Ubiquinone (coenzyme Q) functions as an electron transporter in aerobic respiration and oxidative phosphorylation in the respiratory chain [1]. In addition, many reports suggest that ubiquinone also functions as a lipid-soluble antioxidant in cellular biomembranes, scavenging reactive oxygen species [2][3][4][5]. Indeed, several studies using yeast strains that do not produce ubiquinone suggest that an in vitro function of ubiquinone is to protect against oxidants [6,7]. Another phenotype of such ubiquinone-deficient fission yeast is that they generate high levels of hydrogen sulfide [8][9][10]. As Schizosaccharomyces pombe and other eukaryotes are known to carry sulfide-ubiquinone reductase, an enzyme that oxidizes sulfide via ubiquinone [11], it has been suggested that ubiquinone is linked to sulfide metabolism in many organisms. In addition, it was The isoprenoid chain of ubiquinone (Q) is determined by trans-polyprenyl diphosphate synthase in micro-organisms and presumably in mammals. Because mice and humans produce Q 9 and Q 10 , they are expected to possess solanesyl and decaprenyl diphosphate synthases as the determining enzyme for a type of ubiquinone. Here we show that murine and human solanesyl and decaprenyl diphosphate synthases are heterotetramers composed of newly characterized hDPS1 (mSPS1) and hDLP1 (mDLP1), which have been identified as orthologs of Schizosaccharomyces pombe Dps1 and Dlp1, respectively. Whereas hDPS1 or mSPS1 can complement the S. pombe dps1 disruptant, neither hDLP1 nor mDLP1 could complement the S. pombe dLp1 disruptant. Thus, only hDPS1 and mSPS1 are functional orthologs of SpDps1. Escherichia coli was engineered to express murine and human SpDps1 and ⁄ or SpDlp1 homologs and their ubiquinone types were determined. Whereas transformants expressing a single component produced only Q 8 of E. coli origin, double transformants expressing mSPS1 and mDLP1 or hDPS1 and hDLP1 produced Q 9 or Q 10 , respectively, and an in vitro activity of solanesyl or decaprenyl diphosphate synthase was verified. The complex size of the human and murine long-chain transprenyl diphosphate synthases, as estimated by gel-filtration chromatography, indicates that they consist of heterotetramers. Expression in E. coli of heterologous combinations, namely, mSPS1 and hDLP1 or hDPS1 and mDLP1, generated both Q 9 and Q 10 , indicating both components are involved in determining the ubiquinone side chain. Thus, we identified the components of the enzymes that determine the side chain of ubiquinone in mammals and they resembles the S. pombe, but not plant or Saccharomyces cerevisiae, type of enzyme.Abbreviations
Ubiquinone is an essential component of the electron transfer system in both prokaryotes and eukaryotes and is synthesized from chorismate and polyprenyl diphosphate by eight steps. p-Hydroxybenzoate (PHB) polyprenyl diphosphate transferase catalyzes the condensation of PHB and polyprenyl diphosphate in ubiquinone biosynthesis. We isolated the gene (designated ppt1) encoding PHB polyprenyl diphosphate transferase from Schizosaccharomyces pombe and constructed a strain with a disrupted ppt1 gene. This strain could not grow on minimal medium supplemented with glucose. Expression of COQ2 from Saccharomyces cerevisiae in the defective S. pombe strain restored growth and enabled the cells to produce ubiquinone-10, indicating that COQ2 and ppt1 are functional homologs. The ppt1-deficient strain required supplementation with antioxidants, such as cysteine, glutathione, and ␣-tocopherol, to grow on minimal medium. This suggests that ubiquinone can act as an antioxidant, a premise supported by our observation that the ppt1-deficient strain is sensitive to H 2 O 2 and Cu 2؉. Interestingly, we also found that the ppt1-deficient strain produced a significant amount of H 2 S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S. pombe. Ppt1-green fluorescent protein fusion proteins localized to the mitochondria, indicating that ubiquinone biosynthesis occurs in the mitochondria in S. pombe. Thus, analysis of the phenotypes of S. pombe strains deficient in ubiquinone production clearly demonstrates that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system. Ubiquinone is known to be an electron transporter in the respiratory chain in prokaryotes and eukaryotes. It varies among organisms in the length of its isoprenoid side chain. For example, Saccharomyces cerevisiae uses ubiquinone-6 (UQ-6), Escherichia coli uses UQ-8, and Schizosaccharomyces pombe uses UQ-10 (9, 16, 37). It has been shown that the type of ubiquinone in organisms is determined by the polyprenyl diphosphate synthase enzyme, which catalyzes the condensation reaction of isopentenyl diphosphate with allylic diphosphate to give a defined length of the isoprenoid (22, 26). When polyprenyl diphosphate synthase genes from other sources were expressed in S. cerevisiae and E. coli, the ubiquinone generated was of the same type as that expressed in the donor organism (22)(23)(24)(25)(26). By this method, we successfully produced various ubiquinone species (UQ-5 to UQ-10) in the S. cerevisiae COQ1 mutant (22), which in turn indicates that p-hydroxybenzoate (PHB) polyprenyl diphosphate transferase, which catalyzes the condensation reaction between the isoprenoid side chain and PHB, has a broad substrate specificity. This is supported by consistent observations showing that purified PHB polyprenyl diphosphate transferases from Pseudomonas putida (12, 40) and E. coli (17) have fairly wide substrate specificities in terms of polyprenols. In contrast, PHB g...
The analysis of the structure and function of long chainproducing polyprenyl diphosphate synthase, which synthesizes the side chain of ubiquinone, has largely focused on the prokaryotic enzymes, and little is known about the eukaryotic counterparts. Here we show that decaprenyl diphosphate synthase from Schizosaccharomyces pombe is comprised of a novel protein named Dlp1 acting in partnership with Dps1. Dps1 is highly homologous to other prenyl diphosphate synthases but Dlp1 shares only weak homology with Dps1. We showed that the two proteins must be present simultaneously in Escherichia coli transformants before ubiquinone-10, which is produced by S. pombe but not by E. coli, is generated. Furthermore, the two proteins were shown to form a heterotetrameric complex. This is unlike the prokaryotic counterparts, which are homodimers. The deletion mutant of dlp1 lacked the enzymatic activity of decaprenyl diphosphate synthase, did not produce ubiquinone-10 and had the typical ubiquinone-deficient S. pombe phenotypes, namely hypersensitivity to hydrogen peroxide, the need for antioxidants for growth on minimal medium and an elevated production of H 2 S. Both the dps1 (formerly dps) and dlp1 mutants could generate ubiquinone when they were transformed with a bacterial decaprenyl diphosphate synthase, which functions in its host as a homodimer. This indicates that both dps1 and dlp1 are required for the S. pombe enzymatic activity. Thus, decaprenyl diphosphate from a eukaryotic origin has a heterotetrameric structure that is not found in prokaryotes.
Ubiquinone (UQ), an important component of the electron transfer system, is constituted of a quinone structure and a side chain isoprenoid. The side chain length of UQ differs between microorganisms, and this difference has been used for taxonomic study. In this study, we have addressed the importance of the length of the side chain of UQ for cells, and examined the effect of chain length by producing UQs with isoprenoid chain lengths between 5 and 10 in Saccharomyces cerevisiae. To make the different UQ species, different types of prenyl diphosphate synthases were expressed in a S. cerevisiae COQ1 mutant defective for hexaprenyl diphosphate synthesis. As a result, we found that the original species of UQ (in this case UQ-6) had maximum functionality. However, we found that other species of UQ could replace UQ-6. Thus a broad spectrum of different UQ species are biologically functional in yeast cells, although cells seem to display a preference for their own particular type of UQ.z 1998 Federation of European Biochemical Societies.
Decaprenyl diphosphate (decaprenyl-PP) synthase catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce decaprenyl-PP, which is used for the side chain of ubiquinone (Q)-10. We have cloned the synthase gene, designated ddsA, from Gluconobacter suboxydans and expressed it in Escherichia coli. Sequence analysis revealed the presence of an ORF of 948 bp capable of encoding a 33898-Da polypeptide that displays high similarity (30Ϫ50 %) to other prenyl diphosphate synthases. Expression of the ddsA gene complemented the lethality resulting from a defect in the octaprenyl diphosphate synthase gene of E. coli and produced Q-10, indicating that Q-10 can substitute for the function of Q-8. The His-tagged DdsA protein was purified to characterize its enzymatic properties. This enzyme required detergent (0.05 % Triton X-100) and 10 mM Mg 2ϩ , for full activity. The Michaelis constants for geranyl diphosphate, all-E-farnesyl diphosphate and all-E-geranylgeranyl diphosphate were 7.00, 0.50 and 0.32 µM, respectively. Nine single-amino-acid substitutions were introduced upstream of conserved region II or VI. Most of the mutants showed a considerable decrease in catalytic activity or shortening of the ultimate chain length. However, the A70G mutant produced a longer-chain-length product than wild-type decaprenyl-PP synthase, and the A70Y mutant completely abolished the decaprenyl-PP synthase function, indicating that Ala70 is important for enzyme activity and the determination of the chain-length properties of DdsA.Keywords : isoprenoid ; polyprenyl diphosphate synthase; Gluconobacter suboxydans; ubiquinone-10.Several genes for prenyl diphosphate synthases that synthePrenyl diphosphate synthase catalyzes the condensation of size long-chain isoprenoids from bacteria and yeasts have been isopentenyl diphosphate with allylic diphosphate to give isocloned and characterized. These include the hexaprenyl diphosprenoids of defined length, which are used as precursors in the phate synthase (Coq1) gene from S. cerevisiae [7], the heptasynthesis of steroids, carotenoids, dolichol, prenyl quinones, and prenyl diphosphate synthase genes from Bacillus subtilis [8,9] [2, 3]. The species of isoprenoid quinones, e.g. ubiqui-[3], and the decaprenyl diphosphate (decaprenyl-PP) synthase none (Q), menaquinone and plastoquinone, and its chain length gene from Schizosaccharomyces pombe [12]. are important criteria for the taxonomic study of microorganismsThe length of the product is precisely defined by the nature [4]. However, we have shown that the length of the side chain of the prenyl diphosphate synthase engaged in the reaction. of Q is not a critical factor for electron transfer in microorgaAmino-acid-sequence comparisons of polyprenyl diphosphate nisms, and that chain length is determined solely by the type of synthases have revealed the presence of seven highly conserved polyprenyl diphosphate synthase [5,6]. For example, Escheriregions including two aspartate-rich domains (domain II and dochia...
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