ubiquinone and 4-hydroxybenzoic-acid

Coenzyme Q (ubiquinone or CoQ) is an essential lipid that plays a role in mitochondrial respiratory electron transport and serves as an important antioxidant. In human and yeast cells, CoQ synthesis derives from aromatic ring precursors and the isoprene biosynthetic pathway.

Topics: Ataxia; Genes, Fungal; Genome, Human; Humans; Mitochondrial Diseases; Mitochondrial Proteins; Models, Biological; Muscle Weakness; Mutation; Parabens; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquinone

Topics: Alkyl and Aryl Transferases; Animals; Cells, Cultured; Chemical Phenomena; Chemistry; Chromans; Eukaryotic Cells; Humans; Hydroxybenzoates; Methyltransferases; Mixed Function Oxygenases; Parabens; Prokaryotic Cells; Transferases; Ubiquinone; Vitamin E; Yeasts

Topics: Chromatography, Liquid; NAD; NADH, NADPH Oxidoreductases; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Tandem Mass Spectrometry; Ubiquinone

Ubiquinone (UQ) is a lipophilic electron carrier that functions in the respiratory and photosynthetic electron transfer chains of proteobacteria and eukaryotes. Bacterial UQ biosynthesis is well studied in the gammaproteobacterium Escherichia coli, in which most bacterial UQ-biosynthetic enzymes have been identified. However, these enzymes are not always conserved among UQ-containing bacteria. In particular, the alphaproteobacterial UQ biosynthesis pathways contain many uncharacterized steps with unknown features. In this work, we identified in the alphaproteobacterium Rhodobacter capsulatus a new decarboxylative hydroxylase and named it UbiN. Remarkably, the UbiN sequence is more similar to a salicylate hydroxylase than the conventional flavin-containing UQ-biosynthetic monooxygenases. Under aerobic conditions, R. capsulatus ΔubiN mutant cells accumulate 3-decaprenylphenol, which is a UQ-biosynthetic intermediate. In addition, 3-decaprenyl-4-hydroxybenzoic acid, which is the substrate of UQ-biosynthetic decarboxylase UbiD, also accumulates in ΔubiN cells under aerobic conditions. Considering that the R. capsulatus ΔubiD-X double mutant strain (UbiX produces a prenylated FMN required for UbiD) grows as a wild-type strain under aerobic conditions, these results indicate that UbiN catalyzes the aerobic decarboxylative hydroxylation of 3-decaprenyl-4-hydroxybenzoic acid. This is the first example of the involvement of decarboxylative hydroxylation in ubiquinone biosynthesis. This finding suggests that the C1 hydroxylation reaction is, at least in R. capsulatus, the first step among the three hydroxylation steps involved in UQ biosynthesis. Although the C5 hydroxylation reaction is often considered to be the first hydroxylation step in bacterial UQ biosynthesis, it appears that the R. capsulatus pathway is more similar to that found in mammalians.

Topics: Animals; Escherichia coli; Mammals; Mixed Function Oxygenases; Rhodobacter capsulatus; Ubiquinone

Antrodia cinnamomea, an endemic fungus species of Taiwan, has long been used as a luxurious dietary supplement to enhance liver functions and as a remedy for various cancers. Antroquinonol (AQ), identified from the mycelium of A. cinnamomea, is currently in phase II clinical trials in the USA and Taiwan for the treatment of non-small-cell lung cancer. In the previous studies, we have demonstrated that AQ and 4-acetylantroquinonol B (4-AAQB) utilize orsellinic acid, via polyketide pathway, as the ring precursor, and their biosynthetic sequences are similar to those of coenzyme Q. In order to test 4-hydroxybenzoic acid (4-HBA), synthesized via shikimate pathway, is the ring precursor of AQ analogs, the strategy of metabolic labeling with stable isotopes was applied in this study. Here we have confirmed that 4-HBA serves as the ring precursor for AQ but not a precursor of 4-AAQB. Experimental results indicated that A. cinnamomea preferentially utilizes endogenous 4-HBA via shikimate pathway for AQ biosynthesis. Exogenous tyrosine and phenylalanine can be utilized for AQ biosynthesis when shikimate pathway is blocked by glyphosate. The benzoquinone ring of 4-AAQB is synthesized only via polyketide pathway, but that of AQ is synthesized via both polyketide pathway and shikimate pathway. The precursor-products relationships diagram of AQ and 4-AAQB in A. cinnamomea are proposed based on the experimental findings.

Topics: Antrodia; Molecular Structure; Parabens; Ubiquinone

Muconic acid is a platform chemical and an important intermediate in the degradation process of a series of aromatic compounds. Herein, a plasmid-free synthetic pathway in Pseudomonas chlororaphis HT66 is constructed for the enhanced biosynthesis of muconic acid by connecting endogenous ubiquinone biosynthesis pathway with protocatechuate degradation pathway using chromosomal integration. Instead of being plasmid and inducer dependent, the engineered strains could steadily produce the high muconic acid using glycerol as a carbon source. The engineered strain HT66-MA6 achieved a 3376 mg/L muconic acid production with a yield of 187.56 mg/g glycerol via the following strategies: (1) block muconic acid conversion and enhance muconic acid efflux pumping with phenazine biosynthesis cluster; (2) increase the muconic acid precursors supply through overexpressing the rate-limiting step, and (3) coexpress the "3-dehydroshikimate-derived" route in parallel with the "4-hydroxybenzoic acid-derived" route to create a synthetic "metabolic funnel". Finally, on the basis of the glycerol feeding strategies, the muconic acid yield reached 0.122 mol/mol glycerol. The results suggest that the construction of synthetic pathway with a plasmid-free strategy in P. chlororaphis displays a high biotechnological perspective.

Topics: Adipates; Biosynthetic Pathways; Gene Expression Regulation, Bacterial; Glycerol; Metabolic Engineering; Microorganisms, Genetically-Modified; Parabens; Plasmids; Pseudomonas chlororaphis; Shikimic Acid; Sorbic Acid; Ubiquinone

Land plants possess the unique capacity to derive the benzenoid moiety of the vital respiratory cofactor, ubiquinone (coenzyme Q), from phenylpropanoid metabolism via β-oxidation of

Topics: Arabidopsis; Gene Expression Regulation, Plant; Kaempferols; Parabens; Plants; Solanum lycopersicum; Ubiquinone

Coenzyme Q (Q) is a redox lipid that is central for the energetic metabolism of eukaryotes. The biosynthesis of Q from the aromatic precursor 4-hydroxybenzoic acid (4-HB) is understood fairly well. However, biosynthetic details of how 4-HB is produced from tyrosine remain elusive. Here, we provide key insights into this long-standing biosynthetic problem by uncovering molecular details of the first and last reactions of the pathway in the yeast Saccharomyces cerevisiae, namely the deamination of tyrosine to 4-hydroxyphenylpyruvate by Aro8 and Aro9, and the oxidation of 4-hydroxybenzaldehyde to 4-HB by Hfd1. Inactivation of the HFD1 gene in yeast resulted in Q deficiency, which was rescued by the human enzyme ALDH3A1. This suggests that a similar pathway operates in animals, including humans, and led us to propose that patients with genetically unassigned Q deficiency should be screened for mutations in aldehyde dehydrogenase genes, especially ALDH3A1.

Topics: Aldehyde Dehydrogenase; Benzaldehydes; Biosynthetic Pathways; Gene Deletion; Gene Expression Regulation, Fungal; Humans; Oxidation-Reduction; Parabens; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tyrosine; Ubiquinone

Coenzyme Q10 (CoQ10) plays an indispensable role in ATP generation through oxidative phosphorylation and helps in scavenging superoxides generated during electron transfer reactions. It finds extensive applications specifically related to oxidative damage and metabolic dysfunctions. This article reports the use of a statistical approach to optimize the concentration of key variables for the enhanced production of CoQ10 by Rhodotorula glutinis in a lab-scale fermenter. The culture conditions that promote optimum growth and CoQ10 production were optimized and the interaction of significant variables para-hydroxybenzoic acid (PHB, 819.34 mg/L) and soybean oil (7.78% [v/v]) was studied using response surface methodology (RSM). CoQ10 production increased considerably from 10 mg/L (in control) to 39.2 mg/L in batch mode with RSM-optimized precursor concentration. In the fed-batch mode, PHB and soybean oil feeding strategy enhanced CoQ10 production to 78.2 mg/L.

Topics: Analysis of Variance; Batch Cell Culture Techniques; Bioreactors; Biostatistics; Biotechnology; Models, Theoretical; Parabens; Rhodotorula; Soybean Oil; Ubiquinone

Plastoquinone is a redox active lipid that serves as electron transporter in the bifunctional photosynthetic-respiratory transport chain of cyanobacteria. To examine the role of genes potentially involved in cyanobacterial plastoquinone biosynthesis, we have focused on three Synechocystis sp. PCC 6803 genes likely encoding a chorismate pyruvate-lyase (sll1797) and two 4-hydroxy-3-solanesylbenzoate decarboxylases (slr1099 and sll0936). The functions of the encoded proteins were investigated by complementation experiments with Escherichia coli mutants, by the in vitro enzyme assays with the recombinant proteins, and by the development of Synechocystis sp. single-gene knock-out mutants. Our results demonstrate that sll1797 encodes a chorismate pyruvate-lyase. In the respective knock-out mutant, plastoquinone was hardly detectable, and the mutant required 4-hydroxybenzoate for growth underlining the importance of chorismate pyruvate-lyase to initiate plastoquinone biosynthesis in cyanobacteria. The recombinant Slr1099 protein displayed decarboxylase activity and catalyzed in vitro the decarboxylation of 4-hydroxy-3-prenylbenzoate with different prenyl side chain lengths. In contrast to Slr1099, the recombinant Sll0936 protein did not show decarboxylase activity regardless of the conditions used. Inactivation of the sll0936 gene in Synechocystis sp., however, caused a drastic reduction in the plastoquinone content to levels very similar to those determined in the slr1099 knock-out mutant. This proves that not only slr1099 but also sll0936 is required for plastoquinone synthesis in the cyanobacterium. In summary, our data demonstrate that cyanobacteria produce plastoquinone exclusively via a pathway that is in the first reaction steps almost identical to ubiquinone biosynthesis in E. coli with conversion of chorismate to 4-hydroxybenzoate, which is then prenylated and decarboxylated.

Topics: Carboxy-Lyases; Chorismic Acid; Escherichia coli; Evolution, Molecular; Oxo-Acid-Lyases; Parabens; Photosynthesis; Phylogeny; Plastoquinone; Recombinant Fusion Proteins; Synechocystis; Ubiquinone

Coenzyme Q (CoQ) is a medically valuable compound and a high yielding strain for CoQ will have several benefits for the industrial production of CoQ. To increase the CoQ(8) content of E. coli, we blocked the pathway for the synthesis of menaquinone by deleting the menA gene. The blocking of menaquinone pathway increased the CoQ(8) content by 81 % in E. coli (ΔmenA). To study the CoQ producing potential of E. coli, we employed previous known increasing strategies for systematic metabolic engineering. These include the supplementation with substrate precursors and the co-expression of rate-limiting genes. The co-expression of dxs-ubiA and the supplementation with substrate precursors such as pyruvate (PYR) and parahydroxybenzoic acid (pHBA) increased the content of CoQ(8) in E. coli (ΔmenA) by 125 and 59 %, respectively. Moreover, a 180 % increase in the CoQ(8) content in E. coli (ΔmenA) was realized by the combination of the co-expression of dxs-ubiA and the supplementation with PYR and pHBA. All in all, CoQ(8) content in E. coli increased 4.06 times by blocking the menaquinone pathway, dxs-ubiA co-expression and the addition of sodium pyruvate and parahydroxybenzoic acid to the medium. Results suggested a synergistic effect among different metabolic engineering strategies.

Topics: Alkyl and Aryl Transferases; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Industrial Microbiology; Metabolic Engineering; Parabens; Plasmids; Ubiquinone; Vitamin K 2

Xanthomonas oryzae pv. oryzae, the causal agent of rice bacterial blight, produces membrane-bound yellow pigments, referred to as xanthomonadins. Xanthomonadins protect the pathogen from photodamage and host-induced perioxidation damage. They are also required for epiphytic survival and successful host plant infection. Here, we show that XanB2 encoded by PXO_3739 plays a key role in xanthomonadin and coenzyme Q8 biosynthesis in X. oryzae pv. oryzae PXO99A. A xanB2 deletion mutant exhibits a pleiotropic phenotype, including xanthomonadin deficiency, producing less exopolysaccharide (EPS), lower viability and H2O2 resistance, and lower virulence. We further demonstrate that X. oryzae pv. oryzae produces 3-hydroxybenzoic acid (3-HBA) and 4-hydroxybenzoic acid (4-HBA) via XanB2. 3-HBA is associated with xanthomonadin biosynthesis while 4-HBA is mainly used as a precursor for coenzyme Q (CoQ)8 biosynthesis. XanB2 is the alternative source of 4-HBA for CoQ8 biosynthesis in PXO99A. These findings suggest that the roles of XanB2 in PXO99A are generally consistent with those in X. campestris pv. campestris. The present study also demonstrated that X. oryzae pv. oryzae PXO99A has evolved several specific features in 3-HBA and 4-HBA signaling. First, our results showed that PXO99A produces less 3-HBA and 4-HBA than X. campestris pv. campestris and this is partially due to a degenerated 4-HBA efflux pump. Second, PXO99A has evolved unique xanthomonadin induction patterns via 3-HBA and 4-HBA. Third, our results showed that 3-HBA or 4-HBA positively regulates the expression of gum cluster to promote EPS production in PXO99A. Taken together, the results of this study indicate that XanB2 is a key metabolic enzyme linking xanthomonadin, CoQ, and EPS biosynthesis, which are collectively essential for X. oryzae pv. oryzae pathogenesis.

Topics: Bacterial Proteins; Genetic Complementation Test; Hydrogen Peroxide; Hydroxybenzoates; Oryza; Parabens; Phenotype; Pigmentation; Plant Diseases; Polysaccharides, Bacterial; Sequence Deletion; Ubiquinone; Virulence; Xanthomonas

A three-phase fluidized bed reactor (TPFBR) was designed to evaluate the potential of CoQ(10) production by gel-entrapped Sphingomonas sp. ZUTE03 via a conversion-extract coupled process. In the reactor, the CoQ(10) yield reached 46.99 mg/L after 8 h of conversion; a high-level yield of about 45 mg/L was maintained even after 15 repetitions (8 h/batch). To fully utilize the residual precursor (para-hydroxybenzoic acid, PHB) in the aqueous phase, the organic phase was replaced with new solution containing 70 mg/L solanesol for each 8 h batch. The CoQ(10) yield of each batch was maintained at a level of about 43 mg/L until the PHB ran out. When solid solanesol was fed to the organic phase for every 8 h batch, CoQ(10) could accumulate and reach a yield of 171.52 mg/L. When solid solanesol and PHB were fed to the conversion system after every 8 h batch, the CoQ(10) yield reached 441.65 mg/L in the organic phase after 20 repetitions, suggesting that the conversion-extract coupled process could enhance CoQ(10) production in the TPFBR.

Topics: Bioreactors; Biotechnology; Cells, Immobilized; Culture Media; Parabens; Sphingomonas; Terpenes; Ubiquinone

Most of the Coq proteins involved in coenzyme Q (ubiquinone or Q) biosynthesis are interdependent within a multiprotein complex in the yeast Saccharomyces cerevisiae. Lack of only one Coq polypeptide, as in Δcoq strains, results in the degradation of several Coq proteins. Consequently, Δcoq strains accumulate the same early intermediate of the Q(6) biosynthetic pathway; this intermediate is therefore not informative about the deficient biosynthetic step in a particular Δcoq strain. In this work, we report that the overexpression of the protein Coq8 in Δcoq strains restores steady state levels of the unstable Coq proteins. Coq8 has been proposed to be a kinase, and we provide evidence that the kinase activity is essential for the stabilizing effect of Coq8 in the Δcoq strains. This stabilization results in the accumulation of several novel Q(6) biosynthetic intermediates. These Q intermediates identify chemical steps impaired in cells lacking Coq4 and Coq9 polypeptides, for which no function has been established to date. Several of the new intermediates contain a C4-amine and provide information on the deamination reaction that takes place when para-aminobenzoic acid is used as a ring precursor of Q(6). Finally, we used synthetic analogues of 4-hydroxybenzoic acid to bypass deficient biosynthetic steps, and we show here that 2,4-dihydroxybenzoic acid is able to restore Q(6) biosynthesis and respiratory growth in a Δcoq7 strain overexpressing Coq8. The overexpression of Coq8 and the use of 4-hydroxybenzoic acid analogues represent innovative tools to elucidate the Q biosynthetic pathway.

Topics: Biosynthetic Pathways; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Genetic Complementation Test; Immunoblotting; Mitochondria; Mitochondrial Proteins; Mutation; Parabens; Phosphotransferases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transformation, Genetic; Ubiquinone

The bacterial type III export apparatus is found in the flagellum and in the needle complex of some pathogenic Gram-negative bacteria. In the needle complex its function is to secrete effector proteins for infection into Eukaryotic cells. In the bacterial flagellum it exports specific proteins for the building of the flagellum during its assembly. The export apparatus is composed of about five membrane proteins and three soluble proteins. The mechanism of the export apparatus is not fully understood. The five membrane proteins are well conserved and essential. Here a cross-complementation assay was performed: substituting in the flagellar system of Salmonella one of these membrane proteins, FlhB, by the FlhB ortholog from Aquifex aeolicus (an evolutionary distant hyperthermophilic bacteria) or a chimeric protein (AquSalFlhB) made by the combination of the trans-membrane domain of A. aeolicus FlhB with the cytoplasmic domain of Salmonella FlhB dramatically reduced numbers of flagella and motility. From cells expressing the chimeric AquSalFlhB protein, suppressor mutants with enhanced motility were isolated and the mutations were identified using whole genome sequencing. Gain-of-function mutations were found in the gene encoding FlhA, another membrane protein of the type III export apparatus. Also, mutations were identified in genes encoding 4-hydroxybenzoate octaprenyltransferase, ubiquinone/menaquinone biosynthesis methyltransferase, and 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, which are required for ubiquinone biosynthesis. The mutations were shown by reversed-phase high performance liquid chromatography to reduce the quinone pool of the cytoplasmic membrane. Ubiquinone biosynthesis could be restored for the strain bearing a mutated gene for 4-hydroxybenzoate octaprenyltransferase by the addition of excess exogenous 4-hydroxybenzoate. Restoring the level of ubiquinone reduced flagella biogenesis with the AquSalFlhB chimera demonstrating that the respiratory chain quinone pool is responsible for this phenomenon.

Topics: Bacteria; Bacterial Proteins; Blotting, Western; Flagella; Genes, Bacterial; Genetic Complementation Test; Membrane Proteins; Movement; Parabens; Protein Transport; Recombinant Proteins; Salmonella; Suppression, Genetic; Ubiquinone

In a water-organic solvent, two-phase conversion system, CoQ(10) could be produced directly from solanesol and para-hydroxybenzoic acid (PHB) by free cells of Sphingomonas sp. ZUTE03 and CoQ(10) concentration in the organic solvent phase was significantly higher than that in the cell. CoQ(10) yield reached a maximal value of 60.8 mg l(-1) in the organic phase and 40.6 mg g(-1)-DCW after 8 h. CoQ(10) also could be produced by gel-entrapped cells in the two-phase conversion system. Soybean oil and hexane were found to be key substances for CoQ(10) production by gel-entrapped cells of Sphingomonas sp. ZUTE03. Soybean oil might improve the release of CoQ10 from the gel-entrapped cells while hexane was the suitable solvent to extract CoQ(10) from the mixed phase of aqueous and organic. The gel-entrapped cells could be re-used to produce CoQ(10) by a repeated-batch culture. After 15 repeats, the yield of CoQ(10) kept at a high level of more than 40 mg l(-1). After 8 h conversion under optimized precursor's concentration, CoQ(10) yield of gel-trapped cells reached 52.2 mg l(-1) with a molar conversion rate of 91% and 89.6% (on PHB and solanesol, respectively). This is the first report on enhanced production of CoQ(10) in a two-phase conversion system by gel-entrapped cells of Sphingomonas sp. ZUTE03.

Topics: Cells, Immobilized; Chemical Fractionation; Industrial Microbiology; Parabens; Sphingomonas; Terpenes; Ubiquinone

CoQ is an essential electron carrier in the mitochondrial respiratory chain of both eukaryotes and prokaryotes. It consists of a benzoquinone head group and a hydrophobic polyisoprenoid tail. The genes (COQ1-9) involved in CoQ biosynthesis have been characterized in yeast. In this study, we generated and molecularly characterized a mutant allele of a novel Drosophila gene, sbo, which encodes a protein that is predicted to catalyze the prenylation of p-hydroxybenzoate with the isoprenoid chain during the process of CoQ synthesis. Expression of sbo in yeast rescues the lethality of ∆COQ2 mutant cells, indicating that sbo is a functional homolog of COQ2. HPLC results show that the levels of CoQ(9) and CoQ(10) were significantly reduced in sbo heterozygous adult flies. Furthermore, the mean lifespans of males and females heterozygous for sbo are extended by 12.5% and 30.8%, respectively. Homozygous sbo animals exhibit reduced activities of the insulin/insulin-like growth factor signaling (IIS) pathway. Taken together, we conclude that sbo is an essential gene for Drosophila development, mutation of which leads to an extension of lifespan most likely by altering endogenous CoQ biosynthesis.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Animals; Drosophila melanogaster; Drosophila Proteins; Female; Humans; Larva; Longevity; Male; Mitochondria; Molecular Sequence Data; Mutation; Parabens; Saccharomyces cerevisiae; Sequence Homology; Ubiquinone

Coenzyme Q (Q), an essential component of eukaryotic cells, is synthesized by several enzymes from the precursor 4-hydroxybenzoic acid. Mutations in six of the Q biosynthesis genes cause diseases that can sometimes be ameliorated by oral Q supplementation. We establish here that Coq6, a predicted flavin-dependent monooxygenase, is involved exclusively in the C5-hydroxylation reaction. In an unusual way, the ferredoxin Yah1 and the ferredoxin reductase Arh1 may be the in vivo source of electrons for Coq6. We also show that hydroxylated analogs of 4-hydroxybenzoic acid, such as vanillic acid or 3,4-dihydroxybenzoic acid, restore Q biosynthesis and respiration in a Saccharomyces cerevisiae coq6 mutant. Our results demonstrate that appropriate analogs of 4-hydroxybenzoic acid can bypass a deficient Q biosynthetic enzyme and might be considered for the treatment of some primary Q deficiencies.

Topics: Adrenodoxin; Chromatography, High Pressure Liquid; Ferredoxin-NADP Reductase; Hydroxybenzoates; Hydroxylation; Membrane Proteins; Mutation; Parabens; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquinone; Vanillic Acid

In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q(10) (CoQ(10)), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ(10) precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ(10) was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ(10)-producing E. coli strain resulted in an increase in CoQ(10) content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ(10) content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB.

Topics: 4-Aminobenzoic Acid; Alkyl and Aryl Transferases; Dimethylallyltranstransferase; Escherichia coli; Gene Deletion; Genetic Engineering; Mevalonic Acid; Parabens; Phenols; Polyisoprenyl Phosphates; Promoter Regions, Genetic; Sesquiterpenes; Sphingomonadaceae; Sphingomonas; Terpenes; Ubiquinone; Up-Regulation

Yeast ubiquinone or coenzyme Q(6) (Q(6)) is a redox active lipid that plays a crucial role in the mitochondrial electron transport chain. At least nine proteins (Coq1p-9p) participate in Q(6) biosynthesis from 4-hydroxybenzoate (4-HB). We now show that the mitochondrial ferredoxin Yah1p and the ferredoxin reductase Arh1p are required for Q(6) biosynthesis, probably for the first hydroxylation of the pathway. Conditional Gal-YAH1 and Gal-ARH1 mutants accumulate 3-hexaprenyl-4-hydroxyphenol and 3-hexaprenyl-4-aminophenol. Para-aminobenzoic acid (pABA) is shown to be the precursor of 3-hexaprenyl-4-aminophenol and to compete with 4-HB for the prenylation reaction catalyzed by Coq2p. Yeast cells convert U-((13)C)-pABA into (13)C ring-labeled Q(6), a result that identifies pABA as a new precursor of Q(6) and implies an additional NH(2)-to-OH conversion in Q(6) biosynthesis. Our study identifies pABA, Yah1p, and Arh1p as three actors in Q(6) biosynthesis.

Topics: 4-Aminobenzoic Acid; Adrenodoxin; Aminophenols; Ferredoxin-NADP Reductase; Membrane Proteins; Mitochondria; Mutation; Oxidation-Reduction; Parabens; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquinone

Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable (13)C(6)-isotope of pABA (p- amino[aromatic-(13)C(6)]benzoic acid ([(13)C(6)]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[(13)C(6)]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [(13)C(6)]pABA or [(13)C(6)]4HB generate both (13)C(6)-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product (13)C(6)-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ(6). This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ(6) quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates.

Topics: 4-Aminobenzoic Acid; Biocatalysis; Chorismic Acid; Genes, Fungal; Lipid Metabolism; Lyases; Parabens; Prenylation; Saccharomyces cerevisiae; Ubiquinone

The changes of functional state isolated by Lanhendorf old rat hearts with low content of ubiqinone--coenzyme Q (CoQ) under activation of it endogenous synthesis through administration of precursors--4-hydroxybenzoic acid, methionine and modulator vitamin E were studied. The activation of ubiqinone biosynthesis contribute to cardioprotective effect due to reduce the degree of the ischemia-reperfusion injury in old rat heart, namely the restoration of myocardial contractile function and coronary flow as well as decrease the end diastolic pressure and oxygen cost of the heart compared with control group of the animals during ischemia-reperfusion. Thus the results allow to conclude that the activation of KoQ biosynthesis under administration of it precursors has protective effect in the development of the heart postreperfusion damages in aging.

Topics: Aging; alpha-Tocopherol; Animals; Cardiotonic Agents; Coronary Circulation; Heart; Heart Function Tests; In Vitro Techniques; Male; Methionine; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; Parabens; Rats; Rats, Wistar; Ubiquinone

This research work is devoted to study of the sensitivity of mitochondrial permeability transition pore (MPTP) opening to its inductors--ions Ca2+ (10(-4) mol/l) and oxidant phenylarsine oxide (10(-4) mol/l) and content of ubiquinone (coenzyme Q, CoQ) and vitamin E in heart mitochondria of adult, old (control) and old rats under administration of precursors an modulator of CoQ biosynthesis--4-hydroxybenzoic acid, methionine and modulator of CoQ biosynthesis, namely vitamin E. The results of our research demonstrate that administration of complex of biologically active substances, which are precursors and modulators of CoQ biosynthesis, leads to decrease in the sensitivity of MPTP opening to its inductors and increase of CoQ and vitamin E content in old rats heart mitochondria. Therefore the results obtained lead to a conclusion that increase of CoQ content due to administration of precursors and modulator of its biosynthesis is an effective way in the inhibition of MPTP opening. This approach as well as application of CoQ-containing medicals may be used for correction of mitochondrial dysfunction under various pathologies of cardiovascular system and in aging.

Topics: Aging; Animals; Arsenicals; Calcium; Male; Methionine; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Myocardium; Parabens; Rats; Rats, Wistar; Ubiquinone; Vitamin E

Prenylation of the aromatic intermediate p-hydroxybenzoate (PHB) is a critical step in ubiquinone (UQ) biosynthesis. The enzyme that catalyzes this prenylation reaction is p-hydroxybenzoate polyprenyltransferase (PPT), which substitutes an aromatic proton at the m-position of PHB with a prenyl chain provided by polyprenyl diphosphate synthase. The rice genome contains three PPT candidates that share significant similarity with the yeast PPT (COQ2 gene), and the rice gene showing the highest similarity to COQ2 was isolated by reverse transcription-PCR and designated OsPPT1a. The deduced amino acid sequence of OsPPT1a contained a putative mitochondrial sorting signal at the N-terminus and conserved domains for putative substrate-binding sites typical of PPT protein family members. The subcellular localization of OsPPT1a protein was shown to be mainly in mitochondria based on studies using a green fluorescent protein-PPT fusion. A yeast complementation study revealed that OsPPT1a expression successfully recovered the growth defect of the coq2 mutant. A prenyltransferase assay using recombinant protein showed that OsPPT1a accepted prenyl diphosphates of various chain lengths as prenyl donors, whereas it showed strict substrate specificity for the aromatic substrate PHB as a prenyl acceptor. The apparent K (m) values for geranyl diphosphate and PHB were 59.7 and 6.04 microM, respectively. The requirement by OsPPT1a and COQ2 for divalent cations was also studied, with Mg2+ found to produce the highest enzyme activity. Northern analysis showed that OsPPT1a mRNA was accumulated in all tissues of O. sativa. These results suggest that OsPPT1a is a functional PPT involved in UQ biosynthesis in O. sativa.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; DNA, Plant; Exons; Gene Expression Regulation, Plant; Genes, Plant; Mitochondria; Molecular Sequence Data; Oryza; Parabens; Plant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Plant; Substrate Specificity; Ubiquinone

Polyprenyl 4-hydroxybenzoate transferase (Coq2p) plays a central role in ubiquinone biosynthesis. Coq2p mediates the conjugation of 4-hydroxybenzoate, the benzoquinone ring precursor, with the completed side chain. The activity is most easily assayed by measuring the rate of incorporation of 4-hydroxybenzoate as radiolabeled substrate into polyprenyl 4-hydroxybenzoate. The in vitro assay requires addition of a detergent into the reaction mixture to activate enzyme activity, and Triton X-100 is used for this purpose in the routine assay. We have found that both 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate and sodium cholate, but not sodium deoxycholate, lysophosphatidyl choline, or octylglucoside, significantly stimulate the activity over that measured with Triton X-100. High-performance liquid chromatography analysis of lipid extracts revealed that the increase of specific activity resulted in a similar increase in reaction product, this effect is due not merely to a better lipid extraction but also to the actual stimulation of enzyme activity. With our improved method, we were able to measure Coq2p activity with much greater sensitivity in both fresh and frozen/thawed mitochondria and in crude homogenates obtained from cultured cells. Our method will simplify evaluation of Coq2p activity in scarce biological materials, such as cells obtained from human tissue biopsies, and thus it will facilitate the biochemical characterization of ubiquinone deficiencies.

Topics: Alkyl and Aryl Transferases; Animals; Cells, Cultured; Cholic Acids; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Detergents; HL-60 Cells; Humans; Lipids; Mitochondria, Liver; Octoxynol; Parabens; Rats; Sodium Cholate; Ubiquinone

Ubiquinone (coenzyme Q; CoQ) is the only lipophilic antioxidant that is endogenously synthesized by all organisms. CoQ biosynthesis is determined in vitro by supplying a radiolabeled precursor and, after lipid extraction and CoQ separation by thin-layer chromatography or high-performance liquid chromatography, the radioactivity present in the sample is quantified. In the rapid and simple method described here, we avoid the use of organic solvents by supplying 4-hydroxy-[U-14C]benzoate as radiolabeled precursor and precipitating CoQ with trichloroacetic acid (TCA). After TCA precipitation, all radioactivity was present in the precipitate and CoQ was the only radiolabeled molecule detected. The radioactive material was then solubilized with NaOH and quantified in a scintillation counter.

Topics: Carbon Radioisotopes; Carcinoma, Hepatocellular; Humans; Parabens; Tumor Cells, Cultured; Ubiquinone

By removing the enolpyruvyl group from chorismate, chorismate lyase (CL) produces p-hydroxybenzoate (p-HB) for the ubiquinone biosynthetic pathway. We have analyzed CL by several spectroscopic and chemical techniques and measured its kinetic (kcat=1.7 s(-1), K(m)=29 microM) and product inhibition parameters (K(p)=2.1 microM for p-HB). Protein aggregation, a serious problem with wild type CL, proved to be primarily due to the presence of two surface-active cysteines, whose chemical modification or mutation (to serines) gave greatly improved solution behavior and minor effects on enzyme activity. CL is strongly inhibited by its product p-HB; for this reason activity and inhibition measurements were analyzed by both initial rate and progress curve methods. The results are consistent, but in this case where the stable enzyme-product complex rapidly becomes the predominant form of the enzyme, progress curve methods are more efficient. We also report inhibition measurements with several substrate and product analogs that give information on ligand binding interactions of the active site. The biological function of the unusual product retention remains uncertain, but may involve a mechanism of directed delivery to the membrane-bound enzyme that follows CL in the ubiquinone pathway.

Topics: Anthranilate Synthase; Binding Sites; Chorismate Mutase; Cysteine; Enzyme Stability; Escherichia coli; Kinetics; Oxo-Acid-Lyases; Parabens; Protein Engineering; Serine; Solubility; Ubiquinone

Topics: Animals; Anti-Bacterial Agents; Antifungal Agents; Atovaquone; Female; Naphthoquinones; Oxidoreductases; Parabens; Pneumocystis; Polyenes; Rats; Ubiquinone

The naphthoquinone atovaquone is effective against Plasmodium and Pneumocystis carinii carinii. In Plasmodium, the primary mechanism of drug action is an irreversible binding to the mitochondrial cytochrome bc(1) complex as an analog of ubiquinone. Blockage of the electron transport chain ultimately inhibits de novo pyrimidine biosynthesis since dihydroorotate dehydrogenase, a key enzyme in pyrimidine biosynthesis, is unable to transfer electrons to ubiquinone. In the present study, the effect of atovaquone was examined on Pneumocystis carinii carinii coenzyme Q biosynthesis (rather than electron transport and respiration) by measuring its effect on the incorporation of radiolabeled p-hydroxybenzoate into ubiquinone in vitro. A triphasic dose-response was observed, with inhibition at 10 nM and then stimulation up to 0.2 microM, followed by inhibition at 1 microM. Since other naphthoquinone drugs may also act as analogs of ubiquinone, diospyrin and two of its derivatives were also tested for their effects on ubiquinone biosynthesis in P. carinii carinii. In contrast to atovaquone, these drugs did not inhibit the incorporation of p-hydroxybenzoate into P. carinii carinii ubiquinone.

Topics: Antifungal Agents; Atovaquone; Dose-Response Relationship, Drug; Naphthoquinones; Oxygen Consumption; Parabens; Pneumocystis; Ubiquinone

Promastigotes of Leishmania major contain a ubiquinone which has a side chain made up of nine isoprene subunits (UQ9). Incorporation of radioactivity from [14C] acetate and [14C] mevalonate into ubiquinone as well as the identification of hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase), and mevalonate kinase indicate that the isoprenoid portion of the molecule is synthesized by the acetate-mevalonate pathway as in mammalian cells. Incorporation of [14C] tyrosine into ubiquinone is low, but [14C] parahydroxybenzoic acid is readily incorporated. Distribution of radioactivity from [14C] acetate indicates that about 60-80% is associated with the side chain and about 20% with the ring. Label from parahydroxybenzoic acid is, however, incorporated preferentially into the ring. L. major is capable of synthesizing the aromatic ring of ubiquinone from acetate, parahydroxybenzoate being an important intermediate. In this behaviour it resembles procaryotes. Ubiquinone biosynthetic pathway in L. major thus shares characteristics with mammalian and bacterial systems.

Topics: Acetates; Animals; Cholesterol; Crithidia fasciculata; Glucose; Hydroxymethylglutaryl CoA Reductases; Leishmania major; Mevalonic Acid; Oxidation-Reduction; Parabens; Shikimic Acid; Tyrosine; Ubiquinone

This study investigated the biosynthesis of ubiquinone in isolated and perfused hearts of young and aged rats exposed to ischemia and reperfusion. A first group of hearts was used to determine the changes in coenzyme Q9 (CoQ9) and coenzyme Q10 (CoQ10) concentrations at mitochondrial and microsomal level after 30 min of ischemia (98% reduction of the preischemic flow) and 60 min of reperfusion. A second group was utilized to evaluate the rate of CoQ9 and CoQ10 biosynthesis in the membranes by dissolving two ubiquinone precursors, p-OH-[U-14C]benzoate and mevalonolactone, in the perfusion buffer. The hearts were aerobically perfused for 60 min in the presence of the precursors either immediately after the equilibration period or following 30 min ischemia. The young rat hearts showed a 30% reduction in the mitochondrial levels of CoQ9 after ischemia and reperfusion with respect to the preischemic values (P < 0.05 and P < 0.01, respectively). On the contrary, the mitochondrial CoQ9 content was not modified under these conditions in the aged hearts. At the end of reperfusion, the biosynthesis of mitochondrial CoQ9 and CoQ10 was higher in the young rats (P < 0.05), and lower in the aged rats (P < 0.05), with respect to the aerobic perfusion. In both young and aged rats minor changes in CoQ9 concentrations and biosynthesis were observed at microsomal level. These results indicate that myocardial reperfusion decreases the mitochondrial content of ubiquinone and stimulates CoQ9 biosynthesis in young rats but not in aged rats.

Topics: Acclimatization; Aging; Animals; Heart; In Vitro Techniques; Kinetics; Male; Microsomes; Mitochondria, Heart; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Parabens; Rats; Rats, Wistar; Reference Values; Ubiquinone

Topics: Animals; Blood; Blood Chemical Analysis; Homeostasis; Lasers; Parabens; Rats; Spectrum Analysis; Strophanthins; Time Factors; Ubiquinone

1. The biosynthesis of ubiquinone (UQ) in isolated rat heart under ischemic and hypoxic conditions was investigated. 2. Under ischemic perfusion, a greater amount of biosynthetic intermediates, 3-nonaprenyl and 3-decaprenyl-4-hydroxybenzoate (PPHBs) was accumulated and a smaller amount of UQ-9 and -10 was synthesized when compared with normal conditions. 3. The accumulation of PPHBs was observed without forming UQs during anaerobic perfusion. 4. Hydroxylation which is the following reaction of PPHBs for the biosynthesis of UQ in rat heart, was proceeded by the monooxygenase(s) depending upon the oxygen concentrations.

Topics: Animals; Chromatography, High Pressure Liquid; Coronary Disease; Hydroxylation; Hypoxia; Male; Mitochondria, Heart; Myocardium; Oxygen; Parabens; Pyridines; Rats; Rats, Inbred Strains; Rotenone; Terpenes; Ubiquinone

To investigate the perturbation of ubiquinone biosynthesis by a hypocholesterolemic drug, 3 beta-(2-diethylaminoethoxy)androst-5-en-17-one hydrochloride (U18666A), we measured the incorporation of radioactive mevalonate, methionine, tyrosine, and 4-hydroxybenzoic acid into ubiquinone in glioblastoma cells. These four precursors unanimously showed that ubiquinone biosynthesis was not significantly altered by U18666A, which blocked cholesterol biosynthesis at steps beyond mevalonate formation. The fluctuation of the endogenous mevalonate level had little effect on ubiquinone biosynthesis, implying the relative stability of cellular ubiquinone biosynthesis. Furthermore, exogenously added mevalonate did not have an appreciable effect on ubiquinone biosynthesis. The major ubiquinone produced in rat glioblastoma cells was identified as ubiquinone-9. The mevalonate-derived products accumulated in the U18666A-treated cells differed significantly from those reported in a broken cell study, suggesting the existence of delicate mechanisms regulating the formation of cholesterol intermediates.

Topics: Androstenes; Animals; Anticholesteremic Agents; Cell Line; Glioma; Hydroxybenzoates; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Methionine; Mevalonic Acid; Naphthalenes; Parabens; Rats; Tyrosine; Ubiquinone

Isolated mitochondria from potato tubers, spinach leaves, and daffodil petals from intermediates of the ubiquinone biosynthetic pathway (prenylated 4-hydroxybenzoate, prenylated phenols, and quinoid compounds) from [1-14C]isopentenyl diphosphate and endogenous or exogenous 4-hydroxybenzoate. In contrast [2-14C]mevalonate 5-diphosphate, the immediate precursor of isopentenyl diphosphate was not accepted as a substrate. These results suggest that plant mitochondria have their own prenyltransferase and prenylation system, similar to the plastid compartment which also starts by the use of isopentenyl diphosphate [see Kreuz, K. and Kleinig, H. (1984) Eur. J. Biochem. 141, 531-535].

Topics: Cell Fractionation; Hemiterpenes; Hydroxybenzoates; Mitochondria; Organophosphorus Compounds; Parabens; Plants; Ubiquinone

Topics: Aldehydes; Benzaldehydes; Benzoates; Carbon Isotopes; Metabolism; Parabens; Phenylacetates; Quinones; Radiometry; Research; Rhodospirillum; Rhodospirillum rubrum; Spectrum Analysis; Ubiquinone

Topics: Acetates; Benzoates; Carbon Isotopes; Chromatography; Light; Metabolism; Methionine; Parabens; Quinones; Research; Rhodospirillum; Rhodospirillum rubrum; Ubiquinone