Molecular cloning and mutational analysis of the <i>ddsA</i> gene encoding decaprenyl diphosphate synthase from <i>Gluconobacter suboxydans</i>

Okada, Kazunori; Kainou, Tomohiro; Tanaka, Katsunori; Nakagawa, Tsuyoshi; Matsuda, Hideyuki; Kawamukai, Makoto

doi:10.1046/j.1432-1327.1998.2550052.x

Cited by 82 publications

(65 citation statements)

References 4 publications

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“…Ubiquinone Extraction and Measurement-Ubiquinone was extracted as previously described (34). The crude extract of UQ was analyzed by normal-phase TLC with authentic standard UQ-10.…”

Section: Methodsmentioning

confidence: 99%

A Solanesyl-diphosphate Synthase Localizes in Glycosomes of Trypanosoma cruzi

Ferella

Montalvetti

Rohloff

et al. 2006

Journal of Biological Chemistry

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We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass ϳ 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target.

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“…Ubiquinone Extraction and Measurement-Ubiquinone was extracted as previously described (34). The crude extract of UQ was analyzed by normal-phase TLC with authentic standard UQ-10.…”

Section: Methodsmentioning

confidence: 99%

A Solanesyl-diphosphate Synthase Localizes in Glycosomes of Trypanosoma cruzi

Ferella

Montalvetti

Rohloff

et al. 2006

Journal of Biological Chemistry

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“…To determine this we measured the adult lifespan of N2 worms fed E. coli engineered to produce Q 7 , Q 8 , Q 9 or Q 10 ( Table 1). The designated Q n isoform is the predominant species in each of these E. coli strains (Okada et al, 1996(Okada et al, , 1997(Okada et al, , 1998Jonassen et al, 2003). In order to determine whether the Q isoforms produced might be altered following the extended incubations required for lifespan determination, we determined the quinone content for each of these E. coli strains recovered from NGM plates following incubation conditions that simulate a lifespan experiment.…”

Section: Diets Containing Q 8 Q 9 Q 10 Isoforms Do Not Influence mentioning

confidence: 99%

Altered bacterial metabolism, not coenzyme Q content, is responsible for the lifespan extension in Caenorhabditis elegans fed an Escherichia coli diet lacking coenzyme Q

et al. 2008

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SummaryCoenzyme Q n is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. Caenorhabditis elegans fed Escherichia coli devoid of Q 8 have a significant lifespan extension when compared to C. elegans fed a standard 'Q-replete' E. coli diet. Here we examine possible mechanisms for the lifespan extension caused by the Q-less E. coli diet. A bioassay for Q uptake shows that a water-soluble formulation of Q 10 is effectively taken up by both clk-1 mutant and wild-type nematodes, but does not reverse lifespan extension mediated by the Q-less E. coli diet, indicating that lifespan extension is not due to the absence of dietary Q per se. The enhanced longevity mediated by the Q-less E. coli diet cannot be attributed to dietary restriction, different Qn isoforms, reduced pathogenesis or slowed growth of the Q-less E. coli , and in fact requires E. coli viability. Q-less E. coli have defects in respiratory metabolism. C. elegans fed Q-replete E. coli mutants with similarly impaired respiratory metabolism due to defects in complex V also show a pronounced lifespan extension, although not as dramatic as those fed the respiratory deficient Q-less E. coli diet. The data suggest that feeding respiratory incompetent E. coli , whether Q-less or Q-replete, produces a robust life extension in wild-type C. elegans . We believe that the fermentation-based metabolism of the E. coli diet is an important parameter of C. elegans longevity.

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“…A protocol to assay isoprenoid synthase activity was developed from a combination of the procedures of Cunillera et al (1996), Okada et al (1998Okada et al ( , 2000, and Fujii et al (1982). Briefly, 100 mL reaction mixtures containing cell-free extract (100 mg of protein) in 100 mM potassium phosphate, pH 7.4, 1 mM EDTA, 1 mM 2-mercaptoethanol, 5 mM MgCl 2 , 2.2 nmol of DMAPP, and 4.4 nmol of [4-14 C]IPP were incubated for 1 h at 37°C.…”

Section: In Vitro Isoprenoid Synthase Assaysmentioning

confidence: 99%

Maize cDNAs Expressed in Endosperm Encode Functional Farnesyl Diphosphate Synthase with Geranylgeranyl Diphosphate Synthase Activity

et al. 2006

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Isoprenoids are the most diverse and abundant group of natural products. In plants, farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors to many isoprenoids having essential functions. Terpenoids and sterols are derived from FPP, whereas gibberellins, carotenoids, casbenes, taxenes, and others originate from GGPP. The corresponding synthases (FPP synthase [FPPS] and GGPP synthase [GGPPS]) catalyze, respectively, the addition of two and three isopentenyl diphosphate molecules to dimethylallyl diphosphate. Maize (Zea mays L. cv B73) endosperm cDNAs encoding isoprenoid synthases were isolated by functional complementation of Escherichia coli cells carrying a bacterial gene cluster encoding all pathway enzymes needed for carotenoid biosynthesis, except for GGPPS. This approach indicated that the maize gene products were functional GGPPS enzymes. Yet, the predicted enzyme sequences revealed FPPS motifs and homology with FPPS enzymes. In vitro assays demonstrated that indeed these maize enzymes produced both FPP and GGPP and that the N-terminal sequence affected the ratio of FPP to GGPP. Their functionality in E. coli demonstrated that these maize enzymes can be coupled with a metabolon to provide isoprenoid substrates for pathway use, and suggests that enzyme bifunctionality can be harnessed. The maize cDNAs are encoded by a small gene family whose transcripts are prevalent in endosperm beginning mid development. These maize cDNAs will be valuable tools for assessing the critical structural properties determining prenyl transferase specificity and in metabolic engineering of isoprenoid pathways, especially in cereal crops.

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Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl diphosphate synthase from Gluconobacter suboxydans

Cited by 82 publications

References 4 publications

A Solanesyl-diphosphate Synthase Localizes in Glycosomes of Trypanosoma cruzi

A Solanesyl-diphosphate Synthase Localizes in Glycosomes of Trypanosoma cruzi

Altered bacterial metabolism, not coenzyme Q content, is responsible for the lifespan extension in Caenorhabditis elegans fed an Escherichia coli diet lacking coenzyme Q

Maize cDNAs Expressed in Endosperm Encode Functional Farnesyl Diphosphate Synthase with Geranylgeranyl Diphosphate Synthase Activity

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