2006.05625.x Complete saturation of the geranylgeranyl groups of biosynthetic interme- diates of archaeal membrane lipids is an important reaction that confers chemical stability on the lipids of archaea, which generally inhabit extreme conditions. An enzyme encoded by the AF0464 gene of a hyperthermophi- lic archaeon, Archaeoglobus fulgidus, which is a distant homologue of plant geranylgeranyl reductases and an A. fulgidus menaquinone-specific prenyl reductase [Hemmi H, Yoshihiro T, Shibuya K, Nakayama T, & Nishino T (2005) J Bacteriol 187, 1937–1944], was recombinantly expressed and puri- fied, and its geranylgeranyl reductase activity was examined. The radio HPLC analysis indicated that the flavoenzyme, which binds FAD noncova- lently, showed activity towards lipid-biosynthetic intermediates containing one or two geranylgeranyl groups under anaerobic conditions. It showed a preference for 2,3-di-O-geranylgeranylglyceryl phosphate over 3-O-geranyl- geranylglyceryl phosphate and geranylgeranyl diphosphate in vitro, and did not reduce the prenyl group of respiratory quinones in Escherichia coli cells. The substrate specificity strongly suggests that the enzyme is involved in the biosynthesis of archaeal membrane lipids. GC-MS analysis of the reaction product from 2,3-di-O-geranylgeranylglyceryl phosphate proved that the substrate was converted to archaetidic acid (2,3-di-O-phytanyl- glyceryl phosphate). The archaeal enzyme required sodium dithionite as the electron donor for activity in vitro, similarly to the menaquinone-specific prenyl reductase from the same anaerobic archaeon. On the other hand, in the presence of NADPH (the preferred electron donor for plant homo- logues), the enzyme reaction did not proceed. Abbreviations DGGGP, 2,3-di-O-geranylgeranylglyceryl phosphate; DGGGPS, 2,3-di-O-geranylgeranylglyceryl phosphate synthase; GGGP, 3-O- geranylgeranylglyceryl phosphate; GGGPS, 3-O-geranylgeranylglyceryl phosphate synthase; GGR, geranylgeranyl reductase; GGPP, geranylgeranyl diphosphate; GGPS, geranylgeranyl diphosphate synthase; IPP, isopentenyl diphosphate; PR, prenyl reductase. FEBS Journal 274 (2007) 805–814 ª 2007 The Authors Journal compilation ª 2007 FEBS 805 formed in lipids. Third, the stereochemistry of the gly- cerol moiety is enantiomeric between the archaeal and the typical lipids. Fourth, most thermophilic and meth- anogenic archaea also contain bipolar cyclic lipids (‘tetraether’ lipids), which are probably formed by the dimerization of two ‘diether’ lipids. The biosynthesis of the archaeal membrane lipid has been studied previ- ously (Fig. 1). The precursor of the glycerol moiety, sn-glycerol-1-phosphate, is formed from dihydroxy acetone phosphate [4,5]. On the other hand, the pre- cursor of the prenyl moiety is synthesized from active C 5 isoprene units, for example, isopentenyl diphos- phate (IPP) and dimethylallyl diphosphate, usually by geranylgeranyl diphosphate (GGPP) synthase, which yields the C 20 precursor [6,7], although a few archaea are known to utilize geranylfarnesyl diphosphate syn- thase to synthesize the C 25 precursor [8,9]. The gera- nylgeranyl chains thus produced are then transferred to the sn-3 position of sn-glyceryl-1-phosphate by 3-O-geranylgeranylglyceryl phosphate (GGGP) syn- thase [10–12] and subsequently to the sn-2 position by 2,3-di-O-gerenylgeranylglyceryl phosphate (DGGGP) synthase [13]. The fundamental carbon-oxygen skeleton of archaeal membrane lipids is formed at this point, fol- lowed by various processes, such as modification of polar head groups [14], saturation or cyclization of pre- nyl chains, and the creation of a bipolar cyclic structure [15]. However, although a few enzymes that catalyze polar head modification (i.e. CTP:DGGGP cytidyl- transferase [16] and archaetidylserine synthase [17]) have been found, enzymes catalyzing the other process have not been examined in detail. The complete saturation of prenyl chains would con- fer chemical stability on archaeal membrane lipids. Therefore, the reduction of prenyl chains is generally thought to play an important role in the survival of archaea under extreme conditions, such as high tem- perature or salinity, although partially saturated prenyl chains have been found in some archaea and, interest- ingly, the number of unsaturated double bonds is known to be related to the temperature at which the organism grows [18,19]. Saturated prenyl groups are also found in compounds, other than membrane lipids, in archaea [2,20]. Many archaea produce respiratory quinones (i.e. menaquinone, caldariellaquinone, sulfo- lobusquinone, thermoplasmaquinone, etc.) that contain fully or partially saturated prenyl side-chains. We recently identified an enzyme that catalyzes the satura- tion of the prenyl side-chain of menaquinone in a hyperthermophilic archaeon, Archaeoglobus fulgidus [21]. The enzyme, prenyl reductase (PR), is a distant homologue of geranylgeranyl reductase (GGR) from plants [22,23] and cyanobacteia [24], which catalyzes the saturation of the geranylgeranyl group to produce chlorophyll, tocopherol and probably phyloquinone. The menaquinone-specific PR is a FAD-dependent flavoenzyme. Sodium dithionite, as an electron donor, is required for the reducing reaction to proceed in vitro, but NADPH does not function, although plant homologues are dependent on this reducing agent. In addition, the enzyme does not require a diva- lent metal ion for reaction. We isolated three other genes of GGR homologues from A. fulgidus, none of which have known functions. In this article, we report on the function of an archaeal GGR homologue encoded in the ORF AF0464 (chlP-1), which is efficiently expressed in Escherichia coli, as evidenced by our previous report [21]. The recombinant enzyme was affinity purified, and its activity was assessed in vitro under anaerobic conditions. The enzyme, whose properties are very similar to those of the menaquinone-specific PR, cata- lyzes the conversion of geranylgeranyl groups of Fig. 1. Biosynthetic pathway of archaeal membrane lipids. X denotes a polar head group. When X is phosphate, phosphoserine or phosphoethanolamine, the archaeal lipid with two phytanyl chains is denoted as archaetidic acid, archaetidylserine or archaeti- dylethanolamine, respectively. DGGGP, 2,3-di-O-geranylgeranyl- glyceryl phosphate; DGGGPS, DGGGP synthase; GGGP, 3-O-geranylgeranylglyceryl phosphate; GGGPS, GGGP synthase; GGPP, geranylgeranyl diphosphate; GGPS, GGPP synthase; IPP, isopentenyl diphosphate. Enzymes are indicated in boxes. Archaetidic acid-synthesizing archaeal reductase M. Murakami et al. 806 FEBS Journal 274 (2007) 805–814 ª 2007 The Authors Journal compilation ª 2007 FEBS DGGGP into phytanyl groups. It also acts, although weakly, on the geranylgeranyl group of GGGP and GGPP. These facts strongly suggest the involvement of the enzyme, A. fulgidus GGR, in the biosynthesis of membrane lipids. Results Recombinant expression and purification of an archaeal GGR homologue In our previous study, we reported on the recombinant expression of archaeal GGR homologues encoded in ORFs, namely AF0464 (chlP-1), AF1023 (chlP-2), AF1637 (chlP-3) and AF0648 [21]. The expression of AF0648 resulted in a change in the quinone profile of the host E. coli, which led us to conclude that the menaquinone-specific PR is encoded in AF0648. How- ever, the functions of the other three GGR homo- logues were not clear at that time. Among them, only the recombinant expression product of AF0464 could be obtained from the soluble fraction after centrifuga- tion of the cell lysate, whereas the other two were found in the precipitate. Therefore, in this study, we attempted to purify the recombinant protein encoded in AF0464 in order to understand its function in greater detail. To express the archaeal protein as a fusion with a polyhistidine-tag at its N terminus, a gene fragment, containing AF0464, was cut from the pET3a–AF0464 vector, constructed previously, and inserted into the pET15b vector. E. coli cells were transformed with the resultant plasmids and then cul- tured with the appropriate induction. The expressed protein was purified using a Ni-chelating affinity col- umn chromatography after heat treatment. The purity of the protein was verified by SDS ⁄ PAGE. As shown in Fig. 2, a strong protein band was observed in the crude extract, as well as in the heat-treated enzyme solution. However, the molecular weight of the protein estimated from SDS⁄ PAGE data seems to be slightly smaller than the calculated value, 46978.37. The pro- tein did not specifically bind to a Ni-chelating affinity column and was recovered in the flow-through frac- tion. Edman degradation of the protein gave us the N-terminal amino acid sequence, MYDVVVGA, which clearly showed that the protein arose from the translation from Met at the 24th position of the expec- ted full-length of the archaeal enzyme (except for the polyhistidine-tag). In contrast, a distinct protein of slightly lower mobility, which corresponds reasonably well with the calculated molecular weight, was purified using the affinity column. We used the later, affinity- purified, protein for further characterization because we needed purer protein solution. The UV-visible spec- trum of the concentrated, purified enzyme solution is shown in Fig. 3. In this spectrum, specific peaks for flavin coenzymes were observed at  380 and 440 nm, like the spectrometric analysis of recombinant A. fulgi- dus PR. Thus, we attempted to extract the flavin cofac- tor from the protein by heating in methanol. The extracted compound, which had a yellow color and emits fluorescence under UV light, comigrated with FAD, but not with FMN, on chromatography paper (data not shown). This fact proved that the archaeal protein, at least when recombinantly expressed in E. coli, contains noncovalently bound FAD. By refer- ring to the absorption coefficient for free FAD, 87% of the purified enzyme was estimated to bind FAD. Fig. 2. SDS ⁄ PAGE of the recombinant geranylgeranyl reductase (GGR) homologue from Archaeoglobus fulgidus. Lane 1, standard molecular marker; lane 2, crude extract from BL21(DE3) ⁄ pET15b- AF0464; lane 3, supernatant fraction after heat treatment; lane 4, recombinant GGR homologue purified using Ni-chelating affinity col- umn chromatography. The strongly expressed protein bands in lanes 2 and 3, indicated by an asterisk, are shown to arise from the archaeal protein expressed in a truncated form. Fig. 3. UV-visible spectrum of a geranylgeranyl reductase (GGR) homologue from Archaeoglobus fulgidus. M. Murakami et al. Archaetidic acid-synthesizing archaeal reductase FEBS Journal 274 (2007) 805–814 ª 2007 The Authors Journal compilation ª 2007 FEBS 807 GGR assay using radio HPLC We performed enzyme assays under anaerobic condi- tions because A. fulgidus is an obligate anaerobe and because the menaquinone-specific PR from the archaeon requires anaerobic conditions [21]. Oxygen was removed from the reaction mixture by bubbling with N 2 gas, and sodium dithionite was added to eliminate oxygen completely in the reaction mixture and also to donate electrons for reducing reactions. The radio- labeled products synthesized with the three recombin- ant prenyltransferases [i.e. GGPP synthase (GGPS), GGGP synthase (GGGPS), and DGGGP synthase (DGGGPS)], which mainly contained DGGGP, were first used as substrates for the reductase assay. After the reaction, compounds that contained a phosphate or diphosphate group were hydrolyzed with acid phos- phatase and then extracted with n-pentane to be ana- lyzed by radio HPLC. The elution profiles of the pentane extracts showed the appearance of a new peak, with an elution time longer than that of the hydrolyzed product from DGGGP (Fig. 4A). When 0.1% Triton X-100 was added, several new peaks appeared between the new peak and that of dephos- phorylated DGGGP. To confirm this finding, various concentrations of Triton X-100 were added to the reaction mixture, in which radioactive substrates, syn- thesized from fourfold greater quantities of [1- 14 C]IPP AB DC Fig. 4. Radio HPLC analysis of the products from the geranylgeranyl reductase (GGR) assay. (A) Elution profiles of the radiolabeled com- pounds extracted from the GGR assay mixture, which mainly contained radiolabeled 2,3-di-O-geranylgeranylglyceryl phosphate (DGGGP) as the substrate. The compounds were dephosphorylated with acid phosphatase prior to HPLC analysis. Digeranylgeranylglycerol arose from the dephosphorylation of unreacted DGGGP eluted at  18 min. An asterisk indicates peaks eluted at  10 min that are probably derived from dephosphorylated alcohols from the intermediates of substrate production (i.e. geranylgeraniol and ⁄ or geranylgeranylglycerol). (B) Elu- tion profiles of the radioactive compounds from the GGR assay mixture to which various concentrations of Triton X-100 were added. For the synthesis of the substrates for these assays, a fourfold greater amount of [1- 14 C]IPP was used. An asterisk indicates peaks derived from radioactive compounds brought into the GGR reactions other than DGGGP. Peaks at  10 min are probably derived from geranylgeraniol and ⁄ or geranylgeranylglycerol, whereas the compounds corresponding to peaks at  7 min are unidentified. (C and D) The radio HPLC profiles of the compounds from the GGR assay mixture, in which radiolabeled 3-O-geranylgeranylglyceryl phosphate (GGGP) (C) and geranyl- geranyl diphosphate (GGPP) (D) were the main substrates. Archaetidic acid-synthesizing archaeal reductase M. Murakami et al. 808 FEBS Journal 274 (2007) 805–814 ª 2007 The Authors Journal compilation ª 2007 FEBS and (all-E) farnesyl diphosphate, were used (Fig. 4B). The elution profiles clearly showed that higher deter- gent concentrations increased the intensity of the peaks with shorter elution times. These new peaks were very similar to those observed in the reduction of menaqui- none by A. fulgidus PR, which arise from menaquin- ones with a partially saturated prenyl group. The compounds corresponding to the new peaks appeared to be produced from DGGGP because they eluted suc- cessively after the peak of DGGGP and because the decline in the DGGGP peak corresponded to the appearance of the new peaks. Furthermore, a total of eight new peaks, derived from DGGGP, appeared, strongly suggesting that the peaks correspond with reaction products that have different numbers of dou- ble bonds remaining unsaturated. If so, the peak with the longest elution time, observed in the absence of detergent, would be expected to arise from archaetidic acid, the final product with two phytanyl chains. The addition of 2 mm NADPH, instead of sodium dithio- nite, and also the removal of sodium dithionite, failed to produce such new peaks, suggesting that NADPH does not act as a specific electron donor for the enzyme. This hypothesis was also supported by the facts that the addition of NADPH did not diminish the absorption peak of the enzyme at  440 nm, which is derived from the oxidized-form of FAD, even under anaerobic conditions, and that the enzyme did not reduce NADPH at 55 °C in the presence of oxygen (data not shown). The supplemental addition of 0.5 mm FAD did not significantly enhance the reac- tion, probably because the enzyme is already saturated with FAD, as described above. The addition of 10 mm EDTA to the reaction mixture did not inhibit the reac- tion, indicating that the enzyme does not require a divalent metal ion, as observed in the reaction of the menaquinone-specific PR from A. fulgidus. We next carried out GGR assays, using other radio- active substrates, to determine the substrate specificity of the enzyme. The reaction products of GGPS, and of both GGPS and GGGPS, which mainly contained GGPP and GGGP, respectively, were used as the sub- strates. As shown in Fig. 4C,D, new peaks with longer elution times were also observed in the elution profiles of the reaction with both substrates. These observa- tions suggested that the enzyme is able, at least parti- ally, to reduce both of the substrates, which contain a geranylgeranyl group. However, the enzyme activity for these substrates seemed not to be as high as that for DGGGP because such new product peaks, arising from GGPP or GGGP, were not as obvious as those from DGGGP when the reaction mixture contained both DGGGP and the other substrates (Fig. 4A,B). The product specificity of the enzyme strongly suggests that the enzyme preferentially catalyzes reducing reac- tions to produce archaetidic acid from DGGGP. Product analysis by GC-MS The butanol-extracted products from the GGR assay, in which a nonlabeled substrate, such as DGGGP, GGGP and GGPP, was used, were dephosphorylated, trimethylsilylated and subjected to GC-MS analysis. When DGGGP was used as the substrate, a small peak, with the same retention time at 31.5 min as that of tri- methylsilylated archaeol, extracted from Halobacterium salinarum as an authentic sample, was observed on the chromatogram (Fig. 5). Such a peak could not be found when the enzyme was not present in the reaction mixture. Although only a slight ion peak identical to [M+H] + was observed at m ⁄ z 726, strong peaks at m ⁄ z 710, 621 and 426 were considered to corres- pond with [M-CH 3 ] + , [M-CH 3 OSi(CH 3 ) 3 ] + and [M-C 20 H 41 OH] + , respectively. Moreover, the mass spectrum of the compound corresponding to the peak was almost identical to those of trimethylsilylated authentic archaeol we prepared and previously reported by Teixidor & Grimalt [25], strongly suggesting that DGGGP was converted to archaetidic acid, a common component of the archaeal membrane. DGGGP and partially saturated intermediate products were not detected by GC-MS, which can be explained by a scen- ario in which the production of such intermediates might be negligible because detergent was not added to the reaction mixture and that compounds with double bonds were comparatively labile and therefore decom- posed under the severe conditions used for the detection of archaeol. On the other hand, such new products were not detected when GGGP or GGPP was used in the assay (data not shown), probably because the sat- uration of the geranylgeranyl group did not proceed well. Discussion In this article, we characterized the function of an archaeal homologue of plant GGR and report here that the enzyme is able to reduce geranylgeranyl groups of archaeal membrane lipid precursors. The properties of the enzyme from A. fulgidus are very similar to those of the recently reported menaquinone- specific PR from the same organism [21], which is also homologous to plant GGR, in the following respects. First, these reductases, at least when recombinantly expressed in E. coli, are flavoenzymes that non- covalently bind FAD. Second, they require sodium M. Murakami et al. Archaetidic acid-synthesizing archaeal reductase FEBS Journal 274 (2007) 805–814 ª 2007 The Authors Journal compilation ª 2007 FEBS 809 dithionite, not NADPH, the preferable reducing agent for plant homologues, for in vitro activity. Third, they do not require a divalent metal ion. A. fulgidus GGR prefers DGGGP as the substrate and can convert it to a general component of the archaeal membrane (arch- aetidic acid), which contains two phytanyl chains. The enzyme also accepts GGGP and GGPP as substrates, but the activity for them seems to be much weaker than that for DGGGP. On the other hand, the expres- sion of the enzyme in E. coli was reported to have no effect on the quinone profile of the host, clearly indica- ting that menaquinone and ubiquinone are not prefer- able substrates for the enzyme [21]. These facts strongly suggest the involvement of the enzyme, GGR, in the biosynthesis of membrane lipids in the hyper- thermophilic archaeon A. fulgidus, as predicted in the previous publication. It should be noted here that the reduction of lipid precursors was not catalyzed by the menaquinone-specific PR from A. fulgidus (data not shown). The archaeal enzyme is distinct from plant GGR because the final product of the enzyme contains a phytanyl group, whereas plant GGR cannot saturate all the double bonds of a geranylgeranyl group and finally yields a phytyl group, which retains a double bond at position 2 [22–24]. This difference is very important because the double bond is responsible for the formation of an allylic carbocation during the pre- nyltransfer reaction, which means the phytanyl group cannot be transferred to acceptors by prenyltransf- erases, whereas the phytyl group can. Therefore, the substrate specificity of A. fulgidus GGR is reasonable: if phytanyl diphosphate is produced as a result of the complete reduction of GGPP, it cannot be utilized for the biosynthesis of isoprenoid compounds, such as archeal membrane lipids and respiratory quinones. On the other hand, phytyl diphosphate produced by plant GGR is actually used in the biosynthesis of chloro- phyll, tocopherol and phylloquinone. Morii et al. reported on a CTP:DGGGP cytidyl- transferase from Methanothermobacter thermoautotro- phicus, which catalyzes the modification of the polar head group of archaeal phospholipid [16]. The enzyme can accept DGGGP as a substrate, but cannot utilize archaetidic acid. On the other hand, an archaetidyl- serine synthase from M. thermoautotrophicus accepts both substrates with geranylgeranyl and phtanyl groups (i.e. CDP-2,3-di-O-geranylgeranylglycerol and Fig. 5. GC-MS analysis of the product from the geranylgeranyl reductase (GGR) assay. (A) Chromatogram of butanol-extracted compounds from the GGR assay, in which 2,3-di-O-geranylgeranylglyceryl phosphate (DGGGP) was used as the substrate. Reac- tion products were dephosphorylated and then trimethylsilylated. The peak with an arrowhead had the same retention time as that of an authentic sample, trimethylsilylat- ed archaeol. (B and C) Mass spectra of the peak in (A) and the authentic sample, respectively. Archaetidic acid-synthesizing archaeal reductase M. Murakami et al. 810 FEBS Journal 274 (2007) 805–814 ª 2007 The Authors Journal compilation ª 2007 FEBS CDP-2,3-di-O-phytanylglycerol), respectively, for the formation of phosphatidylserine [17]. (Here, the term ‘phosphatidyl’ denotes phosphoglycerolipids in a broad sense, including archaetidyl phospholipids and their analogues with geranylgeranyl groups in this case.) Thus, the authors conclude, based on the specificities of the enzymes, that the saturation of the geranylgera- nyl groups of archaeal phospholipids occurs at least after the transfer of the cytidyl group. A. fulgidus was reported to contain no detectable phosphatidylserine, but does contain phosphatidylethanolamine [3]. So, if a similar situation exists in the cells of A. fulgidus, GGR from the archaeon should catalyze the saturation of the geranylgeranyl groups of various phospholipid precursors, such as CDP-2,3-di-O-geranylgeranylglyc- erol, 2,3-di-O-geranylgeranylglyceryl phosphoserine and 2,3-di-O-geranylgeranylglyceryl phosphoethanol- amine, as well as DGGGP. However, the A. fulgidus genome still encodes two more homologues, with unknown functions, of GGR [21]. If these homologues also catalyze the reduction of a geranylgeranyl or prenyl group, they and A. fulgidus GGR might have distinct substrate specificities and physiological roles. The activity of the enzymes on the unknown precur- sors of bipolar cyclic lipids (‘tetraether’ lipids), which A. fulgidus also produces, is particularly interesting. While we were writing this article, Nishimura & Eguchi reported on the purification of GGR, which is specific for DGGGP and some other precursors of archaeal phospholipids, from a thermoacidophilic archeaon, Thermoplasma acidophilum [26]. They deter- mined the partial amino acid sequence of the enzyme and concluded that the enzyme is encoded in an ORF, Ta0516m, which is homologous to AF0464. Their results strongly support our findings, although they did not confirm the enzyme activity of the gene expres- sion product. However, they purified the enzyme from the membrane fraction of T. acidophilum, which indi- cates that T. acidophilum GGR is tightly associated with the membrane. On the other hand, A. fulgidus GGR seemed to be soluble, at least when expressed in E. coli, because it could be purified from the superna- tant fraction after heat treatment, which usually makes recombinant membrane proteins precipitate with the membrane fractions of E. coli, even though it had not been solublized with detergents. This characteristic of A. fulgidus GGR is also similar to that of the mena- quinone-specific PR. In fact, these enzymes were pre- dicted to be soluble by sosui, a program for classification and secondary structure prediction of membrane proteins [27] (data not shown). Further- more, T. acidophilum GGR can utilize NAD(P)H as an electron donor, whereas the enzyme from A. fulgi- dus cannot. This fact strongly suggests that A. fulgidus GGR accepts electrons from other specific reducing agents (e.g. cofactor F 420 or redox proteins such as fer- redoxin), in the living cells. Experimental procedures Materials (All-E)-farnesyl diphosphate was donated by K. Ogura and T. Koyama (Tohoku University). Nonlabeled IPP was donated by C. Ohto (Toyota Motor Co, Toyota, Japan). [1- 14 C]IPP was purchased from GE Healthcare (Piscataway, NJ, USA). All other chemicals were of analytical grade. General procedures Restriction enzyme digestions, transformations and other standard molecular biology techniques were carried out as described by Sambrook et al. [28]. Expression and purification of the recombinant enzyme The NdeI–BamHI fragment, containing the ORF AF0464, was cut from the pET3a-derived expression vector, reported previously [21], and inserted into the pET15b vector (Novagen, Darmstadt, Germany). E. coli BL21(DE3), transformed with the new vector, was cultured aerobically in Luria–Bertani broth supplemented with 50 mgÆL )1 ampi- cillin. When the attenuance (D), at 600 nm, of the culture reached 0.6, the transformant cells were induced by treat- ment with 1.0 mm isopropyl thio-b-d-galactoside. After 18 h of additional culture, the cells were harvested and dis- rupted by sonication in HisTrap binding buffer, containing 20 mm potassium phosphate buffer, pH 7.6, 0.5 m NaCl and 100 mm imidazole. The homogenate was centrifuged at 15 000 g for 15 min, and the supernatant was recovered as a crude extract. The crude extract was heated at 55 °C for 1 h, and the denatured proteins were removed by centrifu- gation at 15 000 g for 15 min. The supernatant fraction was recovered as a heat-treated enzyme. The heat-treated enzyme, after filtration through a 0.45 lm membrane, was loaded onto a HisTrap column (GE Healthcare), previously equilibrated with binding buffer. The column was washed with binding buffer, and specifically bound proteins were then eluted with an elution buffer, containing 20 mm potas- sium phosphate buffer, pH 7.6, 0.5 m NaCl and 500 mm imidazole, and used for characterization as purified GGR. The level of protein expression was determined by electro- phoresis on a 12% SDS polyacrylamide gel. UV-visible analysis of the purified enzyme solution (containing 0.25 lgÆlL )1 of the enzyme) was conducted with a Shim- adzu UV-2450 spectrophotometer (Shimadzu, Kyoto, M. Murakami et al. Archaetidic acid-synthesizing archaeal reductase FEBS Journal 274 (2007) 805–814 ª 2007 The Authors Journal compilation ª 2007 FEBS 811 Japan). The absorption coefficient of FAD used to calcu- late the concentration was 11 300 at 450 nm [29]. The con- centration of the protein was quantified by the Bradford method [30]. Prediction of transmembrane regions was per- formed using the sosui program (http://bpnuap.nagoya-u. ac.jp/sosui/). Protein sequencing We performed Edman degradation for sequencing N-ter- minal amino acids of the protein. After SDS ⁄ PAGE, pro- teins were transferred onto a poly(vinylidene difluoride) membrane, and the transferred protein band was clipped out and brought into Edman degradation with a Procise- TM HT protein sequencing system (Applied Biosystems, Framingham, MA, USA). Flavin analysis We concentrated 500 lL of the purified enzyme solution into a volume of  100 lL using a Centricon YM-10 spin filter (Millipore, Billerica, MD, USA), replacing the buffer with water. To the concentrated enzyme solution, 1 mL of methanol was added. The mixture was heated at 100 °C for 15 min, and then centrifuged at 20 000 g for 10 min. The recovered supernatant was evaporated to  10 lL and then spotted onto ADVANTEC 51A chromatography paper (ADVANTEC, Tokyo, Japan) and developed with n-buta- nol ⁄ methanol ⁄ 5% Na 2 HPO 4 (60 : 15 : 30, v ⁄ v ⁄ v). Authen- tic FMN and FAD were chromatographed on the same paper. Spots corresponding to flavins were detected by UV illumination. Preparation of hypothetical substrates for A. fulgidus GGR Enzymatic synthesis of GGPP, GGGP and DGGGP was performed as reported previously [13]. The standard reac- tion mixture contained, in a final volume of 100 lL, 430 pmol of [1- 14 C]IPP (2.1 GBqÆmmol )1 ), 1 nmol of (all-E) farnesyl diphosphate, 0.2 lmol of a-glycerophosphate, 2 lmol of MgCl 2 ,2lmol of sodium phosphate buffer, pH 5.8, and suitable amounts of recombinant enzymes (i.e. Sulfolobus acidocaldarius GGPS, S. solfataricus GGGPS and S. solfataricus DGGGPS). The mixture was incubated at 55 °C for 1 h and then used directly in the GGR assay as the substrate mixture that mainly contains DGGGP. To synthesize the substrate mixtures containing mainly GGPP and GGGP, DGGGPS or both DGGGPS and GGGPS were removed, respectively. For the analysis of the GGR reaction products by mass spectrometry, nonlabeled compounds were enzymatically synthesized and purified. The standard reaction mixture contained, in a final volume of 3 mL, 600 nmol of nonlabe- led IPP, 600 nmol of (all-E) farnesyl diphosphate, 80 lmol of a-glycerophosphate, 30 lmol of MgCl 2 , 300 lmol of 2-molpholinoethanesulfonic acid-NaOH buffer, pH 5.8, and suitable amounts of recombinant prenyltransferases (i.e. GGPS, GGGPS and DGGGPS). The mixture was incuba- ted at 55 °C for 2 h and then extracted with 3 mL of 1-but- anol saturated with H 2 O. After evaporation, the butanol layer was loaded onto a COSMOSIL 5C 4 -AR-300 reverse- phase column (4.6 · 150 mm; Nacalai Tesque, Kyoto, Japan), interfaced with an HPLC system, to purify DGGGP. To recover GGGP and GGPP, DGGGPS and both DGGGPS and GGGPS were removed from the mix- ture, respectively. The compounds were eluted from the col- umn with eluent A (25 mm NH 4 HCO 3 ) isocratically for the first 2.5 min, and then with a linear gradient from 100% eluent A to 100% eluent B (acetonitrile) through 15 min, and finally with eluent B for 12.5 min, at a flow rate of 1mLÆmin )1 . Elution of the products was detected by UV absorption at 210 nm. Radio HPLC assay of GGR All manipulations for the GGR assay were carried out in an anaerobic chamber until the reaction was complete. The standard reaction mixture contained (in a volume of 350 lL) the substrate mixture from the prenyltransferase reaction described above, 200 lmol of 3-molpholinopro- panesulfonic acid-NaOH buffer, pH 7.5, and an appropri- ate amount of purified GGR. Various amounts of Triton X-100 were added, as required. The solutions of all con- tents, except for the enzyme and detergent, were bubbled with N 2 gas to remove oxygen. To the mixture, 50 lmol of sodium dithionite in 50 lLofN 2 -bubbled water was added. The mixture was then incubated at 55 °C for 1 h, and the reaction was stopped by adding 200 lL of a cold, saturated NaCl solution. The mixture was extracted with 600 lL of 1-butanol saturated with H 2 O, and the butanol- extracted compounds were hydrolyzed with potato acid phosphatase (Sigma-Aldrich, St Louis, MO, USA) by the method of Fujii et al. [31]. The resulting alcohols were extracted with n-pentane and analyzed by HPLC with a YMC Pack ODS-A C 18 reverse-phase column (4.6 · 250 mm, 5 lm; YMC Co., Ltd, Kyoto, Japan). The alco- holic compounds were isocratically eluted from the column with methanol ⁄ 2-propanol (7 : 3, v ⁄ v) at a flow rate of 0.5 mLÆmin )1 . Elution of the products was detected by radioactivity measured using a ramona Star radio-HPLC analyzer (Raytest, Straubenhardt, Garmany). The flow rate of the scintillation cocktail was 0.5–2 mLÆmin )1 . Extraction of archaeol from H. salinalium H. salinarum was cultured in 1 L of culture medium, con- taining 5 g of casamino acids, 5 g of yeast extract, 3 g of Archaetidic acid-synthesizing archaeal reductase M. Murakami et al. 812 FEBS Journal 274 (2007) 805–814 ª 2007 The Authors Journal compilation ª 2007 FEBS trisodium citrate, 20 g of MgSO 4 )7H 2 0, 2 g of KCl and 200 g of NaCl, at 37 °C for 3 days, and then harvested by centrifugation. Lipid extraction was performed by the method of Bligh & Dyer [32]. The chloroform layer, con- taining the total lipids, was concentrated by evaporation, and acetone was added to > 20-fold excess. The mixture was stored at 4 °C overnight to precipitate the polar lipids. Excision of polar head groups was performed according to the method of Demizu et al. [33]. The precipitated polar lipids were subjected to acetolysis at 165 °C for 20 h in 5 mL of a mixture of acetic acid ⁄ acetic anhydride (3 : 2, v ⁄ v). After evaporating the solution to dryness, the acetyl- ated lipids were hydrolyzed, by acid methanolysis, at 100 °C for 10 h in 3.5 mL of 5% HCl in methanol solution. After evaporation, the lipids, which mainly contained archaeol, were recovered by partitioning with chloro- form ⁄ methanol ⁄ water (10 : 10 : 9, v ⁄ v ⁄ v). GC-MS analysis The nonlabeled substrates, purified as described above, were used for reaction with GGR. The substrate solution in a glass tube was concentrated by evaporation and then placed in an anaerobic chamber. All manipulations des- cribed below were carried out under anaerobic conditions until the reaction was complete. In the tube of the sub- strate, the standard reaction mixture, in a volume of 2.7 mL, containing 1.5 mmol of 3-molpholinopropanesulf- onic acid-NaOH buffer, pH 7.5, and an appropriate amount of purified GGR, was added. Various amounts of Triton X-100 were added, as required. All solutions, except for the enzyme and detergent, were bubbled with N 2 gas to remove oxygen. To the mixture, 300 lmol of sodium dithi- onite, dissolved in 300 lLofN 2 -bubbled water, was added. The mixture was then incubated at 55 °C for 2 h, and the reaction mixture was extracted with 3 mL of 1-butanol sat- urated with H 2 O. The compounds in the butanol layer were enzymatically dephosphorylated by the method of Fujii et al. [31]. The resulting alcohols were extracted with n-pen- tane, and the pentane layer was completely evaporated. The residual lipids, or the authentic archaeol, were dissolved with 90 lL of anhydrous pyridine. After mixing the pyrid- ine solution with 10 lL of 1-trimehylsilylimidazole (Wako Pure Chemical Industries, Osaka, Japan) for more than 15 min at room temperature, part of the solution was sub- jected to a GC-MS analysis performed with a Hewlett- Packard 6890 gas chromatograph interfaced with a MStation JMS-700 mass spectrometry system (JEOL, Tokyo, Japan). A J&W DB TM- 1 capillary column (30 m · 0.25 mm, d.f. ¼ 0.25 lm) was used for the GC. Samples were injected at 70 °C, and the temperature was increased to 220 °C, at a rate of 50 °CÆmin )1 , and then to 320 °C, at 4 °CÆmin )1 , and held constant for 6 min. HOURSelium was used as the carrier gas. The electron impact-MS was performed at 70 eV with a mass range from m ⁄ z 50–750 and a cycle time 1 s in the positive ion mode. Acknowledgements This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We are grateful to Dr K. Ogura and Dr T. Koyama, Tohoku University, for providing farnesyl diphosphate. We wish to thank Dr C. Ohto, Toyota Motor Co., for donating IPP and dimethyl- ally diphosphate. We are grateful to S. Kitamura, Nagoya University, for his technical assistance with the GC-MS analyses. We also thank Dr Y. Sakagami and Dr M. Ojika for helpful discussions on mass spectro- metry. References 1 De Rosa M & Gambacorta A (1988) The lipids of archaebacteria. Prog Lipid Res 27, 153–175. 2 Gambacorta A, Trincone A, Nicolaus B, Lama L & De Rosa M (1994) Unique features of lipids of Archaea. Syst Appl Microbiol 16, 518–527. 3 Koga Y & Morii H (2005) Recent advances in struc- tural research on ether lipids from archaea including comparative and physiological aspects. Biosci Biotechnol Biochem 69, 2019–2034. 4 Nishihara M & Koga Y (1995) sn-Glycerol-1-phos- phate dehydrogenase in Methanobacterium thermoauto- trophicum: key enzyme in biosynthesis of the enantiomeric glycerophosphate backbone of ether phospholipids of archaebacteria. J Biochem (Tokyo) 117, 933–935. 5 Nishihara M & Koga Y (1997) Purification and proper- ties of sn-glycerol-1-phosphate dehydrogenase from Methanobacterium thermoautotrophicum: characteriza- tion of the biosynthetic enzyme for the enantiomeric glycerophosphate backbone of ether polar lipids of archaea. J Biochem (Tokyo) 122, 572–576. 6 Chen A & Poulter CD (1994) Isolation and characteri- zation of idsA: the gene for the short chain isoprenyl diphosphate synthase from Methanobacterium thermo- autotrophicum. Arch Biochem Biophys 314, 399–404. 7 Ohnuma S-i, Suzuki M & Nishino T (1994) Archaebac- terial ether-linked lipid biosynthetic gene. Expression cloning, sequencing, and characterization of geranylger- anyl-diphosphate synthase. J Biol Chem 269, 14792– 14797. 8 Tachibana A (1994) A novel prenyltransferase, farnesyl- geranyl diphosphate synthase, from the haloalkaliphilic archaeon, Natronobacterium pharaonis. FEBS Lett 341, 291–294. M. Murakami et al. Archaetidic acid-synthesizing archaeal reductase FEBS Journal 274 (2007) 805–814 ª 2007 The Authors Journal compilation ª 2007 FEBS 813 9 Tachibana A, Yano Y, Otani S, Nomura N, Sako Y & Taniguchi M (2000) Novel prenyltransferase gene encoding farnesylgeranyl diphosphate synthase from a hyperthermophilic archaeon, Aeropyrum pernix. Molecu- lar evolution with alteration in product specificity. Eur J Biochem 267, 321–328. 10 Chen A, Zhang D & Poulter CD (1993) (S)-Geranylger- anylglyceryl phosphate synthase. Purification and char- acterization of the first pathway-specific enzyme in archaebacterial membrane lipid biosynthesis. J Biol Chem 268, 21701–21705. 11 Soderberg T, Chen A & Poulter CD (2001) Geranylger- anylglyceryl phosphate synthase. Characterization of the recombinant enzyme from Methanobacterium thermoau- totrophicum. Biochemistry 40, 14847–14854. 12 Nemoto N, Oshima T & Yamagishi A (2003) Purifica- tion and characterization of geranylgeranylglyceryl phosphate synthase from a thermoacidophilic archaeon, Thermoplasma acidophilum. J Biochem (Tokyo) 133, 651–657. 13 Hemmi H, Shibuya K, Takahashi Y, Nakayama T & Nishino T (2004) (S)-2,3-Di-O-geranylgeranylglyceryl phosphate synthase from the thermoacidophilic archaeon Sulfolobus solfataricus. Molecular cloning and characterization of a membrane-intrinsic prenyltransfer- ase involved in the biosynthesis of archaeal ether-linked membrane lipids. J Biol Chem 279, 50197–50203. 14 Daiyasu H, Kuma K, Yokoi T, Morii H, Koga Y & Toh H (2005) A study of archaeal enzymes involved in polar lipid synthesis linking amino acid sequence infor- mation, genomic contexts and lipid composition. Archaea 1, 399–410. 15 Kon T, Nemoto N, Oshima T & Yamagishi A (2002) Effects of a squalene epoxidase inhibitor, terbinafine, on ether lipid biosyntheses in a thermoacidophilic archaeon, Thermoplasma acidophilum. J Bacteriol 184, 1395–1401. 16 Morii H, Nishihara M & Koga Y (2000) CTP: 2,3-di- O-geranylgeranyl-sn-glycero-1-phosphate cytidyltransfer- ase in the methanogenic archaeon Methanothermobacter thermoautotrophicus. J Biol Chem 275, 36568–36574. 17 Morii H & Koga Y (2003) CDP-2,3-Di-O-geranylgera- nyl-sn-glycerol: 1-serine O-archaetidyltransferase (archaetidylserine synthase) in the methanogenic archaeon Methanothermobacter thermautotrophicus . J Bacteriol 185, 1181–1189. 18 Nichols DS, Miller MR, Davies NW, Goodchild A, Raftery M & Cavicchioli R (2004) Cold adaptation in the antarctic archaeon Methanococcoides burtonii involves membrane lipid unsaturation. J Bacteriol 186, 8508–8515. 19 Gibson JA, Miller MR, Davies NW, Neill GP, Nichols DS & Volkman JK (2005) Unsaturated diether lipids in the psychrotrophic archaeon Halorubrum lacusprofundi. Syst Appl Microbiol 28, 19–26. 20 Collins MD & Jones D (1981) Distribution of isopre- noid quinone structural types in bacteria and their taxo- nomic implication. Microbiol Rev 45, 316–354. 21 Hemmi H, Takahashi Y, Shibuya K, Nakayama T & Nishino T (2005) Menaquinone-specific prenyl reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus. J Bacteriol 187, 1937–1944. 22 Keller Y, Bouvier F, d’Harlingue A & Camara B (1998) Metabolic compartmentation of plastid prenyllipid bio- synthesis – evidence for the involvement of a multifunc- tional geranylgeranyl reductase. Eur J Biochem 251, 413–417. 23 Tanaka R, Oster U, Kruse E, Rudiger W & Grimm B (1999) Reduced activity of geranylgeranyl reductase leads to loss of chlorophyll and tocopherol and to par- tially geranylgeranylated chlorophyll in transgenic tobacco plants expressing antisense RNA for geranyl- geranyl reductase. Plant Physiol 120, 695–704. 24 Addlesee HA, Gibson LC, Jensen PE & Hunter CN (1996) Cloning, sequencing and functional assignment of the chlorophyll biosynthesis gene, chlP,ofSynecho- cystis sp. PCC 6803. FEBS Lett 389, 126–130. 25 Teixidor P & Grimalt JO (1992) Gas chromatographic determination of isoprenoid alkylglycerol diethers in archaebacterial cultures and environmental samples. J Chromatogr A 607, 253–259. 26 Nishimura Y & Eguchi T (2006) Biosynthesis of archaeal membrane lipids: digeranylgeranylglyceropho- spholipid reductase of the thermoacidophilic archaeon Thermoplasma acidophilum. J Biochem (Tokyo) 139, 1073–1081. 27 Mitaku S, Hirokawa T & Tsuji T (2002) Amphiphilicity index of polar amino acids as an aid in the characteriza- tion of amino acid preference at membrane–water inter- faces. Bioinformatics 18 , 608–616. 28 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 29 Whitby LG (1953) A new method for preparing flavin- adenine dinucleotide. Biochem J 54, 437–442. 30 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein util- izing the principle of protein-dye binding. Anal Biochem 72, 248–254. 31 Fujii H, Koyama T & Ogura K (1982) Efficient enzy- matic hydrolysis of polyprenyl pyrophosphates. Biochim Biophys Acta 712, 716–718. 32 Bligh EG & Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37, 911–917. 33 Demizu K, Ohtsubo S, Kohno S, Miura I, Nishihara M & Koga Y (1992) Quantitative determination of methano- genic cells based on analysis of ether-linked glycerolipids by high-performance liquid chromatography. J Ferment Bioeng 73, 135–139. Archaetidic acid-synthesizing archaeal reductase M. Murakami et al. 814 FEBS Journal 274 (2007) 805–814 ª 2007 The Authors Journal compilation ª 2007 FEBS . Geranylgeranyl reductase involved in the biosynthesis of archaeal membrane lipids in the hyperthermophilic archaeon Archaeoglobus fulgidus Motomichi Murakami 1 ,. These facts strongly suggest the involvement of the enzyme, GGR, in the biosynthesis of membrane lipids in the hyper- thermophilic archaeon A. fulgidus, as predicted in the previous publication from the thermoacidophilic archaeon Sulfolobus solfataricus. Molecular cloning and characterization of a membrane- intrinsic prenyltransfer- ase involved in the biosynthesis of archaeal ether-linked membrane