Four genes that encode the homologues of plant geranylgeranyl reductase were isolated from a hyperthermophilic archaeon Archaeoglobus fulgidus, which produces menaquinone with a fully saturated heptaprenyl side chain, menaquinone-7(14H). The recombinant expression of one of the homologues in Escherichia coli led to a distinct change in the quinone profile of the host cells, although the homologue is the most distantly related to the geranylgeranyl reductase. The new compounds found in the profile had successively longer elution times than those of ordinary quinones from E. coli, i.e., menaquinone-8 and ubiquinone-8, in high-performance liquid chromatography on a reversed-phase column. Structural analyses of the new compounds by electron impactmass spectrometry indicated that their molecular masses progressively increase relative to the ordinary quinones at a rate of 2 U but that they still contain quinone head structures, strongly suggesting that the compounds are quinones with partially saturated prenyl side chains. In vitro assays with dithionite as the reducing agent showed that the prenyl reductase is highly specific for menaquinone-7, rather than ubiquinone-8 and prenyl diphosphates. This novel enzyme noncovalently binds flavin adenine dinucleotide, similar to geranylgeranyl reductase, but was not able to utilize NAD(P)H as the electron donor, unlike the plant homologue.

Complete saturation of the geranylgeranyl groups of biosynthetic intermediates 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 hyperthermophilic 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 Bacteriol187, 1937–1944], was recombinantly expressed and purified, and its geranylgeranyl reductase activity was examined. The radio HPLC analysis indicated that the flavoenzyme, which binds FAD noncovalently, 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‐geranylgeranylglyceryl 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‐phytanylglyceryl 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 homologues), the enzyme reaction did not proceed.

The core structure of membrane lipids of archaea have some unique properties that permit archaea to be distinguished from the others, i.e. bacteria and eukaryotes. (S)-2,3-Di-O-geranylgeranylglyceryl phosphate synthase, which catalyzes the transfer of a geranylgeranyl group from geranylgeranyl diphosphate to (S)-3-O-geranylgeranylglyceryl phosphate, is involved in the biosynthesis of archaeal membrane lipids. Enzymes of the UbiA prenyltransferase family are known to catalyze the transfer of a prenyl group to various acceptors with hydrophobic ring structures in the biosynthesis of respiratory quinones, hemes, chlorophylls, vitamin E, and shikonin. The thermoacidophilic archaeon Sulfolobus solfataricus was found to encode three homologues of UbiA prenyltransferase in its genome. One of the homologues encoded by SSO0583 was expressed in Escherichia coli, purified, and characterized. Radio-assay and mass spectrometry analysis data indicated that the enzyme specifically catalyzes the biosynthesis of (S)-2,3-di-O-geranylgeranylglyceryl phosphate. The fact that the orthologues of the enzyme are encoded in almost all archaeal genomes clearly indicates the importance of their functions. A phylogenetic tree constructed using the amino acid sequences of some typical members of the UbiA prenyltransferase family and their homologues from S. solfataricus suggests that the two other S. solfataricus homologues, excluding the (S)-2,3-di-Ogeranylgeranylglyceryl phosphate synthase, are involved in the production of respiratory quinone and heme, respectively. We propose here that archaeal prenyltransferases involved in membrane lipid biosynthesis might be prototypes of the protein family and that archaea might have played an important role in the molecular evolution of prenyltransferases.The structures of membrane lipids have some interesting and remarkable properties that enable us to distinguish archaea from other organisms, i.e. eukaryotes and bacteria (1) (Fig. 1). Although the archaeal "diether" membrane lipids are homologues of glycerolipids in other organisms, they differ with respect to the following features: 1) The hydrocarbon moieties of the archaeal lipids are fully reduced C 20 or C 25 prenyl groups, whereas the ordinary glycerolipids contain linear acyl groups. 2) The alkyl groups are attached to glycerol via an ether bond in archaeal lipids, while glycerol and the acyl chains are ester-bonded in the bacterial and eukaryotic glycerolipids.3) The two groups of membrane lipids have opposite chiralities at their glycerol moieties; in short, the glycerol moieties of the archaeal and other glycerolipids are sn-2,3-di-O-alkylated and sn-1,2-di-O-acylated, respectively. Moreover, the existence of circular "tetraether" lipids, which are synthesized from two molecules of diether lipids in methanogenic and thermophilic archaea, emphasizes the uniqueness of the archaeal membrane lipids.The biosynthesis of the core structure of archaeal membrane lipids has been studied to date (Fig. 2). The genes of (all-E) geranylgeranyl diphos...

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In order to determine the enantioselectivity of (S)-2,3-di-O-geranylgeranylglyceryl phosphate synthase (DGGGPS) from the thermoacidophilic archaeon Sulfolobus solfataricus, we developed an efficient enantioselective route to the enantiomeric geranylgeranylglyceryl phosphates (R)-GGGP and (S)-GGGP. Previous routes to these substrates involved enzymatic conversions, due to the lability of the polyprenyl chains towards common phosphorylation reaction conditions. The synthesis described herein employs a mild trimethyl phosphite/carbon tetrabromide oxidative phosphorylation to circumvent this problem. In contrast to previous results suggesting that only (S)-GGGP can act as the prenyl-acceptor substrate, both (R)-GGGP and (S)-GGGP were found to be substrates for DGGGPS.All living organisms have been classified into three primary kingdoms: archaebacteria, eubacteria, and eukaryotes. 1 While the membrane lipids in eubacteria and eukaryotes are composed of glyceryl esters of fatty acids, archaebacterial membrane lipids contain isopranyl glyceryl ethers. 2,3 The core structures of archaeal membrane lipids, including diether and bipolar tetraether lipids, contain fully-reduced C 20 or C 25 prenyl groups. 4,5 Biosynthesis of the isoprene moieties in the core lipids follows the mevalonate pathway used also by eubacteria and eukaryotes. 6-8 The prenyl transfer reactions responsible for building the isoprenoid diphosphates, e.g., geranylgeranyl diphosphate (GGPP), are catalyzed by a family of prenyltransferases. 9-12 As shown in Figure 1, the biogenesis of the core-structure of the archaeal membrane lipids starts with the prenyl transfer reaction catalyzed by (S)-3-Ogeranylgeranylglyceryl phosphate [(S)-GGGP] synthase, which selectively uses (S)-glyceryl phosphate as the prenyl acceptor (Fig. 1). Then, the product is utilized as the presumed acceptor substrate for the biosynthesis of (S)-2,3-Di-O-geranylgeranylglyceryl phosphate (DGGGP), an advanced intermediate of archaeal membrane lipids. 13To date, only an enzyme-assisted synthesis of (S)-GGGP has been reported. 11 An enantiospecific chemical synthesis of both individual enantiomers of GGGP was required to validate this biosynthetic hypothesis, due in part to the acid-sensitive geranylgeranyl group of GGGP. In order to fully characterize the substrate selectivity of DGGGP synthase (DGGGPS), we developed a mild and effective route to the two GGGP enantiomers. GGGP indeed posed significant challenges, and the biological results with the enantiomers were unexpected.The synthesis of (S)-GGGP (8) is summarized in Scheme 1. Treatment of the (2E,6E,10E) -geranylgeraniol 1 with Ph 3 P/CBr 4 at 25 °C afforded geranylgeranyl bromide 2. 14 Next, (S)-solketal was alkylated with geranylgeranyl bromide by using KH as base, to give ether 3 in 73% yield. 11 The reported HCl/THF method to remove the acetonide 13 resulted in a complex mixture containing the desired product in low yield. The desired diol 4 was thus prepared in 75% yield using p-TsOH in methanol. 15,16 In order to obta...