No abstract

Schizosaccharomyces pombe produces ubiquinone-10 whose side chain is thought to be provided by the product generated by decaprenyl diphosphate synthase. To understand the mechanism of ubiquinone biosynthesis in S. pombe, we have cloned the gene encoding decaprenyl diphosphate synthase by the combination of PCR amplification of the fragment and subsequent library screening. The determined DNA sequence of the cloned gene, called dps, revealed that the dps gene encodes a 378-amino-acid protein that has the typical conserved regions observed in many polyprenyl diphosphate synthases. Computer-assisted homology search indicated that Dps is 45 and 33% identical with hexaprenyl diphosphate synthase from Saccharomyces cerevisiae and octaprenyl diphosphate synthase from Escherichia coli, respectively. An S. pombe dps-deficient strain was constructed. This disruptant was not able to synthesize ubiquinone and had no detectable decaprenyl diphosphate synthase activity, indicating that the dps gene is unique and responsible for ubiquinone biosynthesis. The S. pombe dps-deficient strain could not grow on either rich medium supplemented with glycerol or on minimal medium supplemented with glucose. The dps-deficient strain required cysteine or glutathione for full growth on the minimal medium. In addition, the dps-deficient strain is more sensitive to H2O2 and Cu2+ than the wild type. These results suggests a role of ubiquinone as an antioxidant in fission yeast cells.

Different organisms produce different species of isoprenoid quinones, each with its own distinctive length. These differences in length are commonly exploited in microbial classification. The side chain length of quinone is determined by the nature of the polyprenyl diphosphate synthase that catalyzes the reaction. To determine if the side chain length of ubiquinone (UQ) has any distinct role to play in the metabolism of the cells in which it is found, we cloned the solanesyl diphosphate synthase gene (sdsA) from Rhodobacter capsulatus SB1003 and expressed it in Escherichia coli and Saccharomyces cerevisiae. Sequence analysis revealed that the sdsA gene encodes a 325-amino-acid protein which has similarity (27 to 40%) with other prenyl diphosphate synthases. Expression of the sdsA gene complemented a defect in the octaprenyl diphosphate synthase gene of E. coli and the nonrespiratory phenotype resulting from a defect in the hexaprenyl diphosphate synthase gene of S. cerevisiae. Both E. coli and S. cerevisiae expressing the sdsA gene mainly produced solanesyl diphosphate, which resulted in the synthesis of UQ-9 without any noticeable effect on the growth of the cells. Thus, it appears that UQ-9 can replace the function of UQ-8 in E. coli and UQ-6 in S. cerevisiae. Taken together with previous results, the results described here imply that the side chain length of UQ is not a critical factor for the survival of microorganisms.Prenyl diphosphate synthase catalyzes the condensation of isopentenyl diphosphate with allylic diphosphate to give isoprenoids of defined length, which are used as precursors in the synthesis of steroids, carotenoids, dolichol, prenyl quinones, and prenylated proteins (7) (Fig. 1). Short-chain isoprenoids (C 10 to C 20 ) are used as precursors for many different products, while long-chain isoprenoids (C 30 to C 50 ) are only used for the side chains of isoprenoid quinones (Fig. 1). For this reason, polyprenyl diphosphate synthases which produce isoprenoid chains longer than C 30 have not been studied well, compared to prenyl diphosphate synthases that produce short-chain isoprenoids (C 10 to C 20 ). However, species of quinones, e.g., ubiquinone (UQ), menaquinone, and plastoquinone, and the chain lengths of quinones are important criteria for the taxonomic study of microorganisms (3).It was shown that the side chain length of UQ is determined by polyprenyl diphosphate synthase (15, 16). So, the structure and function of individual prenyl diphosphate synthases provide useful information about the species of quinone produced in a given organism. Several genes for prenyl diphosphate synthase that synthesize long-chain isoprenoids from bacteria and yeasts have been cloned and characterized. These include the hexaprenyl diphosphate synthase (Coq1) gene from Saccharomyces cerevisiae (2), the heptaprenyl diphosphate synthase genes from Bacillus subtilis (28,29) and Bacillus stearothermophilus (12), the octaprenyl diphosphate synthase (IspB) gene from Escherichia coli (1), and the decaprenyl diphosph...