ubiquinone-9 and solanesyl-pyrophosphate

Coenzyme Q(10) (CoQ(10)), like other CoQs of various organisms, plays indispensable roles not only in energy generation but also in several other processes required for cells' survival. In this study, a gene encoding for a decaprenyl diphosphate synthase (Rsdds) was cloned from Rhodobacter sphaeroides in Escherichia coli. The in vivo catalytic activity and product specificity of Rsdds were compared with those of a counterpart enzyme from Agrobacterium tumefaciens (Atdds) in E. coli as a heterologous host. In contrast with Atdds, Rsdds showed lower catalytic activity but higher product specificity for CoQ(10) production, as indicated by the amount of CoQ(9) formation. The higher product specificity of Rsdds was also confirmed by utilizing both Rsdds and Atdds for in vitro synthesis of polyprenyl diphosphates. Thin layer chromatography indicated that the Rsdds enzyme resulted in relatively much less solanesyl diphosphate formation. The purified Rsdds catalyzed the addition of isopentenyl diphosphate to dimethyl allyl diphosphate, geranyl diphosphate, omega,E,E-farnesyl diphosphate (FPP), and omega,E,E,E-geranylgeranyl diphosphate as priming substrates. The kinetic parameters of V (max) (pmol/min), K (M) (microM), k (cat) (1/min), and k (cat) /K (M) of the enzyme using FPP as the most appropriate substrate were determined to be 264.6, 13.1, 8.8, and 0.67, respectively.

Topics: Agrobacterium tumefaciens; Alkyl and Aryl Transferases; Chromatography, Thin Layer; Cloning, Molecular; Coenzymes; Diphosphates; Diterpenes; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Hemiterpenes; Kinetics; Molecular Sequence Data; Organophosphorus Compounds; Polyisoprenyl Phosphates; Rhodobacter sphaeroides; Sequence Analysis, DNA; Sesquiterpenes; Substrate Specificity; Ubiquinone

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.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Cloning, Molecular; Dimethylallyltranstransferase; Escherichia coli; Genetic Complementation Test; Molecular Sequence Data; Polyisoprenyl Phosphates; Polymerase Chain Reaction; Rhodobacter capsulatus; Saccharomyces cerevisiae; Sequence Alignment; Transferases; Ubiquinone