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Sample records for cmp-n-acetylneuraminic acid hydroxylase

  1. CMP-N-acetylneuraminic acid hydroxylase: the first cytosolic Rieske iron-sulphur protein to be described in Eukarya.

    PubMed

    Schlenzka, W; Shaw, L; Kelm, S; Schmidt, C L; Bill, E; Trautwein, A X; Lottspeich, F; Schauer, R

    1996-05-06

    Electron paramagnetic resonance (EPR) spectroscopy and analysis of the primary structure of the CMP-N-acetylneuraminic acid hydroxylase revealed that this enzyme is the first iron-sulphur protein of the Rieske type to be found in the cytosol of Eukarya. The dithionite-reduced hydroxylase exhibited an EPR signal known to be characteristic for a Rieske iron-sulphur centre (2Fe-2S), the g-values being 1.78, 1.91 and 2.01, respectively. An analysis of the primary structure of the hydroxylase led to the identification of an amino acid sequence, known to be characteristic for Rieske proteins. Furthermore, possible binding sites for cytochrome b5, the substrate CMP-Neu5Ac and a mononuclear iron centre were also identified.

  2. CMP-N-acetylneuraminic acid synthetase interacts with fragile X related protein 1

    PubMed Central

    Ma, Yun; Tian, Shuai; Wang, Zongbao; Wang, Changbo; Chen, Xiaowei; Li, Wei; Yang, Yang; He, Shuya

    2016-01-01

    Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P-interacting proteins and determine the biological effect of the interaction. The current study identified CMP-N-acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two-hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β-galactosidase assay and growth studies with selective media. Furthermore, co-immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue-specific regulator of GM1 levels in SH-SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS. PMID:27357083

  3. A CMP-N-acetylneuraminic acid synthetase purified from a marine bacterium, Photobacterium leiognathi JT-SHIZ-145.

    PubMed

    Kajiwara, Hitomi; Mine, Toshiki; Miyazaki, Tatsuo; Yamamoto, Takeshi

    2011-01-01

    A cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase was found in a crude extract prepared from Photobacterium leiognathi JT-SHIZ-145, a marine bacterium that also produces a β-galactoside α2,6-sialyltransferase. The CMP-Neu5Ac synthetase was purified from the crude extract of the cells by a combination of anion-exchange and gel filtration column chromatography. The purified enzyme migrated as a single band (60 kDa) on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The activity of the enzyme was maximal at 35 °C at pH 9.0, and the synthetase required Mg(2+) for activity. Although these properties are similar to those of other CMP-Neu5Ac synthetases isolated from bacteria, this synthetase produced not only CMP-Neu5Ac from cytidine triphosphate and Neu5Ac, but also CMP-N-glycolylneuraminic acid from cytidine triphosphate and N-glycolylneuraminic acid, unlike CMP-Neu5Ac synthetase purified from Escherichia coli.

  4. Plant fatty acid hydroxylases

    DOEpatents

    Somerville, Chris; Broun, Pierre; van de Loo, Frank

    2001-01-01

    This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

  5. Low incidence of N-glycolylneuraminic acid in birds and reptiles and its absence in the platypus.

    PubMed

    Schauer, Roland; Srinivasan, G Vinayaga; Coddeville, Bernadette; Zanetta, Jean-Pierre; Guérardel, Yann

    2009-08-17

    The sialic acids of the platypus, birds, and reptiles were investigated with regard to the occurrence of N-glycolylneuraminic (Neu5Gc) acid. They were released from tissues, eggs, or salivary mucin samples by acid hydrolysis, and purified and analyzed by thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry. In muscle and liver of the platypus only N-acetylneuraminic (Neu5Ac) acid was found. The nine bird species studied also did not express N-glycolylneuraminic acid with the exception of an egg, but not tissues, from the budgerigar and traces in poultry. Among nine reptiles, including one turtle, N-glycolylneuraminic acid was only found in the egg and an adult basilisk, but not in a freshly hatched animal. BLAST analysis of the genomes of the platypus, the chicken, and zebra finch against the CMP-N-acetylneuraminic acid hydroxylase did not reveal the existence of a similar protein structure. Apparently monotremes (platypus) and sauropsids (birds and reptiles) cannot synthesize Neu5Gc. The few animals where Neu5Gc was found, especially in eggs, may have acquired this from the diet or by an alternative pathway. Since Neu5Gc is antigenic to man, the observation that this monosaccharide does not or at least only rarely occur in birds and reptiles, may be of nutritional and clinical significance.

  6. Plant fatty acid hydroxylase

    DOEpatents

    Somerville, Chris; van de Loo, Frank

    2000-01-01

    The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds.

  7. Inhibitors of abscisic acid 8'-hydroxylase.

    PubMed

    Cutler, A J; Rose, P A; Squires, T M; Loewen, M K; Shaw, A C; Quail, J W; Krochko, J E; Abrams, S R

    2000-11-07

    Structural analogues of the phytohormone (+)-abscisic acid (ABA) have been synthesized and tested as inhibitors of the catabolic enzyme (+)-ABA 8'-hydroxylase. Assays employed microsomes from suspension-cultured corn cells. Four of the analogues [(+)-8'-acetylene-ABA, (+)-9'-propargyl-ABA, (-)-9'-propargyl-ABA, and (+)-9'-allyl-ABA] proved to be suicide substrates of ABA 8'-hydroxylase. For each suicide substrate, inactivation required NADPH, increased with time, and was blocked by addition of the natural substrate, (+)-ABA. The most effective suicide substrate was (+)-9'-propargyl-ABA (K(I) = 0.27 microM). Several analogues were competitive inhibitors of ABA 8'-hydroxylase, of which the most effective was (+)-8'-propargyl-ABA (K(i) = 1.1 microM). Enzymes in the microsomal extracts also hydroxylated (-)-ABA at the 7'-position at a low rate. This activity was not inhibited by the suicide substrates, showing that the 7'-hydroxylation of (-)-ABA was catalyzed by a different enzyme from that which catalyzed 8'-hydroxylation of (+)-ABA. Based on the results described, a simple model for the positioning of substrates in the active site of ABA 8'-hydroxylase is proposed. In a representative physiological assay, inhibition of Arabidopsis thaliana seed germination, (+)-9'-propargyl-ABA and (+)-8'-acetylene-ABA exhibited substantially stronger hormonal activity than (+)-ABA itself.

  8. The phylogeny of the aromatic amino acid hydroxylases revisited by characterizing phenylalanine hydroxylase from Dictyostelium discoideum.

    PubMed

    Siltberg-Liberles, Jessica; Steen, Ida Helene; Svebak, Randi M; Martinez, Aurora

    2008-12-31

    The social amoeba Dictyostelium discoideum contains only one aromatic amino acid hydroxylase (AAAH) gene compared to at least three in metazoans. As shown in this work this gene codes for a phenylalanine hydroxylase (DictyoPAH) and phylogenetic analysis places this enzyme close to the precursor AAAHs, aiding to define the evolutionary history of the AAAH family. DictyoPAH shows significant similarities to other eukaryote PAH, but it exhibits higher activity with tetrahydrodictyopterin (DH4) than with tetrahydrobiopterin (BH4) as cofactor. DH4 is an abundant tetrahydropterin in D. discoideum while BH4 is the natural cofactor of the AAAHs in mammals. Moreover, DictyoPAH is devoid of the characteristic regulatory mechanisms of mammalian PAH such as positive cooperativity for L-Phe and activation by preincubation with the substrate. Analysis of the few active site substitutions between DictyoPAH and mammalian PAH, including mutant expression analysis, reveals potential structural determinants for allosteric regulation.

  9. A Unique Dual Activity Amino Acid Hydroxylase in Toxoplasma gondii

    PubMed Central

    Gaskell, Elizabeth A.; Smith, Judith E.; Pinney, John W.; Westhead, Dave R.; McConkey, Glenn A.

    2009-01-01

    The genome of the protozoan parasite Toxoplasma gondii was found to contain two genes encoding tyrosine hydroxylase; that produces l-DOPA. The encoded enzymes metabolize phenylalanine as well as tyrosine with substrate preference for tyrosine. Thus the enzymes catabolize phenylalanine to tyrosine and tyrosine to l-DOPA. The catalytic domain descriptive of this class of enzymes is conserved with the parasite enzyme and exhibits similar kinetic properties to metazoan tyrosine hydroxylases, but contains a unique N-terminal extension with a signal sequence motif. One of the genes, TgAaaH1, is constitutively expressed while the other gene, TgAaaH2, is induced during formation of the bradyzoites of the cyst stages of the life cycle. This is the first description of an aromatic amino acid hydroxylase in an apicomplexan parasite. Extensive searching of apicomplexan genome sequences revealed an ortholog in Neospora caninum but not in Eimeria, Cryptosporidium, Theileria, or Plasmodium. Possible role(s) of these bi-functional enzymes during host infection are discussed. PMID:19277211

  10. Glyceryl ether monooxygenase resembles aromatic amino acid hydroxylases in metal ion and tetrahydrobiopterin dependence.

    PubMed

    Watschinger, Katrin; Keller, Markus A; Hermetter, Albin; Golderer, Georg; Werner-Felmayer, Gabriele; Werner, Ernst R

    2009-01-01

    Glyceryl ether monooxygenase is a tetrahydrobiopterin-dependent membrane-bound enzyme which catalyses the cleavage of lipid ethers into glycerol and the corresponding aldehyde. Despite many different characterisation and purification attempts, so far no gene and primary sequence have been assigned to this enzyme. The seven other tetrahydrobiopterin-dependent enzymes can be divided in the family of aromatic amino acid hydroxylases - comprising phenylalanine hydroxylase, tyrosine hydroxylase and the two tryptophan hydroxylases - and into the three nitric oxide synthases. We tested the influences of different metal ions and metal ion chelators on glyceryl ether monooxygenase, phenylalanine hydroxylase and nitric oxide synthase activity to elucidate the relationship of glyceryl ether monooxygenase to these two families. 1,10-Phenanthroline, an inhibitor of non-heme iron-dependent enzymes, was able to potently block glyceryl ether monooxygenase as well as phenylalanine hydroxylase, but had no effect on inducible nitric oxide synthase. Two tetrahydrobiopterin analogues, N(5)-methyltetrahydrobiopterin and 4-aminotetrahydrobiopterin, had a similar impact on glyceryl ether monooxygenase activity, as has already been shown for phenylalanine hydroxylase. These observations point to a close analogy of the role of tetrahydrobiopterin in glyceryl ether monooxygenase and in aromatic amino acid hydroxylases and suggest that glyceryl ether monooxygenase may require a non-heme iron for catalysis.

  11. Benzoic acid 2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic acid biosynthesis

    SciTech Connect

    Leon, J.; Shulaev, V.; Yalpani, N.

    1995-10-24

    Benzoic acid 2-hydroxylase (BA2H) catalyzes the biosynthesis of salicylic acid from benzoic acid. The enzyme has been partially purified and characterized as a soluble protein of 160 kDa. High-efficiency in vivo labeling of salicyclic acid with {sup 18}O{sub 2} suggested that BA2H is an oxygenase that specifically hydroxylates the ortho position of benzoic acid. The enzyme was strongly induced by either tobacco mosaic virus inoculation of benzoic acid infiltration of tobacco leaves and it was inhibited by CO and other inhibitors of cytochrome P450 hydroxylases. The BA2H activity was immunodepleted by antibodies raised against SU2, a soluble cytochrome P450 from Streptomyces griseolus. The anti-SU2 antibodies immunoprecipitated a radiolabeled polypeptide of around 160 kDa from the soluble protein extracts of L-[{sup 35}S]-methionine-fed tobacco leaves. Purified BA2H showed CO-difference spectra with a maximum at 457 nm. These data suggest that BA2H belongs to a novel class of soluble, high molecular weight cytochrome P450 enzymes. 21 refs., 6 figs., 1 tab.

  12. A single amino acid substitution (F363I) converts the regiochemistry of the spearmint (−)-limonene hydroxylase from a C6- to a C3-hydroxylase

    PubMed Central

    Schalk, Michel; Croteau, Rodney

    2000-01-01

    The essential oils of peppermint and spearmint are distinguished by the position of oxygenation on the constituent p-menthane monoterpenes. Peppermint produces monoterpenes bearing an oxygen at C3, whereas spearmint produces monoterpenes bearing an oxygen at C6. Branching of the monoterpene biosynthetic pathways in these species is determined by two distinct cytochrome P450s that catalyze the regiospecific hydroxylation of (−)-4S-limonene at C3 or C6 exclusively. cDNAs encoding the limonene-3-hydroxylase from peppermint and the limonene-6-hydroxylase from spearmint have been isolated, shown to be 70% identical at the amino acid level, and functionally expressed. A combination of domain swapping and reciprocal site-directed mutagenesis between these two enzymes demonstrated that the exchange of a single residue (F363I) in the spearmint limonene-6-hydroxylase led to complete conversion to the regiospecificity and catalytic efficiency of the peppermint limonene-3-hydroxylase. PMID:11050228

  13. Polymer production by Klebsiella pneumoniae 4-hydroxyphenylacetic acid hydroxylase genes cloned in Escherichia coli.

    PubMed Central

    Gibello, A; Ferrer, E; Sanz, J; Martin, M

    1995-01-01

    The expression of Klebsiella pneumoniae hpaA and hpaH genes, which code for 4-hydroxyphenylacetic acid hydroxylase in Escherichia coli K-12 derivative strains, is associated with the production of a dark brown pigment in the cultures. This pigment has been identified as a polymer which shows several of the characteristics reported for microbial melanins and results from the oxidative activity of 4-hydroxyphenylacetic acid hydroxylase on some dihydroxylated compounds to form o-quinones. A dibenzoquinone is formed from the oxidation of different mono- or dihydroxylated aromatic compounds by the enzyme prior to polymerization. We report a hydroxylase activity, other than tyrosinase, that is associated with the synthesis of a bacterial melanin. PMID:8534083

  14. Selectivity and affinity determinants for ligand binding to the aromatic amino acid hydroxylases.

    PubMed

    Teigen, Knut; McKinney, Jeffrey Alan; Haavik, Jan; Martínez, Aurora

    2007-01-01

    Hydroxylation of the aromatic amino acids phenylalanine, tyrosine and tryptophan is carried out by a family of non-heme iron and tetrahydrobiopterin (BH4) dependent enzymes, i.e. the aromatic amino acid hydroxylases (AAHs). The reactions catalyzed by these enzymes are important for biomedicine and their mutant forms in humans are associated with phenylketonuria (phenylalanine hydroxylase), Parkinson's disease and DOPA-responsive dystonia (tyrosine hydroxylase), and possibly neuropsychiatric and gastrointestinal disorders (tryptophan hydroxylase 1 and 2). We attempt to rationalize current knowledge about substrate and inhibitor specificity based on the three-dimensional structures of the enzymes and their complexes with substrates, cofactors and inhibitors. In addition, further insights on the selectivity and affinity determinants for ligand binding in the AAHs were obtained from molecular interaction field (MIF) analysis. We applied this computational structural approach to a rational analysis of structural differences at the active sites of the enzymes, a strategy that can help in the design of novel selective ligands for each AAH.

  15. Genetics Home Reference: fatty acid hydroxylase-associated neurodegeneration

    MedlinePlus

    ... Certain 2-hydroxylated fatty acids are important in forming normal myelin; myelin is the protective covering that ... C, Alshehhi AA, Proukakis C, Sibtain NA, Maier H, Sharifi R, Patton MA, Bashir W, Koul R, ...

  16. Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns.

    PubMed Central

    Cunliffe, C J; Franklin, T J; Gaskell, R M

    1986-01-01

    An assay for prolyl 4-hydroxylase (EC 1.14.11.2) is described which measures succinic acid produced during the decarboxylation of 2-oxoglutaric acid in the presence of poly(L-Pro-Gly-L-Pro). [1-14C]Succinic acid was separated from its precursor 2-oxo[5-14C]glutaric acid by using ion-exchange minicolumns. The contamination of succinic acid by 2-oxoglutaric acid was approx. 1%, and the recovery of succinic acid was 100%. Kinetic parameters of prolyl 4-hydroxylase measured by the assay showed good agreement with published values. Our experience indicates that the measurement of prolyl 4-hydroxylase by the production of succinic acid is especially suited to investigations involving large numbers of assays. PMID:3028379

  17. Salicylic acid 3-hydroxylase regulates Arabidopsis leaf longevity by mediating salicylic acid catabolism

    PubMed Central

    Zhang, Kewei; Halitschke, Rayko; Yin, Changxi; Liu, Chang-Jun; Gan, Su-Sheng

    2013-01-01

    The plant hormone salicylic acid (SA) plays critical roles in plant defense, stress responses, and senescence. Although SA biosynthesis is well understood, the pathways by which SA is catabolized remain elusive. Here we report the identification and characterization of an SA 3-hydroxylase (S3H) involved in SA catabolism during leaf senescence. S3H is associated with senescence and is inducible by SA and is thus a key part of a negative feedback regulation system of SA levels during senescence. The enzyme converts SA (with a Km of 58.29 µM) to both 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-DHBA in vitro but only 2,3-DHBA in vivo. The s3h knockout mutants fail to produce 2,3-DHBA sugar conjugates, accumulate very high levels of SA and its sugar conjugates, and exhibit a precocious senescence phenotype. Conversely, the gain-of-function lines contain high levels of 2,3-DHBA sugar conjugates and extremely low levels of SA and its sugar conjugates and display a significantly extended leaf longevity. This research reveals an elegant SA catabolic mechanism by which plants regulate SA levels by converting it to 2,3-DHBA to prevent SA overaccumulation. The research also provides strong molecular genetic evidence for an important role of SA in regulating the onset and rate of leaf senescence. PMID:23959884

  18. Salicylic acid 3-hydroxylase regulates Arabidopsis leaf longevity by mediating salicylic acid catabolism.

    PubMed

    Zhang, Kewei; Halitschke, Rayko; Yin, Changxi; Liu, Chang-Jun; Gan, Su-Sheng

    2013-09-03

    The plant hormone salicylic acid (SA) plays critical roles in plant defense, stress responses, and senescence. Although SA biosynthesis is well understood, the pathways by which SA is catabolized remain elusive. Here we report the identification and characterization of an SA 3-hydroxylase (S3H) involved in SA catabolism during leaf senescence. S3H is associated with senescence and is inducible by SA and is thus a key part of a negative feedback regulation system of SA levels during senescence. The enzyme converts SA (with a Km of 58.29 µM) to both 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-DHBA in vitro but only 2,3-DHBA in vivo. The s3h knockout mutants fail to produce 2,3-DHBA sugar conjugates, accumulate very high levels of SA and its sugar conjugates, and exhibit a precocious senescence phenotype. Conversely, the gain-of-function lines contain high levels of 2,3-DHBA sugar conjugates and extremely low levels of SA and its sugar conjugates and display a significantly extended leaf longevity. This research reveals an elegant SA catabolic mechanism by which plants regulate SA levels by converting it to 2,3-DHBA to prevent SA overaccumulation. The research also provides strong molecular genetic evidence for an important role of SA in regulating the onset and rate of leaf senescence.

  19. A mammalian fatty acid hydroxylase responsible for the formation of α-hydroxylated galactosylceramide in myelin

    PubMed Central

    Eckhardt, Matthias; Yaghootfam, Afshin; Fewou, Simon N.; Zöller, Inge; Gieselmann, Volkmar

    2005-01-01

    Hydroxylation is an abundant modification of the ceramides in brain, skin, intestinal tract and kidney. Hydroxylation occurs at the sphingosine base at C-4 or within the amide-linked fatty acid. In myelin, hydroxylation of ceramide is exclusively found at the α-C atom of the fatty acid moiety. α-Hydroxylated cerebrosides are the most abundant lipids in the myelin sheath. The functional role of this modification, however, is not known. On the basis of sequence similarity to a yeast C26 fatty acid hydroxylase, we have identified a murine cDNA encoding FA2H (fatty acid 2-hydroxylase). Transfection of FA2H cDNA in CHO cells (Chinese-hamster ovary cells) led to the formation of α-hydroxylated fatty acid containing hexosylceramide. An EGFP (enhanced green fluorescent protein)–FA2H fusion protein co-localized with calnexin, indicating that the enzyme resides in the endoplasmic reticulum. FA2H is expressed in brain, stomach, skin, kidney and testis, i.e. in tissues known to synthesize fatty acid α-hydroxylated sphingolipids. The time course of its expression in brain closely follows the expression of myelin-specific genes, reaching a maximum at 2–3 weeks of age. This is in agreement with the reported time course of fatty acid α-hydroxylase activity in the developing brain. In situ hybridization of brain sections showed expression of FA2H in the white matter. Our results thus strongly suggest that FA2H is the enzyme responsible for the formation of α-hydroxylated ceramide in oligodendrocytes of the mammalian brain. Its further characterization will provide insight into the functional role of α-hydroxylation modification in myelin, skin and other organs. PMID:15658937

  20. The Crystal Structure of an Integral Membrane Fatty Acid α-Hydroxylase*

    PubMed Central

    Zhu, Guangyu; Koszelak-Rosenblum, Mary; Connelly, Sara M.; Dumont, Mark E.; Malkowski, Michael G.

    2015-01-01

    Neuronal electrical impulse propagation is facilitated by the myelin sheath, a compact membrane surrounding the axon. The myelin sheath is highly enriched in galactosylceramide (GalCer) and its sulfated derivative sulfatide. Over 50% of GalCer and sulfatide in myelin is hydroxylated by the integral membrane enzyme fatty acid 2-hydroxylase (FA2H). GalCer hydroxylation contributes to the compact nature of the myelin membrane, and mutations in FA2H result in debilitating leukodystrophies and spastic paraparesis. We report here the 2.6 Å crystal structure of sphingolipid α-hydroxylase (Scs7p), a yeast homolog of FA2H. The Scs7p core is composed of a helical catalytic cap domain that sits atop four transmembrane helices that anchor the enzyme in the endoplasmic reticulum. The structure contains two zinc atoms coordinated by the side chains of 10 highly conserved histidines within a dimetal center located near the plane of the cytosolic membrane. We used a yeast genetic approach to confirm the important role of the dimetal-binding histidines in catalysis and identified Tyr-322 and Asp-323 as critical determinants involved in the hydroxylase reaction. Examination of the Scs7p structure, coupled with molecular dynamics simulations, allowed for the generation of a model of ceramide binding to Scs7p. Comparison of the Scs7p structure and substrate-binding model to the structure of steroyl-CoA desaturase revealed significant differences in the architecture of the catalytic cap domain and location of the dimetal centers with respect to the membrane. These observations provide insight into the different mechanisms of substrate binding and recognition of substrates by the hydroxylase and desaturase enzymes. PMID:26515067

  1. WRINKLED1 Rescues Feedback Inhibition of Fatty Acid Synthesis in Hydroxylase-Expressing Seeds1[OPEN

    PubMed Central

    Browse, John

    2016-01-01

    Previous attempts at engineering Arabidopsis (Arabidopsis thaliana) to produce seed oils containing hydroxy fatty acids (HFA) have resulted in low yields of HFA compared with the native castor (Ricinus communis) plant and caused undesirable effects, including reduced total oil content. Recent studies have led to an understanding of problems involved in the accumulation of HFA in oils of transgenic plants, which include metabolic bottlenecks and a decrease in the rate of fatty acid synthesis. Focusing on engineering the triacylglycerol assembly mechanisms led to modest increases in the HFA content of seed oil, but much room for improvement still remains. We hypothesized that engineering fatty acid synthesis in the plastids to increase flux would facilitate enhanced total incorporation of fatty acids, including HFA, into seed oil. The transcription factor WRINKLED1 (WRI1) positively regulates the expression of genes involved in fatty acid synthesis and controls seed oil levels. We overexpressed Arabidopsis WRI1 in seeds of a transgenic line expressing the castor fatty acid hydroxylase. The proportion of HFA in the oil, the total HFA per seed, and the total oil content of seeds increased to an average of 20.9%, 1.26 µg, and 32.2%, respectively, across five independent lines, compared with 17.6%, 0.83 µg, and 27.9%, respectively, for isogenic segregants. WRI1 and WRI1-regulated genes involved in fatty acid synthesis were up-regulated, providing for a corresponding increase in the rate of fatty acid synthesis. PMID:27208047

  2. Increased cholesterol 7α-hydroxylase expression and size of the bile acid pool in the lactating rat

    PubMed Central

    Wooton-Kee, Clavia Ruth; Cohen, David E.; Vore, Mary

    2008-01-01

    Maximal bile acid secretory rates and expression of bile acid transporters in liver and ileum are increased in lactation, possibly to facilitate increased enterohepatic recirculation of bile acids. We determined changes in the size and composition of the bile acid pool and key enzymes of the bile acid synthetic pathway [cholesterol 7α-hydroxylase (Cyp7a1), sterol 27-hydroxylase (Cyp27a1), and sterol 12α-hydroxylase (Cyp8b1)] in lactating rats relative to female virgin controls. The bile acid pool increased 1.9 to 2.5-fold [postpartum (PP) days 10, 14, and 19–23], compared with controls. A 1.5-fold increase in cholic acids and a 14 to 20% decrease in muricholic acids in lactation significantly increased the hydrophobicity index. In contrast, the hepatic concentration of bile acids and small heterodimer partner mRNA were unchanged in lactation. A 2.8-fold increase in Cyp7a1 mRNA expression at 16 h (10 h of light) demonstrated a shift in the diurnal rhythm at day 10 PP; Cyp7a1 protein expression and cholesterol 7α-hydroxylase activity were significantly increased at this time and remained elevated at day 14 PP but decreased to control levels by day 21 PP. There was an overall decrease in Cyp27a1 mRNA expression and a 20% decrease in Cyp27a1 protein expression, but there was no change in Cyp8b1 mRNA or protein expression at day 10 PP. The increase in Cyp7a1 expression PP provides a mechanism for the increase in the bile acid pool. PMID:18292185

  3. Single turnover kinetics of tryptophan hydroxylase: evidence for a new intermediate in the reaction of the aromatic amino acid hydroxylases.

    PubMed

    Pavon, Jorge Alex; Eser, Bekir; Huynh, Michaela T; Fitzpatrick, Paul F

    2010-09-07

    Tryptophan hydroxylase (TrpH) uses a non-heme mononuclear iron center to catalyze the tetrahydropterin-dependent hydroxylation of tryptophan to 5-hydroxytryptophan. The reactions of the TrpH.Fe(II), TrpH.Fe(II).tryptophan, TrpH.Fe(II).6MePH(4).tryptophan, and TrpH.Fe(II).6MePH(4).phenylalanine complexes with O(2) were monitored by stopped-flow absorbance spectroscopy and rapid quench methods. The second-order rate constant for the oxidation of TrpH.Fe(II) has a value of 104 M(-1) s(-1) irrespective of the presence of tryptophan. Stopped-flow absorbance analyses of the reaction of the TrpH.Fe(II).6MePH(4).tryptophan complex with oxygen are consistent with the initial step being reversible binding of oxygen, followed by the formation with a rate constant of 65 s(-1) of an intermediate I that has maximal absorbance at 420 nm. The rate constant for decay of I, 4.4 s(-1), matches that for formation of the 4a-hydroxypterin product monitored at 248 nm. Chemical-quench analyses show that 5-hydroxytryptophan forms with a rate constant of 1.3 s(-1) and that overall turnover is limited by a subsequent slow step, presumably product release, with a rate constant of 0.2 s(-1). All of the data with tryptophan as substrate can be described by a five-step mechanism. In contrast, with phenylalanine as substrate, the reaction can be described by three steps: a second-order reaction with oxygen to form I, decay of I as tyrosine forms, and slow product release.

  4. Cinnamic acid 4-hydroxylase of sorghum [Sorghum biocolor (L.) Moench] gene SbC4H1 restricts lignin synthesis in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cinnamic acid 4-hydroxylase (C4H) is the first hydroxylase enzyme of the phenylpropanoid pathway, and its content and activity affects the lignin synthesis. In this study, we isolated a C4H gene SbC4H1 from the suppression subtractive hybridization library of brown midrib (bmr) mutants of Sorghum b...

  5. Cloning and functional characterization of CYP94A2, a medium chain fatty acid hydroxylase from Vicia sativa.

    PubMed

    Le Bouquin, R; Pinot, F; Benveniste, I; Salaün, J P; Durst, F

    1999-07-22

    A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A2) was isolated from a Vicia sativa library. CYP94A2 displays 58% sequence identity with CYP94A1, a fatty acid omega-hydroxylase isolated from the same material. Heterologous expression of CYP94A2 in Saccharomyces cerevisiae yeast strain WAT11 shows that it catalyses the hydroxylation of myristic (C14) acid with a K(m(app)) of 4.0 microM and a turnover rate number of 80 min(-1). In addition, lauric (C12) and palmitic (C16) acids were hydroxylated at a ten-fold lower rate, while C18 fatty acids were not oxidized. Remarkably, the regiospecificity of hydroxylation is different for the C12, C14, and C16 fatty acids and appears to be correlated with the length of the carbon chain. Northern blot analysis showed a low level of constitutive expression of CYP94A2 in V. sativa seedlings. In contrast to CYP94A1, transcript accumulation of CYP94A2 was only weakly enhanced in seedlings treated with clofibrate or methyl jasmonate, indicating that both substrate range and gene regulation of the two fatty acid hydroxylases are different.

  6. New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

    PubMed

    Anumula, Kalyan Rao

    2012-07-01

    Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings.

  7. Induction of benzoic acid 2-hydroxylase in virus-inoculated tobacco

    SciTech Connect

    Leon, J.; Yalpani, N.; Raskin, I.; Lawton, M.A. )

    1993-10-01

    Salicylic acid (SA) plays an important role in the induction of plant resistance to pathogens. An accompanying article shows that SA is synthesized via the decarboxylation of cinnamic acid to benzoic acid (BA), which is, in turn, hydroxylated to SA. Leaf extracts of tobacco catalyze the 2-hydroxylation of Ba to SA. The monooxygenase catalyzing this reaction, benzoic acid 2-hydroxylase (BA2H), required NAD(P)H or reduced methyl viologen as an electron donor. BA2H activity was detected in healthy tobacco leaf extracts (1-2 nmol h[sup [minus]1] g[sup [minus]1] fresh weight) and was significantly increased upon inoculation with tobacco mosaic virus (TMV). This increase paralleled the levels of free SA in the leaves. Induction of BA2H activity was restricted to tissue expressing a hypersensitive response at 24[degrees]C. TMV induction of BA2H activity and Sa accumulation were inhibited when inoculated tobacco plants were incubated for 4 d at 32[degrees]C and then transferred to 24[degrees]C, they showed a 15-fold increase in BA2H activity and a 65-fold increase in free SA content compared with healthy plants incubated at 24[degrees]C. Treatment of leaf tissue with the protein synthesis inhibitor cycloheximide blocked the induction of BA2H activity by TMV. The effect of TMV inoculation on BA2H could be duplicated by infiltrating leaf discs of healthy plants with BA. This response was observed even when applied levels of BA were much lower than the levels observed in vivo after virus inoculation. Feeding tobacco leaves with phenylalanine, cinnamic acid, or o-coumaric acid (putative precursors of SA) failed to trigger the induction of BA2H activity. BA2H appears to be a pathogen-inducible protein with an important regulatory role in SA accumulation during the development of induced resistance to TMV in tobacco. 33 refs., 6 figs., 3 tabs.

  8. Liver disease in infancy caused by oxysterol 7 α-hydroxylase deficiency: successful treatment with chenodeoxycholic acid.

    PubMed

    Dai, Dongling; Mills, Philippa B; Footitt, Emma; Gissen, Paul; McClean, Patricia; Stahlschmidt, Jens; Coupry, Isabelle; Lavie, Julie; Mochel, Fanny; Goizet, Cyril; Mizuochi, Tatsuki; Kimura, Akihiko; Nittono, Hiroshi; Schwarz, Karin; Crick, Peter J; Wang, Yuqin; Griffiths, William J; Clayton, Peter T

    2014-09-01

    A child of consanguineous parents of Pakistani origin developed jaundice at 5 weeks and then, at 3 months, irritability, a prolonged prothrombin time, a low albumin, and episodes of hypoglycaemia. Investigation showed an elevated alanine aminotransferase with a normal γ-glutamyl-transpeptidase. Analysis of urine by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) showed that the major peaks were m/z 480 (taurine-conjugated 3β-hydroxy-5-cholenoic acid) and m/z 453 (sulphated 3β-hydroxy-5-cholenoic acid). Analysis of plasma by gas chromatography-mass spectrometry (GC-MS) showed increased concentrations of 3β-hydroxy-5-cholenoic acid, 3β-hydroxy-5-cholestenoic acid and 27-hydroxycholesterol, indicating oxysterol 7 α-hydroxylase deficiency. The patient was homozygous for a mutation (c.1249C>T) in CYP7B1 that alters a highly conserved residue in oxysterol 7 α-hydroxylase (p.R417C) - previously reported in a family with hereditary spastic paraplegia type 5. On treatment with ursodeoxycholic acid (UDCA), his condition was worsening, but on chenodeoxycholic acid (CDCA), 15 mg/kg/d, he improved rapidly. A biopsy (after 2 weeks on CDCA), showed a giant cell hepatitis, an evolving micronodular cirrhosis, and steatosis. The improvement in liver function on CDCA was associated with a drop in the plasma concentrations and urinary excretions of the 3β-hydroxy-Δ5 bile acids which are considered hepatotoxic. At age 5 years (on CDCA, 6 mg/kg/d), he was thriving with normal liver function. Neurological development was normal apart from a tendency to trip. Examination revealed pes cavus but no upper motor neuron signs. The findings in this case suggest that CDCA can reduce the activity of cholesterol 27-hydroxylase - the first step in the acidic pathway for bile acid synthesis.

  9. Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsis plants that express a fatty acyl hydroxylase cDNA from castor bean.

    PubMed Central

    Broun, P; Somerville, C

    1997-01-01

    A cDNA encoding the oleate 12-hydroxylase from castor bean (Ricinus communis L.) has previously been shown to direct the synthesis of small amounts of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in seeds of transgenic tobacco plants. Expression of the cDNA under control of the Brassica napus napin promoter in transgenic Arabidopsis thaliana plants resulted in the accumulation of up to 17% of seed fatty acids as ricinoleate and two novel fatty acids that have been identified by gas chromatography-mass spectrometry as lesquerolic (14-hydroxyeicos-cis-11-enoic acid) and densipolic (12-hydroxyoctadec-cis-9,15-dienoic acid) acids. Traces of auricolic acid were also observed. These results suggest that either the castor hydroxylase can utilize oleic acid and eicosenoic acid as substrates for ricinoleic and lesquerolic acid biosynthesis, respectively, or Arabidopsis contains an elongase that accepts ricinoleic acid as a substrate. These observations are also consistent with indirect biochemical evidence that an n-3 desaturase is capable of converting ricinoleic acid to densipolic acid. Expression of the castor hydroxylase also caused enhanced accumulation of oleic acid and a corresponding decrease in the levels of polyunsaturated fatty acids. Since the steady-state level of mRNA for the oleate-12 desaturase was not affected, it appears that the presence of the hydroxylase, directly or indirectly, causes posttranscriptional inhibition of desaturation. PMID:9085577

  10. A conservative amino acid substitution alters the regiospecificity of CYP94A2, a fatty acid hydroxylase from the plant Vicia sativa.

    PubMed

    Kahn, R A; Le Bouquin, R; Pinot, F; Benveniste, I; Durst, F

    2001-07-15

    Fatty acid omega-hydroxylation is involved in the biosynthesis of the plant cuticle, formation of plant defense signaling molecules, and possibly in the rapid catabolism of free fatty acids liberated under stress conditions. CYP94A2 is a cytochrome P450-dependent medium-chain fatty acid hydroxylase that was recently isolated from Vicia sativa. Contrary to CYP94A1 and CYP86A1, two other fatty acid hydroxylases previously characterized in V. sativa and Arabidopsis thaliana, CYP94A2 is not a strict omega-hydroxylase, but exhibits chain-length-dependent regioselectivity of oxidative attack. Sequence alignments of CYP94A2 with CYP94A1 and molecular modeling studies suggested that F494, located in SRS-6 (substrate recognition site) was involved in substrate recognition and positioning. Indeed, a conservative amino acid substitution at that position markedly altered the regiospecificity of CYP94A2. The observed shift from omega toward omega-1 hydroxylation was prominent with lauric acid as substrate and declined with increasing fatty acid chain length.

  11. Isoform of castor oleate hydroxylase

    DOEpatents

    Shanklin, John; Whittle, Edward J.

    2005-12-13

    The present invention relates to oleate hydroxylase genes, proteins, and methods of their use. The present invention also relates to methods of using the oleate hydroxylase genes and proteins, including in their expression in transgenic organisms and in the production of hydroxylated fatty acids.

  12. The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers.

    PubMed

    Zhang, Shengnan; Hinck, Andrew P; Fitzpatrick, Paul F

    2015-08-25

    Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10-50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains.

  13. The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat

    PubMed Central

    Verma, Shiv K.; Dubey, Jitender P.

    2017-01-01

    The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2 encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rodents less fearful of their definitive Felidae hosts. However, L-DOPA is also a structural precursor of melanins, secondary quinones, and dityrosine protein crosslinks, which are produced by many species. For example, dityrosine crosslinks are abundant in the oocyst walls of Eimeria and T. gondii, although their structural role has not been demonstrated, Here, we investigated the biology of AAH knockout parasites in the sexual reproductive cycle within cats. We found that ablation of the AAH genes resulted in reduced infection in the cat, lower oocyst yields, and decreased rates of sporulation. Our findings suggest that the AAH genes play a predominant role during infection in the gut of the definitive feline host. PMID:28288194

  14. Cyanide restores N gene-mediated resistance to tobacco mosaic virus in transgenic tobacco expressing salicylic acid hydroxylase

    PubMed

    Chivasa; Carr

    1998-09-01

    Salicylhydroxamic acid (SHAM), an inhibitor of alternative oxidase (AOX), blocks salicylic acid-induced resistance to tobacco mosaic virus (TMV) but does not inhibit pathogenesis-related PR-1 protein synthesis or resistance to fungal and bacterial pathogens. We found that the synthetic resistance-inducing chemical 2, 6-dichloroisonicotinic acid also induced Aox transcript accumulation and SHAM-sensitive resistance to TMV. The respiratory inhibitors antimycin A and KCN also induced Aox transcript accumulation and resistance to TMV but did not induce PR-1 accumulation. Tobacco plants of the TMV-resistant cultivar Samsun NN transformed with the salicylic acid hydroxylase (nahG) gene could no longer restrict virus to the inoculation site, resulting in spreading necrosis instead of discrete necrotic lesions. Treatment with KCN restored TMV localization and normal lesion morphology. SHAM antagonized this effect, allowing virus escape and spreading necrosis to resume. The results demonstrate the importance of the SHAM-sensitive (potentially AOX-dependent) signal transduction pathway in mediating virus localization early in the hypersensitive response.

  15. Hydroxytyrosol from tyrosol using hydroxyphenylacetic acid-induced bacterial cultures and evidence of the role of 4-HPA 3-hydroxylase.

    PubMed

    Liebgott, Pierre-Pol; Amouric, Agnès; Comte, Alexia; Tholozan, Jean-Luc; Lorquin, Jean

    2009-12-01

    Hydroxytyrosol (HTyr) is a potent natural antioxidant found in olive mill wastewaters. Bacterial conversion of 4-tyrosol (2-(4-hydroxyphenyl)-ethanol) to HTyr was reported in a limited number of bacterial species including Pseudomonas aeruginosa. In this work, we studied this conversion, taking as a model the newly isolated Halomonas sp. strain HTB24. It was first hypothesized that the enzyme responsible for 4-tyrosol hydroxylation in HTyr was a 4-hydroxyphenylacetic acid 3-hydroxylase (HPAH, EC 1.14.13.3), previously known to convert 4-hydroxyphenylacetic acid (4-HPA) into 3,4-dihydroxyphenylacetic acid (3,4-DHPA) in P. aeruginosa. Cloning and expression of hpaB (oxygenase component) and hpaC (reductase component) genes from P. aeruginosa confirmed this hypothesis. Furthermore, using cultures of HTB24 containing 4-tyrosol, it was shown that 4-HPA accumulation preceded 4-tyrosol hydroxylation. We further demonstrated that the synthesis of HPAH activity was induced by 4-HPA, with the latter compound being formed from 4-tyrosol oxidation by aryl-dehydrogenases. Interestingly, similar results were obtained with other 4-HPA-induced bacteria, including P. aeruginosa, Serratia marcescens, Escherichia coli, Micrococcus luteus and other Halomonas, thus demonstrating general hydroxylating activity of 4-tyrosol by the HPAH enzyme. E. coli W did not have aryl-dehydrogenase activity and hence were unable to oxidize 4-tyrosol to 4-HPA and HTyr to 3,4-DHPA, making this bacterium a good candidate for achieving better HTyr production.

  16. Species-specific mechanisms for cholesterol 7alpha-hydroxylase (CYP7A1) regulation by drugs and bile acids.

    PubMed

    Handschin, Christoph; Gnerre, Carmela; Fraser, David J; Martinez-Jimenez, Celia; Jover, Ramiro; Meyer, Urs A

    2005-02-01

    The gene encoding cholesterol 7alpha-hydroxylase (CYP7A1) is tightly regulated in order to control intrahepatic cholesterol and bile acid levels. Ligands of the xenobiotic-sensing pregnane X receptor inhibit CYP7A1 expression. To retrace the evolution of the molecular mechanisms underlying CYP7A1 inhibition, we used a chicken hepatoma cell system that retains the ability to be induced by phenobarbital and other drugs. Whereas bile acids regulate CYP7A1 via small heterodimer partner and liver receptor homolog-1, mRNA expression of these nuclear receptors is unchanged by xenobiotics. Instead, drugs repress chicken hepatic nuclear factor 4alpha (HNF4alpha) transcript levels concomitant with a reduction in CYP7A1 expression. Importantly, no reduction of HNF4alpha levels is found in mouse liver in vivo and in human primary hepatocyte cultures, respectively. Thus, besides the importance of HNF4alpha in CYP7A1 regulation in all species, birds and mammals use different signaling pathways to adjust CYP7A1 levels after exposure to xenobiotics.

  17. Specific accumulation of CYP94A1 transcripts after exposure to gaseous benzaldehyde: induction of lauric acid ω-hydroxylase activity in Vicia sativa exposed to atmospheric pollutants.

    PubMed

    Kastner, P E; Le Calvé, S; Diss, L; Sauveplane, V; Franke, R; Schreiber, L; Pinot, F

    2011-01-01

    The effects of air pollutants such as aldehydes, ozone, nitrogen dioxide and benzene on fatty acid ω-hydroxylase activity in Vicia sativa microsomes have been investigated. Four days old etiolated V. sativa seedlings were exposed to different concentrations of selected pollutants for varying exposure times. Growing etiolated V. sativa seedlings in air containing the gaseous benzaldehyde (150 nM) led to an 8-fold enhancement of lauric acid ω-hydroxylase activity in microsomes of treated plants compared to controls grown in pure air (96 ± 10 versus 12 ± 2 pmol/min/mg protein, respectively). The induction increased with increasing gas phase concentrations (10-1300 nM) and the maximum of activity was measured after 48 h of exposure. Northern blot analysis revealed that this induction occurred via transcriptional activation of the gene coding for CYP94A1. The absence of CYP94A2 and CYP94A3 transcription activation together with the missing effect on epoxide hydrolases activities indicate the specificity of CYP94A1 induction by benzaldehyde. Exposure to nitrogen dioxide, ozone and formaldehyde also stimulated lauric acid ω-hydroxylases activity while exposure to benzene did not show any effect.

  18. Caffeic acid phenethyl ester is a potent inhibitor of HIF prolyl hydroxylase: structural analysis and pharmacological implication.

    PubMed

    Choi, Daekyu; Han, Jeongoh; Lee, Youna; Choi, Jungyun; Han, Songyi; Hong, Sungchae; Jeon, Hyunchu; Kim, Young Mi; Jung, Yunjin

    2010-09-01

    Caffeic acid phenethyl ester (CAPE) is an active component of propolis from honeybee. We investigated a potential molecular mechanism underlying a CAPE-mediated protective effect against ischemia/reperfusion (I/R) injury and analyzed the structure contributing to the CAPE effect. CAPE induced hypoxia-inducible factor-1 (HIF-1) alpha protein, concomitantly transactivating the HIF-1 target genes vascular endothelial growth factor and heme oxygenase-1, which play a protective role in I/R injury. CAPE delayed the degradation of HIF-1alpha protein in cells, which occurred by inhibition of HIF prolyl hydroxylase (HPH), the key enzyme for von Hippel-Lindau-dependent HIF-1alpha degradation. CAPE inhibition of HPH and induction of HIF-1alpha protein were neutralized by an elevated dose of iron. The catechol moiety, a chelating group, is essential for HPH inhibition, while hydrogenation of the double bond (-C=C-) in the Michael reaction acceptor markedly reduced potency. Removal of the phenethyl moiety of CAPE (substitution with the methyl moiety) severely deteriorated its inhibitory activity for HPH. Our data suggest that a beneficial effect of CAPE on I/R injury may be ascribed to the activation of HIF-1 pathway via inhibition of HPH and reveal that the chelating moiety of CAPE acted as a pharmacophore while the double bond and phenethyl moiety assisted in inhibiting HPH.

  19. Induction of the fatty acid 2-hydroxylase (FA2H) gene by Δ(9)-tetrahydrocannabinol in human breast cancer cells.

    PubMed

    Takeda, Shuso; Harada, Mari; Su, Shengzhong; Okajima, Shunsuke; Miyoshi, Hiroko; Yoshida, Kazutaka; Nishimura, Hajime; Okamoto, Yoshiko; Amamoto, Toshiaki; Watanabe, Kazuhito; Omiecinski, Curtis J; Aramaki, Hironori

    2013-01-01

    To investigate gene(s) being regulated by ∆(9)-tetrahydrocannabinol (∆(9)-THC), we performed DNA microarray analysis of human breast cancer MDA-MB-231 cells, which are poorly differentiated breast cancer cells, treated with ∆(9)-THC for 48 hr at an IC50 concentration of approximately 25 µM. Among the highly up-regulated genes (> 10-fold) observed, fatty acid 2-hydroxylase (FA2H) was significantly induced (17.8-fold). Although the physiological role of FA2H has not yet been fully understood, FA2H has been shown to modulate cell differentiation. The results of Oil Red O staining after ∆(9)-THC exposure showed the distribution of lipid droplets (a sign of the differentiated phenotype) in cells. Taken together, the results obtained here indicate that FA2H is a novel ∆(9)-THC-regulated gene, and that ∆(9)-THC induces differentiation signal(s) in poorly differentiated MDA-MB-231 cells.

  20. Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE1[OPEN

    PubMed Central

    Van de Wouwer, Dorien; Decou, Raphaël; Audenaert, Dominique; Nguyen, Long

    2016-01-01

    Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization. PMID:27485881

  1. Retinoic acid-related orphan receptor α regulates diurnal rhythm and fasting induction of sterol 12α-hydroxylase in bile acid synthesis.

    PubMed

    Pathak, Preeti; Li, Tiangang; Chiang, John Y L

    2013-12-27

    Sterol 12α-hydroxylase (CYP8B1) is required for cholic acid synthesis and plays a critical role in intestinal cholesterol absorption and pathogenesis of cholesterol gallstone, dyslipidemia, and diabetes. In this study we investigated the underlying mechanism of fasting induction and circadian rhythm of CYP8B1 by a cholesterol-activated nuclear receptor and core clock gene retinoic acid-related orphan receptor α (RORα). Fasting stimulated, whereas restricted-feeding reduced expression of CYP8B1 mRNA and protein. However, fasting and feeding had little effect on the diurnal rhythm of RORα mRNA expression, but fasting increased RORα protein levels by cAMP-activated protein kinase A-mediated phosphorylation and stabilization of the protein. Adenovirus-mediated gene transduction of RORα to mice strongly induced CYP8B1 expression, and increased liver cholesterol and 12α-hydroxylated bile acids in the bile acid pool and serum. A reporter assay identified a functional RORα response element in the CYP8B1 promoter. RORα recruited cAMP response element-binding protein-binding protein (CBP) to stimulate histone acetylation on the CYP8B1 gene promoter. In conclusion, RORα is a key regulator of diurnal rhythm and fasting induction of CYP8B1, which regulates bile acid composition and serum and liver cholesterol levels. Antagonizing RORα activity may be a therapeutic strategy for treating inflammatory diseases such as non-alcoholic fatty liver disease and type 2 diabetes.

  2. Use of plant fatty acyl hydroxylases to produce hydroxylated fatty acids and derivatives in plants

    DOEpatents

    Somerville, C.; Loo, F. van de

    1997-09-16

    The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds. 35 figs.

  3. Use of plant fatty acyl hydroxylases to produce hydroxylated fatty acids and derivatives in plants

    DOEpatents

    Somerville, Chris; van de Loo, Frank

    1998-01-01

    The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds.

  4. Use of plant fatty acyl hydroxylases to produce hydroxylated fatty acids and derivatives in plants

    DOEpatents

    Somerville, Chris; van de Loo, Frank

    2002-01-01

    The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds.

  5. Use of plant fatty acyl hydroxylases to produce hydroxylated fatty acids and derivatives in plants

    DOEpatents

    Somerville, C.; Loo, F. van de

    1998-09-01

    The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds. 35 figs.

  6. Use of plant fatty acyl hydroxylases to produce hydroxylated fatty acids and derivatives in plants

    DOEpatents

    Somerville, Chris; van de Loo, Frank

    1997-01-01

    The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds.

  7. Abscinazole-E3M, a practical inhibitor of abscisic acid 8′-hydroxylase for improving drought tolerance

    PubMed Central

    Takeuchi, Jun; Okamoto, Masanori; Mega, Ryosuke; Kanno, Yuri; Ohnishi, Toshiyuki; Seo, Mitsunori; Todoroki, Yasushi

    2016-01-01

    Abscisic acid (ABA) is an essential phytohormone that regulates plant water use and drought tolerance. However, agricultural applications of ABA have been limited because of its rapid inactivation in plants, which involves hydroxylation of ABA by ABA 8′-hydroxylase (CYP707A). We previously developed a selective inhibitor of CYP707A, (−)-Abz-E2B, by structurally modifying S-uniconazole, which functions as an inhibitor of CYP707A and as a gibberellin biosynthetic enzyme. However, its synthetic yield is too low for practical applications. Therefore, we designed novel CYP707A inhibitors, Abz-T compounds, that have simpler structures in which the 1,2,3-triazolyl ring of (−)-Abz-E2B has been replaced with a triple bond. They were successfully synthesised in shorter steps, resulting in greater yields than that of (−)-Abz-E2B. In the enzymatic assays, one of the Abz-T compounds, (−)-Abz-E3M, acted as a strong and selective inhibitor of CYP707A, similar to (−)-Abz-E2B. Analysis of the biological effects in Arabidopsis revealed that (−)-Abz-E3M enhanced ABA’s effects more than (−)-Abz-E2B in seed germination and in the expression of ABA-responsive genes. Treatment with (−)-Abz-E3M induced stomatal closure and improved drought tolerance in Arabidopsis. Furthermore, (−)-Abz-E3M also increased the ABA response in rice and maize. Thus, (−)-Abz-E3M is a more practical and effective inhibitor of CYP707A than (−)-Abz-E2B. PMID:27841331

  8. Phenylalanine hydroxylase deficiency.

    PubMed

    Mitchell, John J; Trakadis, Yannis J; Scriver, Charles R

    2011-08-01

    Phenylalanine hydroxylase deficiency is an autosomal recessive disorder that results in intolerance to the dietary intake of the essential amino acid phenylalanine. It occurs in approximately 1:15,000 individuals. Deficiency of this enzyme produces a spectrum of disorders including classic phenylketonuria, mild phenylketonuria, and mild hyperphenylalaninemia. Classic phenylketonuria is caused by a complete or near-complete deficiency of phenylalanine hydroxylase activity and without dietary restriction of phenylalanine most children will develop profound and irreversible intellectual disability. Mild phenylketonuria and mild hyperphenylalaninemia are associated with lower risk of impaired cognitive development in the absence of treatment. Phenylalanine hydroxylase deficiency can be diagnosed by newborn screening based on detection of the presence of hyperphenylalaninemia using the Guthrie microbial inhibition assay or other assays on a blood spot obtained from a heel prick. Since the introduction of newborn screening, the major neurologic consequences of hyperphenylalaninemia have been largely eradicated. Affected individuals can lead normal lives. However, recent data suggest that homeostasis is not fully restored with current therapy. Treated individuals have a higher incidence of neuropsychological problems. The mainstay of treatment for hyperphenylalaninemia involves a low-protein diet and use of a phenylalanine-free medical formula. This treatment must commence as soon as possible after birth and should continue for life. Regular monitoring of plasma phenylalanine and tyrosine concentrations is necessary. Targets of plasma phenylalanine of 120-360 μmol/L (2-6 mg/dL) in the first decade of life are essential for optimal outcome. Phenylalanine targets in adolescence and adulthood are less clear. A significant proportion of patients with phenylketonuria may benefit from adjuvant therapy with 6R-tetrahydrobiopterin stereoisomer. Special consideration must be

  9. Structural and Functional Characteristics of Oxysterol 7α-Hydroxylase with Amino-Acid Substitution R486C and Their Relation to the Appearance of Neurodegenerative Diseases

    NASA Astrophysics Data System (ADS)

    Dichenko, Ya. V.; Yantsevich, A. V.; Usanov, S. A.

    2015-03-01

    The influence of the amino-acid substitution Arg486Cys on the conformational stability of recombinant cytochrome P450 7B1 (CYP7B1, oxysterol 7α-hydroxylase) was studied. The single base change was shown to decrease the free energy of the transition of the heme-protein from its native state to a denatured one, which pointed to a lower thermodynamic stability for the mutant form of the enzyme. This could be the cause of the metabolic disruption of neurosteroids and, as a consequence, the appearance of neurodegenerative diseases.

  10. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF.

    PubMed

    Koivunen, Peppi; Hirsilä, Maija; Remes, Anne M; Hassinen, Ilmo E; Kivirikko, Kari I; Myllyharju, Johanna

    2007-02-16

    The stability and transcriptional activity of the hypoxia-inducible factors (HIFs) are regulated by two oxygen-dependent events that are catalyzed by three HIF prolyl 4-hydroxylases (HIF-P4Hs) and one HIF asparaginyl hydroxylase (FIH). We have studied possible links between metabolic pathways and HIF hydroxylases by analyzing the abilities of citric acid cycle intermediates to inhibit purified human HIF-P4Hs and FIH. Fumarate and succinate were identified as in vitro inhibitors of all three HIF-P4Hs, fumarate having K(i) values of 50-80 microM and succinate 350-460 microM, whereas neither inhibited FIH. Oxaloacetate was an additional inhibitor of all three HIF-P4Hs with K(i) values of 400-1000 microM and citrate of HIF-P4H-3, citrate being the most effective inhibitor of FIH with a K(i) of 110 microM. Culturing of cells with fumarate diethyl or dimethyl ester, or a high concentration of monoethyl ester, stabilized HIF-1alpha and increased production of vascular endothelial growth factor and erythropoietin. Similar, although much smaller, changes were found in cultured fibroblasts from a patient with fumarate hydratase (FH) deficiency and upon silencing FH using small interfering RNA. No such effects were seen upon culturing of cells with succinate diethyl or dimethyl ester. As FIH was not inhibited by fumarate, our data indicate that the transcriptional activity of HIF is quite high even when binding of the coactivator p300 is prevented. Our data also support recent suggestions that the increased fumarate and succinate levels present in the FH and succinate dehydrogenase-deficient tumors, respectively, can inhibit the HIF-P4Hs with consequent stabilization of HIF-alphas and effects on tumor pathology.

  11. The Arabidopsis cytochrome P450 CYP86A1 encodes a fatty acid ω-hydroxylase involved in suberin monomer biosynthesis

    PubMed Central

    Höfer, Rene; Briesen, Isabel; Beck, Martina; Pinot, Franck; Schreiber, Lukas; Franke, Rochus

    2008-01-01

    The lipophilic biopolyester suberin forms important boundaries to protect the plant from its surrounding environment or to separate different tissues within the plant. In roots, suberin can be found in the cell walls of the endodermis and the hypodermis or periderm. Apoplastic barriers composed of suberin accomplish the challenge to restrict water and nutrient loss and prevent the invasion of pathogens. Despite the physiological importance of suberin and the knowledge of the suberin composition of many plants, very little is known about its biosynthesis and the genes involved. Here, a detailed analysis of the Arabidopsis aliphatic suberin in roots at different developmental stages is presented. This study demonstrates some variability in suberin amount and composition along the root axis and indicates the importance of ω-hydroxylation for suberin biosynthesis. Using reverse genetics, the cytochrome P450 fatty acid ω-hydroxylase CYP86A1 (At5g58860) has been identified as a key enzyme for aliphatic root suberin biosynthesis in Arabidopsis. The corresponding horst mutants show a substantial reduction in ω-hydroxyacids with a chain length hydroxylase of root suberized tissue. Detailed expression studies revealed a strong root specificity and a localized expression in the root endodermis. Transgenic expression of CYP86A1 fused to GFP distributed CYP86A1 to the endoplasmic reticulum, indicating that suberin monomer biosynthesis takes place in this sub-cellular compartment before intermediates are exported in the apoplast. PMID:18544608

  12. CYP94A1, a plant cytochrome P450-catalyzing fatty acid omega-hydroxylase, is selectively induced by chemical stress in Vicia sativa seedlings.

    PubMed

    Benveniste, Irène; Bronner, Roberte; Wang, Yong; Compagnon, Vincent; Michler, Pierre; Schreiber, Lukas; Salaün, Jean-Pierre; Durst, Francis; Pinot, Franck

    2005-08-01

    CYP94A1 is a cytochrome P450 (P450) catalyzing fatty acid (FA) omega-hydroxylation in Vicia sativa seedlings. To study the physiological role of this FA monooxygenase, we report here on its regulation at the transcriptional level (Northern blot). Transcripts of CYP94A1, as those of two other P450-dependent FA hydroxylases (CYP94A2 and CYP94A3) from V. sativa, are barely detectable during the early development of the seedlings. CYP94A1 transcripts, in contrast to those of the two other isoforms, are rapidly (less than 20 min) and strongly (more than 100 times) enhanced after treatment by clofibrate, an hypolipidemic drug in animals and an antiauxin (p-chlorophenoxyisobutyric acid) in plants, by auxins (2,4-dichlorophenoxyacetic acid and indole-3-acetic acid), by an inactive auxin analog (2,3-dichlorophenoxyacetic acid), and also by salicylic acid. All these compounds activate CYP94A1 transcription only at high concentrations (50-500 microM range). In parallel, these high levels of clofibrate and auxins modify seedling growth and development. Therefore, the expression of CYP94A1 under these conditions and the concomitant morphological and cytological modifications would suggest the implication of this P450 in a process of plant defense against chemical injury.

  13. Physiological function of the Pseudomonas putida PpG6 (Pseudomonas oleovorans) alkane hydroxylase: monoterminal oxidation of alkanes and fatty acids.

    PubMed Central

    Nieder, M; Shapiro, J

    1975-01-01

    Pseudomonas putida PpG6 is able to utilize purified n-alkanes of six to ten carbon atoms for growth. It can also grow on the primary terminal oxidation products of these alkanes and on 1-dodecanol but not on the corresponding 2-ketones or 1,6-hexanediol, adipic acid, or pimelic acid. Revertible point mutants can be isolated which have simultaneously lost the ability to grow on all five n-alkane growth substrates but which can still grow on octanol or nonanol. An acetate-negative mutant defective in isocitrate lysase activity is unable to grow on even-numbered alkanes and fatty acids. Analysis of double mutants defective in acetate and propionate or in acetate and glutarate metabolism shows that alkane carbon is assimilated only via acetyl-coenzyme A and propionyl-coenzyme A. These results support the following conclusions: (i) The n-alkane growth specificity of P. putida PpG6 is due to the substrate specificity of whole-cell alkane hydroxylation; (ii) there is a single alkane hydroxylase enzyme complex; (iii) the physiological role of this complex is to initiate the monoterminal oxidation of alkane chains; and (iv) straight-chain fatty acids from butyric through nonanoic are degraded exclusively by beta-oxidation from the carboxyl end of the molecule. PMID:804473

  14. Methyl jasmonate induces lauric acid omega-hydroxylase activity and accumulation of CYP94A1 transcripts but does not affect epoxide hydrolase activities in vicia sativa seedlings

    PubMed

    Pinot; Benveniste; Sala n JP; Durst

    1998-12-01

    Treatment of etiolated Vicia sativa seedlings by the plant hormone methyl jasmonate (MetJA) led to an increase of cytochrome P450 content. Seedlings that were treated for 48 h in a 1 mM solution of MetJA stimulated omega-hydroxylation of 12:0 (lauric acid) 14-fold compared with the control (153 versus 11 pmol min-1 mg-1 protein, respectively). Induction was dose dependent. The increase of activity (2.7-fold) was already detectable after 3 h of treatment. Activity increased as a function of time and reached a steady level after 24 h. Northern-blot analysis revealed that the transcripts coding for CYP94A1, a fatty acid omega-hydroxylase, had already accumulated after 1 h of exposure to MetJA and was maximal between 3 and 6 h. Under the same conditions, a study of the enzymatic hydrolysis of 9,10-epoxystearic acid showed that both microsomal and soluble epoxide hydrolase activities were not affected by MetJA treatment.

  15. Optimization of naringenin and p-coumaric acid hydroxylation using the native E. coli hydroxylase complex, HpaBC.

    PubMed

    Jones, J Andrew; Collins, Shannon M; Vernacchio, Victoria R; Lachance, Daniel M; Koffas, Mattheos A G

    2016-01-01

    Flavonoids are a growing class of bioactive natural products with distinct and interesting bioactivity both in vitro and in vivo. The extraction of flavonoids from plant sources is limited by their low natural abundance and commonly results in a mixture of products that are difficult to separate. However, due to recent advances, the microbial production of plant natural products has developed as a promising alternative for flavonoid production. Through optimization of media, induction temperature, induction point, and substrate delay time, we demonstrate the highest conversion of naringenin to eriodictyol (62.7 ± 2.7 mg/L) to date, using the native E. coli hydroxylase complex, HpaBC. We also show the first evidence of in vivo HpaBC activity towards the monohydroxylated flavan-3-ol afzelechin with catechin product titers of 34.7 ± 1.5 mg/L. This work confirms the wide applicability of HpaBC towards realizing efficient de novo production of various orthohydroxylated flavonoids and flavonoid derived products in E. coli.

  16. Cloning and characterization of Lonicera japonica p-coumaroyl ester 3-hydroxylase which is involved in the biosynthesis of chlorogenic acid.

    PubMed

    Pu, Gaobin; Wang, Peng; Zhou, Bingqian; Liu, Zhenhua; Xiang, Fengning

    2013-01-01

    Lonicera japonica is used in Chinese medicine as a source of antioxidants, primarily flavonoids, and a phenolic acid chlorogenic acid (CGA). Here we report the isolation and characterization of the full-length cDNA of LjC3H, a gene encoding p-coumaroyl ester 3-hydroxylase, an enzyme involved in CGA synthesis. Phylogenetic analysis indicated that is protein belongs to the CYP98A subfamily, and homology modeling revealed that its structure resembles that of other cytochrome P450 family proteins. Southern blot analysis indicated that more than one copy of sequences homologous to LjC3H is present in the L. japonica genome. Heterologous expression of LjC3H cDNA in Escherichia coli allowed an in vitro assay of LjC3H to be performed. This experiment revealed that the enzyme favors p-coumaroylshikimate over p-coumaroylquinate as substrate. LjC3H transcript abundance was increased both by treatment of the leaves with methyl jasmonate and by exposure to UV-B radiation. The CGA levels in the leaves of L. japonica were positively correlated with LjC3H transcript abundance.

  17. Glucagon and cAMP inhibit cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in human hepatocytes: discordant regulation of bile acid synthesis and gluconeogenesis.

    PubMed

    Song, Kwang-Hoon; Chiang, John Y L

    2006-01-01

    The gene encoding cholesterol 7alpha-hydroxylase (CYP7A1) is tightly regulated to control bile acid synthesis and maintain lipid homeostasis. Recent studies in mice suggest that bile acid synthesis is regulated by the fasted-to-fed cycle, and fasting induces CYP7A1 gene expression in parallel to the induction of peroxisome proliferators-activated receptor gamma co-activator 1alpha (PGC-1alpha) and phosphoenolpyruvate carboxykinase (PEPCK). How glucagon regulates CYP7A1 gene expression in the human liver is not clear. Here we show that glucagon and cyclic adenosine monophosphate (cAMP) strongly repressed CYP7A1 mRNA expression in human primary hepatocytes. Reporter assays confirmed that cAMP and protein kinase A (PKA) inhibited human CYP7A1 gene transcription, in contrast to their stimulation of the PEPCK gene. Mutagenesis analysis identified a PKA-responsive region located within the previously identified HNF4alpha binding site in the human CYP7A1 promoter. Glucagon and cAMP increased HNF4alpha phosphorylation and reduced the amount of HNF4alpha present in CYP7A1 chromatin. Our findings suggest that glucagon inhibited CYP7A1 gene expression via PKA phosphorylation of HNF4alpha, which lost its ability to bind the CYP7A1 gene and resulted in inhibition of human CYP7A1 gene transcription. In conclusion, this study unveils a species difference in nutrient regulation of the human and mouse CYP7A1 gene and suggests a discordant regulation of bile acid synthesis and gluconeogenesis by glucagon in human livers during fasting.

  18. Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning

    PubMed Central

    GAO, Hanchao; ZHAO, Chengjiang; XIANG, Xi; LI, Yong; ZHAO, Yanli; LI, Zesong; PAN, Dengke; DAI, Yifan; HARA, Hidetaka; COOPER, David K.C.; CAI, Zhiming; MOU, Lisha

    2016-01-01

    Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications. PMID:27725344

  19. Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.

    PubMed

    Gao, Hanchao; Zhao, Chengjiang; Xiang, Xi; Li, Yong; Zhao, Yanli; Li, Zesong; Pan, Dengke; Dai, Yifan; Hara, Hidetaka; Cooper, David K C; Cai, Zhiming; Mou, Lisha

    2017-02-16

    Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

  20. Comparison of the Function and Expression of CYP26A1 and CYP26B1, the two Retinoic Acid Hydroxylases

    PubMed Central

    Topletz, Ariel R.; Thatcher, Jayne E.; Zelter, Alex; Lutz, Justin D.; Tay, Suzanne; Nelson, Wendel L.; Isoherranen, Nina

    2011-01-01

    All-trans-retinoic acid (atRA) is an important signaling molecule in all chordates. The cytochrome P450 enzymes CYP26 are believed to partially regulate cellular concentrations of atRA via oxidative metabolism and hence affect retinoid homeostasis and signaling. CYP26A1 and CYP26B1 are atRA hydroxylases that catalyze formation of similar metabolites in cell systems. However, they have only 40% sequence similarity suggesting differences between the two enzymes. The aim of this study was to determine whether CYP26A1 and CYP26B1 have similar catalytic activity, form different metabolites from atRA and are expressed in different tissues in adults. The mRNA expression of CYP26A1 and CYP26B1 correlated between human tissues except for human cerebellum in which CYP26B1 was the predominant CYP26 and liver in which CYP26A1 dominated. Quantification of CYP26A1 and CYP26B1 protein in human tissues was in agreement with the mRNA expression and showed correlation between the two isoforms. Qualitatively, recombinant CYP26A1 and CYP26B1 formed the same primary and sequential metabolites from atRA. Quantitatively, CYP26B1 had a lower Km (19nM) and Vmax (0.8pmol/min/pmol) than CYP26A1 (Km=50nM and Vmax=10pmol/min/pmol) for formation of 4-OH-RA. The major atRA metabolites 4-OH-RA, 18-OH-RA and 4-oxo-RA were all substrates of CYP26A1 and CYP26B1, and CYP26A1 had a 2–10 fold higher catalytic activity towards all substrates tested. This study shows that CYP26A1 and CYP26B1 are qualitatively similar RA hydroxylases with overlapping expression profiles. CYP26A1 has higher catalytic activity than CYP26B1 and seems to be responsible for metabolism of atRA in tissues that function as a barrier for atRA exposure. PMID:22020119

  1. Bile acids and cytokines inhibit the human cholesterol 7 alpha-hydroxylase gene via the JNK/c-jun pathway in human liver cells.

    PubMed

    Li, Tiangang; Jahan, Asmeen; Chiang, John Y L

    2006-06-01

    Cholesterol 7 alpha-hydroxylase (CYP7A1) of the bile acid biosynthesis pathway is suppressed by bile acids and inflammatory cytokines. Bile acids are known to induce inflammatory cytokines to activate the mitogen-activated protein kinase/c-Jun N-terminal kinase (JNK) signaling pathway that inhibits CYP7A1 gene transcription. c-Jun has been postulated to mediate bile acid inhibition of CYP7A1. However, the c-Jun target involved in the regulation of CYP7A1 is unknown. Human primary hepatocytes and HepG2 cells were used as models to study chenodeoxycholic acid (CDCA) and interleukin-1 beta (IL-1 beta) regulation of human CYP7A1 gene expression via real-time polymerase chain reaction, reporter assays, co-immunoprecipitation and chromatin immunocipitation (ChIP) assays. IL-1 beta and CDCA reduced CYP7A1 but induced c-Jun messenger RNA expression in human primary hepatocytes. IL-1beta inhibited human CYP7A1 reporter activity via the HNF4 alpha binding site. A JNK-specific inhibitor blocked the inhibitory effect of IL-1 beta on HNF4 alpha expression and CYP7A1 reporter activity. c-Jun inhibited HNF4 alpha and PPARgamma coactivator-1 alpha (PGC-1 alpha) coactivation of CYP7A1 reporter activity, whereas a dominant negative c-Jun did not. Co-immunoprecipitation and ChIP assays revealed that IL-1 beta and CDCA reduced HNF4 alpha bound to the CYP7A1 chromatin, and that c-Jun interacted with HNF4 alpha and blocked HNF4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin. In conclusion, IL-1 beta and CDCA inhibit HNF4 alpha but induce c-Jun, which in turn blocks HNF 4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin and results in inhibition of CYP7A1 gene transcription. The JNK/c-Jun signaling pathway inhibits bile acid synthesis and protects hepatocytes against the toxic effect of inflammatory agents.

  2. Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius

    PubMed Central

    Sadeghi, Mahnaz; Dehghan, Sara; Fischer, Rainer; Wenzel, Uwe; Vilcinskas, Andreas; Kavousi, Hamid Reza; Rahnamaeian, Mohammad

    2013-01-01

    Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3–6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6–24 h and 3–6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance. PMID:24309561

  3. Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius.

    PubMed

    Sadeghi, Mahnaz; Dehghan, Sara; Fischer, Rainer; Wenzel, Uwe; Vilcinskas, Andreas; Kavousi, Hamid Reza; Rahnamaeian, Mohammad

    2013-11-01

    Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3-6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6-24 h and 3-6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance.

  4. Phenylalanine hydroxylase misfolding and pharmacological chaperones.

    PubMed

    Underhaug, Jarl; Aubi, Oscar; Martinez, Aurora

    2012-01-01

    Phenylketonuria (PKU) is a loss-of-function inborn error of metabolism. As many other inherited diseases the main pathologic mechanism in PKU is an enhanced tendency of the mutant phenylalanine hydroxylase (PAH) to misfold and undergo ubiquitin-dependent degradation. Recent alternative approaches with therapeutic potential for PKU aim at correcting the PAH misfolding, and in this respect pharmacological chaperones are the focus of increasing interest. These compounds, which often resemble the natural ligands and show mild competitive inhibition, can rescue the misfolded proteins by stimulating their renaturation in vivo. For PKU, a few studies have proven the stabilization of PKU-mutants in vitro, in cells, and in mice by pharmacological chaperones, which have been found either by using the tetrahydrobiopterin (BH(4)) cofactor as query structure for shape-focused virtual screening or by high-throughput screening of small compound libraries. Both approaches have revealed a number of compounds, most of which bind at the iron-binding site, competitively with respect to BH(4). Furthermore, PAH shares a number of ligands, such as BH(4), amino acid substrates and inhibitors, with the other aromatic amino acid hydroxylases: the neuronal/neuroendocrine enzymes tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPHs). Recent results indicate that the PAH-targeted pharmacological chaperones should also be tested on TH and the TPHs, and eventually be derivatized to avoid unwanted interactions with these other enzymes. After derivatization and validation in animal models, the PAH-chaperoning compounds represent novel possibilities in the treatment of PKU.

  5. Evidence for Modified Mechanisms of Chloroethene Oxidation in Pseudomonas butanovora Mutants Containing Single Amino Acid Substitutions in the Hydroxylase α-Subunit of Butane Monooxygenase▿

    PubMed Central

    Halsey, Kimberly H.; Doughty, David M.; Sayavedra-Soto, Luis A.; Bottomley, Peter J.; Arp, Daniel J.

    2007-01-01

    The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of Pseudomonas butanovora containing single amino acid substitutions in the α-subunit of butane monooxygenase hydroxylase (BMOH-α) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes (CEs) were reduced in mutant strain G113N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-cis-DCE and TCE oxidation, and strain G113N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-cis-DCE epoxide during oxidation of 1,2-cis-DCE, only about 50% of the 1,2-cis-DCE oxidized by strain G113N was detected as the epoxide. Evidence was obtained that 1,2-cis-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-cis-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain G113N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent O2 uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular

  6. Identification of Tazarotenic Acid as the First Xenobiotic Substrate of Human Retinoic Acid Hydroxylase CYP26A1 and CYP26B1.

    PubMed

    Foti, Robert S; Isoherranen, Nina; Zelter, Alex; Dickmann, Leslie J; Buttrick, Brian R; Diaz, Philippe; Douguet, Dominique

    2016-05-01

    Cytochrome P450 (CYP) 26A1 and 26B1 are heme-containing enzymes responsible for metabolizing all-trans retinoic acid (at-RA). No crystal structures have been solved, and therefore homology models that provide structural information are extremely valuable for the development of inhibitors of cytochrome P450 family 26 (CYP26). The objectives of this study were to use homology models of CYP26A1 and CYP26B1 to characterize substrate binding characteristics, to compare structural aspects of their active sites, and to support the role of CYP26 in the metabolism of xenobiotics. Each model was verified by dockingat-RA in the active site and comparing the results to known metabolic profiles ofat-RA. The models were then used to predict the metabolic sites of tazarotenic acid with results verified by in vitro metabolite identification experiments. The CYP26A1 and CYP26B1 homology models predicted that the benzothiopyranyl moiety of tazarotenic acid would be oriented toward the heme of each enzyme and suggested that tazarotenic acid would be a substrate of CYP26A1 and CYP26B1. Metabolite identification experiments indicated that CYP26A1 and CYP26B1 oxidatively metabolized tazarotenic acid on the predicted moiety, with in vitro rates of metabolite formation by CYP26A1 and CYP26B1 being the highest across a panel of enzymes. Molecular analysis of the active sites estimated the active-site volumes of CYP26A1 and CYP26B1 to be 918 Å(3)and 977 Å(3), respectively. Overall, the homology models presented herein describe the enzyme characteristics leading to the metabolism of tazarotenic acid by CYP26A1 and CYP26B1 and support a potential role for the CYP26 enzymes in the metabolism of xenobiotics.

  7. Allosteric regulation of phenylalanine hydroxylase.

    PubMed

    Fitzpatrick, Paul F

    2012-03-15

    The liver enzyme phenylalanine hydroxylase is responsible for conversion of excess phenylalanine in the diet to tyrosine. Phenylalanine hydroxylase is activated by phenylalanine; this activation is inhibited by the physiological reducing substrate tetrahydrobiopterin. Phosphorylation of Ser16 lowers the concentration of phenylalanine for activation. This review discusses the present understanding of the molecular details of the allosteric regulation of the enzyme.

  8. Biosynthesis of Germacrene A Carboxylic Acid in Chicory Roots. Demonstration of a Cytochrome P450 (+)-Germacrene A Hydroxylase and NADP+-Dependent Sesquiterpenoid Dehydrogenase(s) Involved in Sesquiterpene Lactone Biosynthesis

    PubMed Central

    de Kraker, Jan-Willem; Franssen, Maurice C. R.; Dalm, Marcella C. F.; de Groot, Aede; Bouwmeester, Harro J.

    2001-01-01

    Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-Germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates β-elemene with a modest degree of enantioselectivity. PMID:11299372

  9. Identification and characterization of phenol hydroxylase from phenol-degrading Candida tropicalis strain JH8.

    PubMed

    Long, Yan; Yang, Sheng; Xie, Zhixiong; Cheng, Li

    2014-09-01

    The gene phhY encoding phenol hydroxylase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The gene phhY contained an open reading frame of 2130 bp encoding a polypeptide of 709 amino acid residues. From its sequence analysis, it is a member of a family of flavin-containing aromatic hydroxylases and shares 41% amino acid identity with phenol hydroxylase from Trichosporon cutaneum. The recombinant phenol hydroxylase exists as a homotetramer structure with a native molecular mass of 320 kDa. Recombinant phenol hydroxylase was insensitive to pH treatment; its optimum pH was at 7.6. The optimum temperature for the enzyme was 30 °C, and its activity was rapidly lost at temperatures above 60 °C. Under the optimal conditions with phenol as substrate, the K(m) and V(max) of recombinant phenol hydroxylase were 0.21 mmol·L(-1) and 0.077 μmol·L(-1)·min(-1), respectively. This is the first paper presenting the cloning and expression in E. coli of the phenol hydroxylase gene from C. tropicalis and the characterization of the recombinant phenol hydroxylase.

  10. The effects of pollen, propolis, and caffeic acid phenethyl ester on tyrosine hydroxylase activity and total RNA levels in hypertensive rats caused by nitric oxide synthase inhibition: experimental, docking and molecular dynamic studies.

    PubMed

    Ekhteiari Salmas, Ramin; Durdagi, Serdar; Gulhan, Mehmet Fuat; Duruyurek, Merve; Abdullah, Huda I; Selamoglu, Zeliha

    2017-02-15

    The objective of the present study was to evaluate the effects of propolis, pollen, and caffeic acid phenethyl ester (CAPE) on tyrosine hydroxylase (TH) activity and total RNA levels of Nω-nitro-L-arginine methyl ester (L-NAME) inhibition of nitric oxide synthase in the heart, adrenal medulla, and hypothalamus of hypertensive male Sprague dawley rats. The TH activity in the adrenal medulla, heart, and hypothalamus of the rats was significantly increased in the L-NAME group vs. control (p < 0.05). Treatment with L-NAME led to a significant increase in blood pressure (BP) in the L-NAME group compared to control (p < 0.05). These data suggest that propolis, pollen, and CAPE may mediate diminished TH activity in the heart, adrenal medulla, and hypothalamus in hypertensive rats. The decreased TH activity may be due to the modulation and synthesis of catecholamines and BP effects. In addition, the binding mechanism of CAPE within the catalytic domain of TH was investigated by means of molecular modeling approaches. These data suggest that the amino acid residues, Glu429 and Ser354 of TH may play a pivotal role in the stabilization of CAPE within the active site as evaluated by molecular dynamics (MD) simulations. Gibbs binding free energy (ΔGbinding) of CAPE in complex with TH was also determined by post-processing MD analysis approaches (i.e. Poisson-Boltzmann Surface Area (MM-PBSA) method).

  11. Red clover coumarate 3'-hydroxylase (CYP98A44) is capable of hydroxylating p-coumaroyl-shikimate but not p-coumaroyl-malate: implications for the biosynthesis of phaselic acid.

    PubMed

    Sullivan, Michael L; Zarnowski, Robert

    2010-01-01

    Red clover (Trifolium pratense) leaves accumulate several mumol of phaselic acid [2-O-caffeoyl-L-malate] per gram fresh weight. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases (PPO) prevents breakdown of forage protein during storage. Forages like alfalfa (Medicago sativa) lack both foliar PPO activity and o-diphenols. Consequently, breakdown of their protein upon harvest and storage results in economic losses and release of excess nitrogen into the environment. Understanding how red clover synthesizes o-diphenols such as phaselic acid will help in the development of forages utilizing this natural system of protein protection. We have proposed biosynthetic pathways in red clover for phaselic acid that involve a specific hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase. It is unclear whether the transfer reaction to malate to form phaselic acid involves caffeic acid or p-coumaric acid and subsequent hydroxylation of the resulting p-coumaroyl-malate. The latter would require a coumarate 3'-hydroxylase (C3'H) capable of hydroxylating p-coumaroyl-malate, an activity not previously described. Here, a cytochrome P450 C3'H (CYP98A44) was identified and its gene cloned from red clover. CYP98A44 shares 96 and 79% amino acid identity with Medicago truncatula and Arabidopsis thaliana C3'H proteins that are capable of hydroxylating p-coumaroyl-shikimate and have been implicated in monolignol biosynthesis. CYP98A44 mRNA is expressed in stems and flowers and to a lesser extent in leaves. Immune serum raised against CYP98A44 recognizes a membrane-associated protein in red clover stems and leaves and cross-reacts with C3'H proteins from other species. CYP98A44 expressed in Saccharomyces cerevisiae is capable of hydroxylating p-coumaroyl-shikimate, but not p-coumaroyl-malate. This finding indicates that in red clover, phaselic acid is likely formed by transfer of a caffeoyl moiety to malic acid, although the existence of a second C

  12. Genetics Home Reference: 21-hydroxylase deficiency

    MedlinePlus

    ... The CYP21A2 gene provides instructions for making an enzyme called 21-hydroxylase. This enzyme is found in the adrenal glands , where it ... by a shortage (deficiency) of the 21-hydroxylase enzyme. When 21-hydroxylase is lacking, substances that are ...

  13. Prostaglandin E2 promotes hepatic bile acid synthesis by an E prostanoid receptor 3-mediated hepatocyte nuclear receptor 4α/cholesterol 7α-hydroxylase pathway in mice.

    PubMed

    Yan, Shuai; Tang, Juan; Zhang, Yuyao; Wang, Yuanyang; Zuo, Shengkai; Shen, Yujun; Zhang, Qianqian; Chen, Di; Yu, Yu; Wang, Kai; Duan, Sheng-Zhong; Yu, Ying

    2017-03-01

    Prostaglandin E2 (PGE2 ) is an important lipid mediator of inflammation. However, whether and how PGE2 regulates hepatic cholesterol metabolism remains unknown. We found that expression of the PGE2 receptor, E prostanoid receptor 3 (EP3) expression is remarkably increased in hepatocytes in response to hyperlipidemic stress. Hepatocyte-specific deletion of EP3 receptor (EP3(hep-/-) ) results in hypercholesterolemia and augments diet-induced atherosclerosis in low-density lipoprotein receptor knockout (Ldlr(-/-) ) mice. Cholesterol 7α-hydroxylase (CYP7A1) is down-regulated in livers of EP3(hep-/-) Ldlr(-/-) mice, leading to suppressed hepatic bile acid (BA) biosynthesis. Mechanistically, hepatic-EP3 deficiency suppresses CYP7A1 expression by elevating protein kinase A (PKA)-dependent Ser143 phosphorylation of hepatocyte nuclear receptor 4α (HNF4α). Disruption of the PKA-HNF4α interaction and BA sequestration rescue impaired BA excretion and ameliorated atherosclerosis in EP3(hep-/-) Ldlr(-/-) mice.

  14. Pharmacological characterization of 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), a potent and selective hypoxia-inducible factor prolyl hydroxylase inhibitor.

    PubMed

    Barrett, Terrance D; Palomino, Heather L; Brondstetter, Theresa I; Kanelakis, Kimon C; Wu, Xiaodong; Haug, Peter V; Yan, Wen; Young, Andrew; Hua, Hong; Hart, Juliet C; Tran, Da-Thao; Venkatesan, Hariharan; Rosen, Mark D; Peltier, Hillary M; Sepassi, Kia; Rizzolio, Michele C; Bembenek, Scott D; Mirzadegan, Tara; Rabinowitz, Michael H; Shankley, Nigel P

    2011-06-01

    The hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) enzymes represent novel targets for the treatment of anemia, ulcerative colitis, and ischemic and metabolic disease inter alia. We have identified a novel small-molecule inhibitor of PHD, 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), through structure-based drug design methods. The pharmacology of JNJ-42041935 was investigated in enzyme, cellular, and whole-animal systems and was compared with other compounds described in the literature as PHD inhibitors. JNJ-42041935, was a potent (pK(I) = 7.3-7.9), 2-oxoglutarate competitive, reversible, and selective inhibitor of PHD enzymes. In addition, JNJ-42041935 was used to compare the effect of selective inhibition of PHD to intermittent, high doses (50 μg/kg i.p.) of an exogenous erythropoietin receptor agonist in an inflammation-induced anemia model in rats. JNJ-42041935 (100 μmol/kg, once a day for 14 days) was effective in reversing inflammation-induced anemia, whereas erythropoietin had no effect. The results demonstrate that JNJ-42041935 is a new pharmacological tool, which can be used to investigate PHD inhibition and demonstrate that PHD inhibitors offer great promise for the treatment of inflammation-induced anemia.

  15. Δ(9)-THC modulation of fatty acid 2-hydroxylase (FA2H) gene expression: possible involvement of induced levels of PPARα in MDA-MB-231 breast cancer cells.

    PubMed

    Takeda, Shuso; Ikeda, Eriko; Su, Shengzhong; Harada, Mari; Okazaki, Hiroyuki; Yoshioka, Yasushi; Nishimura, Hajime; Ishii, Hiroyuki; Kakizoe, Kazuhiro; Taniguchi, Aya; Tokuyasu, Miki; Himeno, Taichi; Watanabe, Kazuhito; Omiecinski, Curtis J; Aramaki, Hironori

    2014-12-04

    We recently reported that Δ(9)-tetrahydrocannabinol (Δ(9)-THC), a major cannabinoid component in Cannabis Sativa (marijuana), significantly stimulated the expression of fatty acid 2-hydroxylase (FA2H) in human breast cancer MDA-MB-231 cells. Peroxisome proliferator-activated receptor α (PPARα) was previously implicated in this induction. However, the mechanisms mediating this induction have not been elucidated in detail. We performed a DNA microarray analysis of Δ(9)-THC-treated samples and showed the selective up-regulation of the PPARα isoform coupled with the induction of FA2H over the other isoforms (β and γ). Δ(9)-THC itself had no binding/activation potential to/on PPARα, and palmitic acid (PA), a PPARα ligand, exhibited no stimulatory effects on FA2H in MDA-MB-231 cells; thus, we hypothesized that the levels of PPARα induced were involved in the Δ(9)-THC-mediated increase in FA2H. In support of this hypothesis, we herein demonstrated that; (i) Δ(9)-THC activated the basal transcriptional activity of PPARα in a concentration-dependent manner, (ii) the concomitant up-regulation of PPARα/FA2H was caused by Δ(9)-THC, (iii) PA could activate PPARα after the PPARα expression plasmid was introduced, and (iv) the Δ(9)-THC-induced up-regulation of FA2H was further stimulated by the co-treatment with L-663,536 (a known PPARα inducer). Taken together, these results support the concept that the induced levels of PPARα may be involved in the Δ(9)-THC up-regulation of FA2H in MDA-MB-231 cells.

  16. Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L. ) cell suspension cultures

    SciTech Connect

    Kuehnl, T.K.; Koch, U.; Heller, W.; Wellmann, E.

    1987-10-01

    Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsible for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway.

  17. Mutagenesis of a specificity-determining residue in tyrosine hydroxylase establishes that the enzyme is a robust phenylalanine hydroxylase but a fragile tyrosine hydroxylase.

    PubMed

    Daubner, S Colette; Avila, Audrey; Bailey, Johnathan O; Barrera, Dimitrios; Bermudez, Jaclyn Y; Giles, David H; Khan, Crystal A; Shaheen, Noel; Thompson, Janie Womac; Vasquez, Jessica; Oxley, Susan P; Fitzpatrick, Paul F

    2013-02-26

    The aromatic amino acid hydroxylases tyrosine hydroxylase (TyrH) and phenylalanine hydroxylase (PheH) have essentially identical active sites; however, PheH is nearly incapable of hydroxylating tyrosine, while TyrH can readily hydroxylate both tyrosine and phenylalanine. Previous studies have indicated that Asp425 of TyrH is important in determining the substrate specificity of that enzyme [Daubner, S. C., Melendez, J., and Fitzpatrick, P. F. (2000) Biochemistry 39, 9652-9661]. Alanine-scanning mutagenesis of amino acids 423-427, a mobile loop containing Asp425, shows that only mutagenesis of Asp425 alters the activity of the enzyme significantly. Saturation mutagenesis of Asp425 results in large (up to 10(4)) decreases in the V(max) and V(max)/K(tyr) values for tyrosine hydroxylation, but only small decreases or even increases in the V(max) and V(max)/K(phe) values for phenylalanine hydroxylation. The decrease in the tyrosine hydroxylation activity of the mutant proteins is due to an uncoupling of tetrahydropterin oxidation from amino acid hydroxylation with tyrosine as the amino acid substrate. In contrast, with the exception of the D425W mutant, the extent of coupling of tetrahydropterin oxidation and amino acid hydroxylation is unaffected or increases with phenylalanine as the amino acid substrate. The decrease in the V(max) value with tyrosine as the substrate shows a negative correlation with the hydrophobicity of the amino acid residue at position 425. The results are consistent with a critical role of Asp425 being to prevent a hydrophobic interaction that results in a restricted active site in which hydroxylation of tyrosine does not occur.

  18. Cinnamic acid 4-hydroxylase mechanism-based inactivation by psoralen derivatives: cloning and characterization of a C4H from a psoralen producing plant-Ruta graveolens-exhibiting low sensitivity to psoralen inactivation.

    PubMed

    Gravot, Antoine; Larbat, Romain; Hehn, Alain; Lièvre, Karine; Gontier, Eric; Goergen, Jean Louis; Bourgaud, Frédéric

    2004-02-01

    Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) complete cDNA was cloned from the leaves of Ruta graveolens, a psoralen producing plant. The recombinant enzyme (classified CYP73A32) was expressed in Saccharomyces cerevisiae. Mechanism-based inactivation was investigated using various psoralen derivatives. Only psoralen and 8-methoxypsoralen were found to inactivate C4H. The inactivation was dependent on the presence of NADPH, time of pre-incubation, and inhibitor concentration. Inactivation stoichiometry was 0.9 (+/-0.2) for CYP73A1 and 1.1 (+/-0.2) for CYP73A32. SDS-PAGE analysis demonstrated that [3H]psoralen was irreversibly bound to the C4H apoprotein. K(i) and k(inact) for psoralen and 8-methoxypsoralen inactivation on the two C4H revealed a lower sensitivity for CYP73A32 compared to CYP73A1. Inactivation kinetics were also determined for CYP73A10, a C4H from another furocoumarin-producing plant, Petroselinum crispum. This enzyme was found to behave like CYP73A32, with a weak sensitivity to psoralen and 8-MOP inactivation. Cinnamic acid hydroxylation is a key step in the biosynthesis of phenylpropanoid compounds, psoralen derivatives included. Our results suggest a possible evolution of R. graveolens and P. crispum C4H that might tolerate substantial levels of psoralen derivatives in the cytoplasmic compartment without a depletive effect on C4H and the general phenylpropanoid metabolism.

  19. Characterization of the β-Carotene Hydroxylase Gene DSM2 Conferring Drought and Oxidative Stress Resistance by Increasing Xanthophylls and Abscisic Acid Synthesis in Rice1[C][W][OA

    PubMed Central

    Du, Hao; Wang, Nili; Cui, Fei; Li, Xianghua; Xiao, Jinghua; Xiong, Lizhong

    2010-01-01

    Drought is a major limiting factor for crop production. To identify critical genes for drought resistance in rice (Oryza sativa), we screened T-DNA mutants and identified a drought-hypersensitive mutant, dsm2. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative β-carotene hydroxylase (BCH). BCH is predicted for the biosynthesis of zeaxanthin, a carotenoid precursor of abscisic acid (ABA). The amounts of zeaxanthin and ABA were significantly reduced in two allelic dsm2 mutants after drought stress compared with the wild type. Under drought stress conditions, the mutant leaves lost water faster than the wild type and the photosynthesis rate, biomass, and grain yield were significantly reduced, whereas malondialdehyde level and stomata aperture were increased in the mutant. The mutant is also hypersensitive to oxidative stresses. The mutant had significantly lower maximal efficiency of photosystem II photochemistry and nonphotochemical quenching capacity than the wild type, indicating photoinhibition in photosystem II and decreased capacity for eliminating excess energy by thermal dissipation. Overexpression of DSM2 in rice resulted in significantly increased resistance to drought and oxidative stresses and increases of the xanthophylls and nonphotochemical quenching. Some stress-related ABA-responsive genes were up-regulated in the overexpression line. DSM2 is a chloroplast protein, and the response of DSM2 to environmental stimuli is distinctive from the other two BCH members in rice. We conclude that the DSM2 gene significantly contributes to control of the xanthophyll cycle and ABA synthesis, both of which play critical roles in the establishment of drought resistance in rice. PMID:20852032

  20. Genetics Home Reference: dopamine beta-hydroxylase deficiency

    MedlinePlus

    ... Genetics Home Health Conditions dopamine beta-hydroxylase deficiency dopamine beta-hydroxylase deficiency Enable Javascript to view the ... boxes. Download PDF Open All Close All Description Dopamine beta (β)-hydroxylase deficiency is a condition that ...

  1. Unresponsiveness to tetrahydrobiopterin of phenylalanine hydroxylase deficiency.

    PubMed

    Ponzone, Alberto; Porta, Francesco; Mussa, Alessandro; Alluto, Alessandra; Ferraris, Silvio; Spada, Marco

    2010-05-01

    Conflicting results have been reported concerning the efficacy of tetrahydrobiopterin (BH4), the cofactor of phenylalanine hydroxylase, for reducing phenylalanine (Phe) concentration in phenylketonuria (PKU). We aimed to test quantitatively the effects of BH4 in PKU patients. Seven fully characterized patients were selected among a population of 130 PKU subjects as harboring PKU mutations predicted as BH4 responsive and previously considered responsive to a cofactor challenge. They received a simple Phe (100 mg/kg) and 2 combined Phe (100 mg/kg) and BH4 (20 mg/kg) oral loading tests. Cofactor was administered either before or after the amino acid. The concentrations of Phe, tyrosine (Tyr), and biopterin were measured over 24 hours after loading. The comparative analysis of the loading tests showed that in all patients plasma Phe concentrations peaked within 3 hours, and fell within 24 hours by about 50% in benign, 20% in mild, and 15% in severe phenylalanine hydroxylase deficiency regardless of BH4 administration. A consistent or moderate increase of plasma Tyr, again independent of the cofactor challenge, was observed only in the less severe forms of PAH deficiency. Mean blood biopterin concentration increased 6 times after simple Phe and 34 to 39 times after combined loading tests. The administration of BH4 does not alter Phe and Tyr metabolism in PKU patients. The clearance of plasma Phe after oral loading and, as well as Tyr production, is not related to cofactor challenge but to patient's phenotype. The assessment of BH4 responsiveness by the methods so far used is not reliable, and the occurrence of BH4-responsive forms of PKU still has to be definitely proven.

  2. Decreased Expression of Cholesterol 7α-Hydroxylase and Altered Bile Acid Metabolism in Apobec-1−/− Mice Lead to Increased Gallstone Susceptibility*

    PubMed Central

    Xie, Yan; Blanc, Valerie; Kerr, Thomas A.; Kennedy, Susan; Luo, Jianyang; Newberry, Elizabeth P.; Davidson, Nicholas O.

    2009-01-01

    Quantitative trait mapping in mice identified a susceptibility locus for gallstones (Lith6) spanning the Apobec-1 locus, the structural gene encoding the RNA-specific cytidine deaminase responsible for production of apolipoprotein B48 in mammalian small intestine and rodent liver. This observation prompted us to compare dietary gallstone susceptibility in Apobec-1−/− mice and congenic C57BL/6 wild type controls. When fed a lithogenic diet (LD) for 2 weeks, 90% Apobec-1−/− mice developed solid gallstones in comparison with 16% wild type controls. LD-fed Apobec-1−/− mice demonstrated increased biliary cholesterol secretion as well as increased cholesterol saturation and bile acid hydrophobicity indices. These changes occurred despite a relative decrease in cholesterol absorption in LD-fed Apobec-1−/− mice. Among the possible mechanisms to account for this phenotype, expression of Cyp7a1 mRNA and protein were significantly decreased in chow-fed Apobec-1−/− mice, decreasing further in LD-fed animals. Cyp7a1 transcription in hepatocyte nuclei, however, was unchanged in Apobec-1−/− mice, excluding transcriptional repression as a potential mechanism for decreased Cyp7a1 expression. We demonstrated that APOBEC-1 binds to AU-rich regions of the 3′-untranslated region of the Cyp7a1 transcript, containing the UUUN(A/U)U consensus motif, using both UV cross-linking to recombinant APOBEC-1 and in vivo RNA co-immunoprecipitation. In vivo Apobec-1-dependent modulation of Cyp7a1 expression was further confirmed following adenovirus-Apobec-1 administration to chow-fed Apobec-1−/− mice, which rescued Cyp7a1 gene expression. Taken together, the findings suggest that the AU-rich RNA binding-protein Apobec-1 mediates post-transcriptional regulation of murine Cyp7a1 expression and influences susceptibility to diet-induced gallstone formation. PMID:19386592

  3. Identification of phenylalanine 3-hydroxylase for meta-tyrosine biosynthesis.

    PubMed

    Zhang, Wenjun; Ames, Brian D; Walsh, Christopher T

    2011-06-21

    Phenylalanine hydroxylase (PheH) is an iron(II)-dependent enzyme that catalyzes the hydroxylation of aromatic amino acid l-phenylalanine (L-Phe) to l-tyrosine (L-Tyr). The enzymatic modification has been demonstrated to be highly regiospecific, forming proteinogenic para-Tyr (p-Tyr) exclusively. Here we biochemically characterized the first example of a phenylalanine 3-hydroxylase (Phe3H) that catalyzes the synthesis of meta-Tyr (m-Tyr) from Phe. Subsequent mutagenesis studies revealed that two residues in the active site of Phe3H (Cys187 and Thr202) contribute to C-3 rather than C-4 hydroxylation of the phenyl ring. This work sets the stage for the mechanistic and structural study of regiospecific control of the substrate hydroxylation by PheH.

  4. Production of hydroxylated fatty acids in genetically modified plants

    DOEpatents

    Somerville, Chris; Broun, Pierre; van de Loo, Frank; Boddupalli, Sekhar S.

    2011-08-23

    This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

  5. Production of hydroxylated fatty acids in genetically modified plants

    DOEpatents

    Somerville, Chris; Broun, Pierre; van de Loo, Frank; Boddupalli, Sekhar S.

    2005-08-30

    This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

  6. XAFS of human tyrosine hydroxylase

    NASA Astrophysics Data System (ADS)

    Meyer, W.; Haavik, J.; Winkler, H.; Trautwein, A. X.; Nolting, H.-F.

    1995-02-01

    Tyrosine hydroxylase (TH) catalyses the rate-limiting step (hydroxylation of tyrosine to form dihydroxyphenylalanine) in the biosynthetic pathway leading to the catecholamines dopamine, noradrenaline and adrenaline. The human enzyme (hTH) is present in four isoforms, generated by splicing of pre-mRNA. The purified apoenzyme (metal free) binds stoichiometric amounts of iron. The incorporation of Fe(II) results in a rapid and up to 40-fold increase of activity [1]. Besides the coordination of the metal centers in native enzyme we studied the purported inhibition of TH by its immediate products. So we analysed Fe-hTH isoform 1 native as well as oxidized with dopamine and Co-hTH isoform 2.

  7. Pulsed EPR study of amino acid and tetrahydropterin binding in a tyrosine hydroxylase nitric oxide complex: evidence for substrate rearrangements in the formation of the oxygen-reactive complex.

    PubMed

    Krzyaniak, Matthew D; Eser, Bekir E; Ellis, Holly R; Fitzpatrick, Paul F; McCracken, John

    2013-11-26

    Tyrosine hydroxylase is a nonheme iron enzyme found in the nervous system that catalyzes the hydroxylation of tyrosine to form l-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. Catalysis requires the binding of three substrates: tyrosine, tetrahydrobiopterin, and molecular oxygen. We have used nitric oxide as an O₂ surrogate to poise Fe(II) at the catalytic site in an S = 3/2, {FeNO}⁷ form amenable to EPR spectroscopy. ²H-electron spin echo envelope modulation was then used to measure the distance and orientation of specifically deuterated substrate tyrosine and cofactor 6-methyltetrahydropterin with respect to the magnetic axes of the {FeNO}⁷ paramagnetic center. Our results show that the addition of tyrosine triggers a conformational change in the enzyme that reduces the distance from the {FeNO}⁷ center to the closest deuteron on 6,7-²H-6-methyltetrahydropterin from >5.9 Å to 4.4 ± 0.2 Å. Conversely, the addition of 6-methyltetrahydropterin to enzyme samples treated with 3,5-²H-tyrosine resulted in reorientation of the magnetic axes of the S = 3/2, {FeNO}⁷ center with respect to the deuterated substrate. Taken together, these results show that the coordination of both substrate and cofactor direct the coordination of NO to Fe(II) at the active site. Parallel studies of a quaternary complex of an uncoupled tyrosine hydroxylase variant, E332A, show no change in the hyperfine coupling to substrate tyrosine and cofactor 6-methyltetrahydropterin. Our results are discussed in the context of previous spectroscopic and X-ray crystallographic studies done on tyrosine hydroxylase and phenylalanine hydroxylase.

  8. Steroid 21 hydroxylase deficiency congenital adrenal hyperplasia.

    PubMed

    Nimkarn, Saroj; Lin-Su, Karen; New, Maria I

    2011-10-01

    Steroid 21 hydroxylase deficiency is the most common form of congenital adrenal hyperplasia (CAH). The severity of this disorder depends on the extent of impaired enzymatic activity, which is caused by various mutations of the 21 hydroxylase gene. This article reviews adrenal steroidogenesis and the pathophysiology of 21 hydroxylase deficiency. The three forms of CAH are then discussed in terms of clinical presentation, diagnosis and treatment, and genetic basis. Prenatal diagnosis and treatment are also reviewed. The goal of therapy is to correct the deficiency in cortisol secretion and suppress androgen overproduction. Glucocorticoid replacement has been the mainstay of treatment for CAH, but new treatment strategies continue to be developed and studied.

  9. Structural insights into the regulation of aromatic amino acid hydroxylation.

    PubMed

    Fitzpatrick, Paul F

    2015-12-01

    The aromatic amino acid hydroxylases phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase are homotetramers, with each subunit containing a homologous catalytic domain and a divergent regulatory domain. The solution structure of the regulatory domain of tyrosine hydroxylase establishes that it contains a core ACT domain similar to that in phenylalanine hydroxylase. The isolated regulatory domain of tyrosine hydroxylase forms a stable dimer, while that of phenylalanine hydroxylase undergoes a monomer-dimer equilibrium, with phenylalanine stabilizing the dimer. These solution properties are consistent with the regulatory mechanisms of the two enzymes, in that phenylalanine hydroxylase is activated by phenylalanine binding to an allosteric site, while tyrosine hydroxylase is regulated by binding of catecholamines in the active site.

  10. CYP4 enzymes as potential drug targets: focus on enzyme multiplicity, inducers and inhibitors, and therapeutic modulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthase and fatty acid ω-hydroxylase activities.

    PubMed

    Edson, Katheryne Z; Rettie, Allan E

    2013-01-01

    The Cytochrome P450 4 (CYP4) family of enzymes in humans is comprised of thirteen isozymes that typically catalyze the ω-oxidation of endogenous fatty acids and eicosanoids. Several CYP4 enzymes can biosynthesize 20- hydroxyeicosatetraenoic acid, or 20-HETE, an important signaling eicosanoid involved in regulation of vascular tone and kidney reabsorption. Additionally, accumulation of certain fatty acids is a hallmark of the rare genetic disorders, Refsum disease and X-ALD. Therefore, modulation of CYP4 enzyme activity, either by inhibition or induction, is a potential strategy for drug discovery. Here we review the substrate specificities, sites of expression, genetic regulation, and inhibition by exogenous chemicals of the human CYP4 enzymes, and discuss the targeting of CYP4 enzymes in the development of new treatments for hypertension, stroke, certain cancers and the fatty acid-linked orphan diseases.

  11. Functional characterization of salicylate hydroxylase from the fungal endophyte Epichloë festucae

    PubMed Central

    Ambrose, Karen V.; Tian, Zipeng; Wang, Yifei; Smith, Jordan; Zylstra, Gerben; Huang, Bingru; Belanger, Faith C.

    2015-01-01

    Epichloë spp. are symbiotic fungal endophytes of many cool season grasses. The presence of the fungal endophytes often confers insect, drought, and disease tolerance to the host grasses. The presence of the fungal endophytes within the host plants does not elicit host defense responses. The molecular basis for this phenomenon is not known. Epichloë festucae, the endophyte of Festuca rubra, expresses a salicylate hydroxylase similar to NahG from the bacterium Pseudomonas putida. Few fungal salicylate hydroxylase enzymes have been reported. The in planta expression of an endophyte salicylate hydroxylase raised the possibility that degradation of plant-produced salicylic acid is a factor in the mechanism of how the endophyte avoids eliciting host plant defenses. Here we report the characterization of the E. festucae salicylate hydroxylase, designated Efe-shyA. Although the fungal enzyme has the expected activity, based on salicylic acid levels in endophyte-free and endophyte-infected plants it is unlikely that expression of the endophyte salicylate hydroxylase is a factor in the lack of a host defense response to the presence of the fungal endophyte. PMID:26055188

  12. Functional characterization of salicylate hydroxylase from the fungal endophyte Epichloë festucae.

    PubMed

    Ambrose, Karen V; Tian, Zipeng; Wang, Yifei; Smith, Jordan; Zylstra, Gerben; Huang, Bingru; Belanger, Faith C

    2015-06-09

    Epichloë spp. are symbiotic fungal endophytes of many cool season grasses. The presence of the fungal endophytes often confers insect, drought, and disease tolerance to the host grasses. The presence of the fungal endophytes within the host plants does not elicit host defense responses. The molecular basis for this phenomenon is not known. Epichloë festucae, the endophyte of Festuca rubra, expresses a salicylate hydroxylase similar to NahG from the bacterium Pseudomonas putida. Few fungal salicylate hydroxylase enzymes have been reported. The in planta expression of an endophyte salicylate hydroxylase raised the possibility that degradation of plant-produced salicylic acid is a factor in the mechanism of how the endophyte avoids eliciting host plant defenses. Here we report the characterization of the E. festucae salicylate hydroxylase, designated Efe-shyA. Although the fungal enzyme has the expected activity, based on salicylic acid levels in endophyte-free and endophyte-infected plants it is unlikely that expression of the endophyte salicylate hydroxylase is a factor in the lack of a host defense response to the presence of the fungal endophyte.

  13. Production of hydroxylated fatty acids in genetically modified plants

    DOEpatents

    Somerville, Chris; Broun, Pierre; van de Loo, Frank

    2001-01-01

    This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants.

  14. Phenylalanine hydroxylase deficiency: diagnosis and management guideline.

    PubMed

    Vockley, Jerry; Andersson, Hans C; Antshel, Kevin M; Braverman, Nancy E; Burton, Barbara K; Frazier, Dianne M; Mitchell, John; Smith, Wendy E; Thompson, Barry H; Berry, Susan A

    2014-02-01

    Phenylalanine hydroxylase deficiency, traditionally known as phenylketonuria, results in the accumulation of phenylalanine in the blood of affected individuals and was the first inborn error of metabolism to be identified through population screening. Early identification and treatment prevent the most dramatic clinical sequelae of the disorder, but new neurodevelopmental and psychological problems have emerged in individuals treated from birth. The additional unanticipated recognition of a toxic effect of elevated maternal phenylalanine on fetal development has added to a general call in the field for treatment for life. Two major conferences sponsored by the National Institutes of Health held >10 years apart reviewed the state of knowledge in the field of phenylalanine hydroxylase deficiency, but there are no generally accepted recommendations for therapy. The purpose of this guideline is to review the strength of the medical literature relative to the treatment of phenylalanine hydroxylase deficiency and to develop recommendations for diagnosis and therapy of this disorder. Evidence review from the original National Institutes of Health consensus conference and a recent update by the Agency for Healthcare Research and Quality was used to address key questions in the diagnosis and treatment of phenylalanine hydroxylase deficiency by a working group established by the American College of Medical Genetics and Genomics. The group met by phone and in person over the course of a year to review these reports, develop recommendations, and identify key gaps in our knowledge of this disorder. Above all, treatment of phenylalanine hydroxylase deficiency must be life long, with a goal of maintaining blood phenylalanine in the range of 120-360 µmol/l. Treatment has predominantly been dietary manipulation, and use of low protein and phenylalanine medical foods is likely to remain a major component of therapy for the immediate future. Pharmacotherapy for phenylalanine

  15. Identification of the flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes from Antarctic moss and their regulation during abiotic stress.

    PubMed

    Liu, Shenghao; Ju, Jianfang; Xia, Guangmin

    2014-06-10

    Flavonoids are ubiquitous plant secondary metabolites, and their hydroxylation pattern determines their color, stability, and antioxidant capacity. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of cytochrome P450 protein (P450) family, the flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3',5'H). However, they are still not well documented in lower plants such as bryophytes. We report the identification of gene encoding F3'H, F3',5'H from Antarctic moss Pohlia nutans and their transcriptional regulation under different stress conditions. Totally, sixteen cDNAs were isolated from P. nutans by RT-PCR and RACE techniques, all of which were predicted to code for F3'Hs or F3',5'Hs based on their annotations of Blast results. Amino acid alignment showed that they possessed the featured conserved domains of flavonoid hydroxylase, including proline-rich "hinge" region, EXXR motif, oxygen binding pocket motif, heme binding domain and substrate recognition sites. Phylogenetic analysis indicated that moss F3'Hs and F3',5'Hs were highly conserved and have independent evolution from the monocots, dicots and ferns. Meanwhile, real-time PCR analysis revealed that the expression profiling of flavonoid hydroxylase genes was influenced by diverse abiotic stresses including cold, salinity, drought or UV-B radiation and plant hormone abscisic acid (ABA) or jasmonic acid (JA) treatment. Since 3',4',5'-hydroxylated flavonoid-derivatives may serve a multitude of functions, including antioxidant activity and UV filters, the evolution and expression profile of flavonoid hydroxylase probably reflect the adaptive value of Antarctic moss in the acclimation of polar environment.

  16. Mechanism of inhibition of lysyl hydroxylase activity by the organophosphates malathion and malaoxon.

    PubMed

    Samimi, A; Last, J A

    2001-11-01

    Direct inhibition of lysyl hydroxylase by malathion and malaoxon was observed in an in vitro enzyme assay with recombinant lysyl hydroxylase expressed via a baculoviral system. The IC50 values for malathion and malaoxon were estimated to be approximately 60 and 45 mM, respectively. Additional kinetic studies showed this inhibition to be competitive or partially competitive with respect to the synthetic (collagen) peptide, partially uncompetitive with respect to Fe(2+), and partially noncompetitive with respect to ascorbic acid. The calculated values for the K(i) were consistent with the IC50 values. Allosteric effects were not found for any of the cofactors tested, the peptide substrate, or the inhibitors. Interactions were found to be unimolecular for lysyl hydroxylase and its substrate and cofactors as well as for the inhibitors malathion and malaoxon. A computer search of a protein structure database showed an unexpected region of partial homology between the active site sequence of acetylcholinesterase and a segment of lysyl hydroxylase, suggesting a possible molecular basis for these observations. These results suggest the possibility of a novel and hitherto unexpected class of inhibitors of lysyl hydroxylase, based on the organophosphate structure, that might be of value for testing as antifibrotic drugs.

  17. An Oleate Hydroxylase from the Fungus Claviceps purpurea: Cloning, Functional Analysis, and Expression in Arabidopsis[OA

    PubMed Central

    Meesapyodsuk, Dauenpen; Qiu, Xiao

    2008-01-01

    Claviceps purpurea, a fungal pathogen responsible for ergot diseases in many agriculturally important cereal crops, produces high levels of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. It has been believed for many years that the biosynthesis of this fatty acid in C. purpurea involves a hydration process with linoleic acid as the substrate. Using degenerate polymerase chain reaction, we cloned a gene from the sclerotia encoding an enzyme (CpFAH) that has high sequence similarity to the C. purpurea oleate desaturase, but only low similarity to plant oleate hydroxylases. Functional analysis of CpFAH in yeast (Saccharomyces cerevisiae) indicated it acted predominantly as a hydroxylase, introducing hydroxyl groups at the 12-position of oleic acid and palmitoleic acid. As well, it showed Δ12 desaturase activities on 16C and 18C monounsaturated fatty acids and, to a much lesser extent, ω3 desaturase activities on ricinoleic acid. Heterologous expression of CpFAH under the guidance of a seed-specific promoter in Arabidopsis (Arabidopsis thaliana) wild-type and mutant (fad2/fae1) plants resulted in the accumulation of relatively higher levels of hydroxyl fatty acids in seeds. These data indicate that the biosynthesis of ricinoleic acid in C. purpurea is catalyzed by the fungal desaturase-like hydroxylase, and CpFAH, the first Δ12 oleate hydroxylase of nonplant origin, is a good candidate for the transgenic production of hydroxyl fatty acids in oilseed crops. PMID:18467452

  18. Purification and characterization of p-coumaroyl-D-glucose hydroxylase of sweet potato (Ipomoea batatas) roots.

    PubMed

    Tanaka, M; Kojima, M

    1991-01-01

    p-Coumaroyl-D-glucose hydroxylase in sweet potato (Ipomoea batatas Lam.) has been purified to apparent electrophoretic homogeneity using a combination of anion-and cation-exchange, hydrophobic and gel filtration chromatography. The purified enzyme was a monomer with a molecular weight of 33,000 and pI of 8.3. The purified enzyme showed not only hydroxylase activity but also polyphenol oxidase activity. L-Ascorbic acid was the best electron donor for the hydroxylation reaction, which had an optimum pH of 7.0. The enzyme hydroxylated p-coumaroyl-D-glucose, p-coumaric acid, and p-cresol but did not act on o-coumaric acid, m-coumaric acid, 4-hydroxy-3-methoxycinnamic acid, p-hydroxybenzoic acid or L-tyrosine. While the enzyme utilized p-coumaroyl-D-glucose and p-coumaric acid equally at pH 7.0, it hydroxylated only p-coumaroyl-D-glucose at pH 5.5. The enzyme oxidized diphenols such as D,L-(3,4-dihydroxyphenyl) alanine and caffeic acid, but exhibited no clear pH optimum in this reaction characteristic of polyphenol oxidase. Both the hydroxylase and the polyphenol oxidase activities were strongly inhibited by beta-mercaptoethanol, diethyldithiocarbamate, KCN, and p-coumaric acid (in concentrations higher than 5 mM). Ammonium sulfate and sodium chloride activated the hydroxylase activity but not the polyphenol oxidase activity of the enzyme. The enzyme activity and L-ascorbic acid contents changed in a manner suggesting their involvements in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.

  19. Are striatal tyrosine hydroxylase interneurons dopaminergic?

    PubMed

    Xenias, Harry S; Ibáñez-Sandoval, Osvaldo; Koós, Tibor; Tepper, James M

    2015-04-22

    Striatal GABAergic interneurons that express the gene for tyrosine hydroxylase (TH) have been identified previously by several methods. Although generally assumed to be dopaminergic, possibly serving as a compensatory source of dopamine (DA) in Parkinson's disease, this assumption has never been tested directly. In TH-Cre mice whose nigrostriatal pathway had been eliminated unilaterally with 6-hydroxydopamine, we injected a Cre-dependent virus coding for channelrhodopsin-2 and enhanced yellow fluorescent protein unilaterally into the unlesioned midbrain or bilaterally into the striatum. Fast-scan cyclic voltammetry in striatal slices revealed that both optical and electrical stimulation readily elicited DA release in control striata but not from contralateral striata when nigrostriatal neurons were transduced. In contrast, neither optical nor electrical stimulation could elicit striatal DA release in either the control or lesioned striata when the virus was injected directly into the striatum transducing only striatal TH interneurons. This demonstrates that striatal TH interneurons do not release DA. Fluorescence immunocytochemistry in enhanced green fluorescent protein (EGFP)-TH mice revealed colocalization of DA, l-amino acid decarboxylase, the DA transporter, and vesicular monoamine transporter-2 with EGFP in midbrain dopaminergic neurons but not in any of the striatal EGFP-TH interneurons. Optogenetic activation of striatal EGFP-TH interneurons produced strong GABAergic inhibition in all spiny neurons tested. These results indicate that striatal TH interneurons are not dopaminergic but rather are a type of GABAergic interneuron that expresses TH but none of the other enzymes or transporters necessary to operate as dopaminergic neurons and exert widespread GABAergic inhibition onto direct and indirect spiny neurons.

  20. Molecular evaluation of a spearmint mutant altered in the expression of limonene hydroxylases that direct essential oil monoterpene biosynthesis.

    PubMed

    Bertea, Cinzia; Schalk, Michel; Mau, Christopher J D; Karp, Frank; Wildung, Mark R; Croteau, Rodney

    2003-12-01

    Gamma irradiation of Scotch spearmint created a mutant line, 643-10-74, which has an altered essential oil reminiscent of peppermint because the monoterpene metabolites in the oil glands of the mutant are predominantly oxygenated at the C3 position of the p-menthane ring instead of the C6 position normally found in spearmint. The limonene hydroxylase genes responsible for directing the regiochemistry of oxygenation were cloned from Scotch spearmint and mutant 643 and expressed in Escherichia coli. The limonene bydroxylase from the wild-type parent hydroxylated the C6 position while the enzyme from the mutant oxygenated the C3 position. Comparison of the amino acid sequences with other limonene hydroxylases showed that the mutant enzyme was more closely related to the peppermint limonene-3-hydroxylases than to the spearmint limonene-6-hydroxylases. Because of the sequence differences between the Scotch spearmint and mutant 643 limonene hydroxylases, it is most likely that the mutation did not occur within the structural gene for limonene hydroxylase but rather at a regulatory site within the genome that controls the expression of one or the other regiospecific variants.

  1. An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog.

    PubMed Central

    van de Loo, F J; Broun, P; Turner, S; Somerville, C

    1995-01-01

    Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 and with approximately 67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution. Images Fig. 3 Fig. 4 Fig. 5 PMID:7624314

  2. An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog

    SciTech Connect

    Van De Loo, F.J.; Broun, P.; Turner, S.; Somerville, C.

    1995-07-18

    Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 and with {approx}67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution. 26 refs., 6 figs., 1 tab.

  3. RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua

    PubMed Central

    Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar

    2016-01-01

    Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H. PMID:27220407

  4. Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase.

    PubMed

    Li, Jun; Ilangovan, Udayar; Daubner, S Colette; Hinck, Andrew P; Fitzpatrick, Paul F

    2011-01-15

    The hydroxylation of phenylalanine to tyrosine by the liver enzyme phenylalanine hydroxylase is regulated by the level of phenylalanine. Whether there is a distinct allosteric binding site for phenylalanine outside of the active site has been unclear. The enzyme contains an N-terminal regulatory domain that extends through Thr117. The regulatory domain of rat phenylalanine hydroxylase was expressed in Escherichia coli. The purified protein behaves as a dimer on a gel filtration column. In the presence of phenylalanine, the protein elutes earlier from the column, consistent with a conformational change in the presence of the amino acid. No change in elution is seen in the presence of the non-activating amino acid proline. ¹H-¹⁵N HSQC NMR spectra were obtained of the ¹⁵N-labeled protein alone and in the presence of phenylalanine or proline. A subset of the peaks in the spectrum exhibits chemical shift perturbation in the presence of phenylalanine, consistent with binding of phenylalanine at a specific site. No change in the NMR spectrum is seen in the presence of proline. These results establish that the regulatory domain of phenylalanine hydroxylase can bind phenylalanine, consistent with the presence of an allosteric site for the amino acid.

  5. Phenylalanine hydroxylase (PAH) from the lower eukaryote Leishmania major.

    PubMed

    Lye, Lon-Fye; Kang, Song Ok; Nosanchuk, Joshua D; Casadevall, Arturo; Beverley, Stephen M

    2011-01-01

    Aromatic amino acid hydroxylases (AAAH) typically use tetrahydrobiopterin (H(4)B) as the cofactor. The protozoan parasite Leishmania major requires biopterin for growth and expresses strong salvage and regeneration systems to maintain H(4)B levels. Here we explored the consequences of genetic manipulation of the sole L. major phenylalanine hydroxylase (PAH) to explore whether it could account for the Leishmania H(4)B requirement. L. major PAH resembles AAAHs of other organisms, bearing eukaryotic-type domain organization, and conservation of key catalytic residues including those implicated in pteridine binding. A pah(-) null mutant and an episomal complemented overexpressing derivative (pah-/+PAH) were readily obtained, and metabolic labeling studies established that PAH was required to hydroxylate Phe to Tyr. Neither WT nor overexpressing lines were able to hydroxylate radiolabeled tyrosine or tryptophan, nor to synthesize catecholamines. WT but not pah(-) parasites showed reactivity with an antibody to melanin when grown with l-3,4-dihydroxyphenylalanine (L-DOPA), although the reactive product is unlikely to be melanin sensu strictu. WT was auxotrophic for Phe, Trp and Tyr, suggesting that PAH activity was insufficient to meet normal Tyr requirements. However, pah(-) showed an increased sensitivity to Tyr deprivation, while the pah(-)/+PAH overexpressor showed increased survival and could be adapted to grow well without added Tyr. pah(-) showed no alterations in H(4)B-dependent differentiation, as established by in vitro metacyclogenesis, or survival in mouse or macrophage infections. Thus Leishmania PAH may mitigate but not alleviate Tyr auxotrophy, but plays no essential role in the steps of the parasite infectious cycle. These findings suggest PAH is unlikely to explain the Leishmania requirement for biopterin.

  6. Molecular characterization of 25 Chinese pedigrees with 21-hydroxylase deficiency.

    PubMed

    Yu, Yongguo; Wang, Jian; Huang, Xiaodong; Wang, Ying; Yang, Peirong; Li, Juan; Tsuei, Sian Hsiang-Te; Shen, Yongnian; Fu, Qihua

    2011-03-01

    Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders mainly caused by a defect in the steroid 21-hydroxylase gene (CYP21A2). In this study, we investigated the molecular defects of 25 Chinese pedigrees with 21-hydroxylase deficiency (21-OHD). Diagnosis of the probands in the families was based on their typical clinical presentations, such as inborn ambiguous genitalia, or early onset of salt wasting and biochemical metabolite abnormalities. All 10 exons and exon-intron boundaries of the CYP21A2 gene were amplified from the genomic DNA of the probands and then analyzed by direct sequencing. The phenotypes of the 26 patients from 25 pedigrees were classified as the classical form of 21-OHD. One novel mutation (c.1223 G>T) and 13 recurrent mutations of CYP21A2 were identified in the 25 pedigrees by genetic analysis. The novel c.1223 G>T mutation results in the substitution of arginine by leucine at amino acid position 408 (p.Arg408Leu). The most frequent mutation alleles were IVS2-13A/C>G (14/52) and I172N (11/52), followed by chimeric mutations (10/52). Forty six of 52 mutated alleles resulted from pseudogene conversion and 6 of 52 from random mutations. The spectrum of CYP21A2 mutation in our study was slightly different from those previously reported in Chinese and in other ethnic groups of the world. Although microconversion events were the main cause of mutations in the CYP21 gene, random mutations with a common origin can also be the reason for 21-OHD.

  7. Tyrosine hydroxylase in the european eel (Anguilla anguilla): cDNA cloning, brain distribution, and phylogenetic analysis.

    PubMed

    Boularand, S; Biguet, N F; Vidal, B; Veron, M; Mallet, J; Vincent, J D; Dufour, S; Vernier, P

    1998-08-01

    We report the isolation of a full-length eel tyrosine hydroxylase (TH) cDNA that is characterized by a long 3' untranslated region and by a diversity restricted to the 3' end owing to the differential use of three polyadenylation signals. The longest eel TH mRNA was distinctive in the presence of four pentameric elements (AUUUA) in the AU-rich 3' noncoding region. Such a diversity could provide the basis of posttranscriptional or translational regulation of eel TH gene expression. Comparison of the eel TH sequence with those of other aromatic amino acid hydroxylases (TH, tryptophan hydroxylase, and phenylalanine hydroxylase) and phylogenetic analysis confirmed that the N-terminal regulatory domain is highly divergent, contrasting with the conservation of the catalytic core of the enzyme. Molecular phylogenies including the available sequences of the three hydroxylase genes suggested that the duplication of their common ancestor occurred before the emergence of arthropods. The regional expression of the eel TH mRNA was studied by semiquantitative PCR, northern blots, and in situ hybridization and compared with the immunocytochemical localization of TH protein. The data showed that TH mRNA is mostly expressed in the olfactory and hypothalamic areas, whereas sparse TH-expressing cell bodies are present in the telencephalic region and brainstem. No labeling was detected in the mesencephalic area, in striking contrast with that found in amphibians and amniotes.

  8. 4-Coumaroyl coenzyme A 3-hydroxylase activity from cell cultures of Lithospermum erythrorhizon and its relationship to polyphenol oxidase.

    PubMed

    Wang, Z X; Li, S M; Löscher, R; Heide, L

    1997-11-15

    A 4-coumaroyl-CoA 3-hydroxylase activity was purified 4600-fold from cell cultures of Lithospermum erythrorhizon. The enzyme showed a molecular mass of 42,400 +/- 1700 Da in gel chromatography and required ascorbate, NADH, or NADPH as cofactors. 4-Coumaroyl-CoA, 4-coumarate, p-cresol, and several other phenolic substances, but not tyrosine, were accepted as substrates for the hydroxylation. Besides hydroxylase activity, the enzyme showed diphenol oxidase activity. Both activities were inhibited by diethyldithiocarbamate or beta-mercaptoethanol, although at different concentrations. The enzyme showed striking similarity to a 4-coumaroyl-glucose 3-hydroxylase from sweet potato (Ipomoe batatas) roots, which has reportedly been purified to homogeneity and identified as a specific enzyme of chlorogenic acid biosynthesis. Close examination and comparison to a commercially available polyphenol oxidase, however, suggest that the enzyme activities purified from both Lithospermum and sweet potato are polyphenol oxidases rather than specific enzymes of secondary metabolism.

  9. Cytochrome P450 ω-Hydroxylases in Inflammation and Cancer

    PubMed Central

    Johnson, Amanda L.; Edson, Katheryne Z.; Totah, Rheem A.; Rettie, Allan E.

    2015-01-01

    Cytochrome P450-dependent ω-hydroxylation is a prototypic metabolic reaction of CYP4 family members that is important for the elimination and bioactivation of not only therapeutic drugs, but also endogenous compounds, principally fatty acids. Eicosanoids, derived from arachidonic acid, are key substrates in the latter category. Human CYP4 enzymes, mainly CYP4A11, CYP4F2, and CYP4F3B, hydroxylate arachidonic acid at the omega position to form 20-HETE, which has important effects in tumor progression and on angiogenesis and blood pressure regulation in the vasculature and kidney. CYP4F3A in myeloid tissue catalyzes the ω-hydroxylation of leukotriene B4 to 20-hydroxy leukotriene B4, an inactivation process that is critical for the regulation of the inflammatory response. Here, we review the enzymology, tissue distribution, and substrate selectivity of human CYP4 ω-hydroxylases and their roles as catalysts for the formation and termination of the biological effects of key eicosanoid metabolites in inflammation and cancer progression. PMID:26233909

  10. Regulation of collagen biosynthesis by ascorbic acid: a review.

    PubMed Central

    Pinnell, S. R.

    1985-01-01

    L-ascorbic acid is an essential cofactor for lysyl hydroxylase and prolyl hydroxylase, enzymes essential for collagen biosynthesis. In addition, L-ascorbic acid preferentially stimulates collagen synthesis in a manner which appears unrelated to the effect of L-ascorbic acid on hydroxylation reactions. This reaction is stereospecific and unrelated to intracellular degradation of collagen. The effect apparently occurs at a transcriptional or translational level, since L-ascorbic acid preferentially stimulates collagen-specific mRNA. In addition, it stimulates lysyl hydroxylase activity but inhibits prolyl hydroxylase activity in human skin fibroblasts in culture. PMID:3008449

  11. Red Clover Coumarate 3'-Hydroxylase (CYP98A44) is Capable of Hydroxylating P-Coumaroyl-Shikimate but not P-Coumaroyl-Malate: Implications for the Biosynthesis of Phaselic Acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red clover (Trifolium pratense) leaves accumulate several µmol of phaselic acid [2-O-caffeoyl-L-malate] per gram fresh weight. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forages like alfalfa (Medicag...

  12. Adenovirus-mediated transfer of a gene encoding cholesterol 7 alpha-hydroxylase into hamsters increases hepatic enzyme activity and reduces plasma total and low density lipoprotein cholesterol.

    PubMed Central

    Spady, D K; Cuthbert, J A; Willard, M N; Meidell, R S

    1995-01-01

    Clinical interventions that accelerate conversion of cholesterol to bile acids reduce circulating low density lipoprotein (LDL) cholesterol concentrations. The initial and rate-limiting step in the bile acid biosynthetic pathway is catalyzed by hepatic cholesterol 7 alpha-hydroxylase. To examine the effects of transient primary overexpression of this enzyme on sterol metabolism and lipoprotein transport, we constructed a recombinant adenovirus in which a cDNA encoding rat 7 alpha-hydroxylase is expressed from the human cytomegalovirus immediate-early promoter (AdCMV7 alpha). Syrian hamsters administered AdCMV7 alpha intravenously accumulated transgene-specific mRNA in the liver and demonstrated a dose-dependent increase in hepatic microsomal 7 alpha-hydroxylase activity. The increased conversion of cholesterol to bile acids resulted in a compensatory increase in hepatic cholesterol synthesis. In addition, overexpression of 7 alpha-hydroxylase reduced the rate of LDL cholesterol entry into the plasma space and, in animals maintained on a Western-type diet, restored hepatic LDL receptor expression. As a consequence, plasma LDL concentrations fell by approximately 60% in animals maintained on control diet and by approximately 75% in animals consuming a Western-type diet. Plasma high density lipoprotein cholesterol levels were reduced to a lesser degree. These results demonstrate that transient upregulation of bile acid synthesis by direct transfer of a 7 alpha-hydroxylase gene favorably alters circulating lipoprotein profiles and suggest one potential molecular target for genetic strategies aimed at reducing cardiovascular risk. Images PMID:7635963

  13. Acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in hepatic microsomes from induced mice.

    PubMed

    Lewandowski, M; Chui, Y C; Levi, P; Hodgson, E

    1991-02-01

    A simple and sensitive method for the separation of 14C-labelled acetanilide, 4-hydroxyacetanilide, 3-hydroxyacetanilide and 2-hydroxyacetanilide was developed using thin-layer chromatography. This separation is the basis for the assay of acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in liver microsomes from DBA2/N male mice that had been treated with phenobarbital, 3-methylcholanthrene, isosafrole or n-butylbenzodioxole. Microsomes were incubated with [14C]acetanilide and extracted with benzene and ethyl acetate. The extract was applied to silica gel plates and developed with a hexane/isopropanol/ammonium hydroxide/water solvent system. The radiolabelled phenolic metabolites and the parent compound were detected using a Berthold Automatic TLC Linear Analyzer. Although the 4-hydroxylated metabolite was the primary product detected, this method can be used to detect other phenolic metabolites.

  14. Regulation by retinoids of luteinizing hormone/chorionic gonadotropin receptor, cholesterol side-chain cleavage cytochrome P-450, 3 beta-hydroxysteroid dehydrogenase/delta (5-4)-isomerase and 17 alpha-hydroxylase/C17-20 lyase cytochrome P-450 messenger ribonucleic acid levels in the K9 mouse Leydig cell line.

    PubMed

    Lefèvre, A; Rogier, E; Astraudo, C; Duquenne, C; Finaz, C

    1994-12-01

    Vitamin A is a potent regulator of testicular function. We have reported that retinol (R) and retinoic acid (RA) induced a down regulation of luteinizing hormone/human chorionic gonadotropin (LH/CG) binding sites in K9 Leydig cells. In the present study we evaluated the effect of R and RA on LH/CG receptors, cholesterol side-chain cleavage cytochrome P-450 (P-450 scc), 17 alpha-hydroxylase/C17-20 lyase (P-450 17 alpha) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) mRNA levels in K9 mouse Leydig cells. To validate K9 cells as a model for studying Leydig cell steroidogenesis at the molecular level, we first investigated the effect of hCG on mRNA levels of the steroidogenic enzymes. P-450 scc, 3 beta HSD and P-450 17 alpha were expressed constitutively. The addition of 10 ng/ml hCG enhanced mRNA levels for the three genes within 2 h. Maximal accumulation of P-450 scc, P-450 17 alpha and 3 beta HSD mRNA in treated cells represents a 2.5-, 8.5- and 4-fold increase over control values, respectively. P-450 17 alpha expression reached a maximum by 4 h and then declined rapidly to return to control value by 24 h. The pattern of LH/CG receptor mRNAs in K9 cells was very similar to that of MA10 Leydig cells and showed six transcripts of 1.1, 1.6, 1.9, 2.6, 4.2 and 7.0 kb. Treatment of cells with R or RA resulted in a time- and dose-dependent decrease in all six species.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Membrane-bound hydroxylases in elicitor-treated bean cells. Rapid induction of the synthesis of prolyl hydroxylase and a putative cytochrome P-450.

    PubMed

    Bolwell, G P; Dixon, R A

    1986-08-15

    Treatment of cell-suspension cultures of bean (Phaseolus vulgaris cv. Canadian Wonder) with an elicitor preparation heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum resulted in rapid changes in the activities of two microsomal oxygenases, cinnamic acid 4-hydroxylase, involved in accumulation of wall-bound phenolics and phytoalexins, and proline 2-oxoglutarate dioxygenase (prolyl hydroxylase) involved in the post-translational modification of hydroxyproline-rich glycoproteins. An anti-(cytochrome P-450) monoclonal antibody, originally raised against rat cytochrome P-450 isoform c, has been shown to bind to bean microsomes and recognise in Western blots an Mr-48,000 polypeptide, which comigrates with a haeme-containing protein on SDS/polyacrylamide gel analysis and which has been tentatively identified as a cytochrome P-450 capable of the hydroxylation of cinnamic acid. A preparation of proline 2-oxoglutarate dioxygenase purified to homogeneity was used to immunise rabbits for the production of antiserum. An elicitor-induced polypeptide of Mr 65,000 was identified as prolyl hydroxylase while an antigenically related polypeptide of Mr 60,000 was also immunoprecipitated but not induced by elicitor treatment. Use of the two antibodies has demonstrated rapid transient increases in the synthesis of the Mr 48,000 and Mr 65,000 oxygenases in vivo and for mRNAs as measured in in vitro translations, particularly for the putative cytochrome P-450. These increases slightly precede corresponding changes in the synthesis of the soluble enzyme phenylalanine ammonia-lyase, in common with which these oxygenases probably share a mechanism of gene activation underlying the increased activities seen in response to elicitor treatment.

  16. Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies.

    PubMed

    Sakowski, Stacey A; Geddes, Timothy J; Thomas, David M; Levi, Edi; Hatfield, James S; Kuhn, Donald M

    2006-04-26

    Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin. Once thought to be a single-gene product, TPH is now known to exist in two isoforms-TPH1 is found in the pineal and gut, and TPH2 is selectively expressed in brain. Heretofore, probes used for localization of TPH protein or mRNA could not distinguish between the TPH isoforms because of extensive homology shared by them at the nucleotide and amino acid level. We have produced monospecific polyclonal antibodies against TPH1 and TPH2 using peptide antigens from nonoverlapping sequences in the respective proteins. These antibodies allow the differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation, and immunocytochemical staining of tissue sections from brain and gut. TPH1 and TPH2 antibodies do not cross-react with either tyrosine hydroxylase or phenylalanine hydroxylase. Analysis of mouse tissues confirms that TPH1 is the predominant form expressed in pineal gland and in P815 mastocytoma cells with a molecular weight of 51 kDa. TPH2 is the predominant enzyme form expressed in brain extracts from mesencephalic tegmentum, striatum, and hippocampus with a molecular weight of 56 kDa. Antibody specificity against TPH1 and TPH2 is retained across mouse, rat, rabbit, primate, and human tissues. Antibodies that distinguish between the isoforms of TPH will allow studies of the differential regulation of their expression in brain and periphery.

  17. 24-Hydroxylase in Cancer: Impact on Vitamin D-based Anticancer Therapeutics

    PubMed Central

    Luo, Wei; Hershberger, Pamela A.; Trump, Donald L.; Johnson, Candace S.

    2013-01-01

    The active vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in regulating calcium homeostasis and bone mineralization. 1,25(OH)2D3 also modulates cellular proliferation and differentiation in a variety of cell types. 24-hydroxylase, encoded by the CYP24A1 gene, is the key enzyme which converts 1,25(OH)2D3 to less active calcitroic acid. Nearly all cell types express 24-hydroxylase, the highest activity being observed in the kidney. There is increasing evidence linking the incidence and prognosis of certain cancers to low serum 25 (OH)D3 levels and high expression of vitamin D 24-hydroxylase supporting the idea that elevated CYP24A1 expression may stimulate degradation of vitamin D metabolites including 25-(OH)D3 and 1,25(OH)2D3. The over expression of CYP24A1 in cancer cells may be a factor affecting 1,25(OH)2D3 bioavailability and anti-proliferative activity pre-clinically and clinically. The combination of 1,25(OH)2D3 with CYP24A1 inhibitors enhances 1,25(OH)2D3 mediated signaling and anti-proliferative effects and may be useful in overcoming effects of aberrant CYP24 expression. PMID:23059474

  18. Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio)

    PubMed Central

    Nowicki, Magda; Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

    2015-01-01

    Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine’s metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol’s locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug. PMID:26366782

  19. Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio).

    PubMed

    Nowicki, Magda; Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

    2015-11-01

    Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine's metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol's locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.

  20. An additional substrate binding site in a bacterial phenylalanine hydroxylase.

    PubMed

    Ronau, Judith A; Paul, Lake N; Fuchs, Julian E; Corn, Isaac R; Wagner, Kyle T; Liedl, Klaus R; Abu-Omar, Mahdi M; Das, Chittaranjan

    2013-09-01

    Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes oxidation of phenylalanine to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH has a regulatory domain in which binding of the substrate leads to allosteric activation of the enzyme. However, the existence of PAH regulation in evolutionarily distant organisms, for example some bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum, a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site 15.7 Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 μM for phenylalanine. Under the same conditions, ITC revealed no detectable binding for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of amino acid residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) led to impaired binding, consistent with the presence of distal site binding in solution. Although kinetic analysis revealed that the distal site mutants suffer discernible loss of their catalytic activity, X-ray crystallographic analysis of Y155A and F258A, the two mutants with the most noticeable decrease in activity, revealed no discernible change in the structure of their active sites, suggesting that the effect of distal binding may result from protein dynamics in solution.

  1. Collagen Prolyl Hydroxylases are Essential for Breast Cancer Metastasis

    PubMed Central

    Gilkes, Daniele M.; Chaturvedi, Pallavi; Bajpai, Saumendra; Wong, Carmen Chak-Lui; Wei, Hong; Pitcairn, Stephen; Hubbi, Maimon E.; Wirtz, Denis; Semenza, Gregg L.

    2013-01-01

    Metastasis is the leading cause of death among patients with breast cancer. Understanding the role of the extracellular matrix in the metastatic process may lead to the development of improved therapies for cancer patients. Intratumoral hypoxia is found in the majority of breast cancers and is associated with an increased risk of metastasis and patient mortality. Here we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We show that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers, resulting in enhanced invasion and metastasis to lymph nodes and lungs. Lastly, we establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies, and demonstrate that ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, decreases tumor fibrosis and metastasis in a mouse model of breast cancer. PMID:23539444

  2. Intricate regulation of tyrosine hydroxylase activity and gene expression.

    PubMed

    Kumer, S C; Vrana, K E

    1996-08-01

    Tyrosine hydroxylase catalyzes the rate-limiting step in the biosynthesis of the catecholamines dopamine, norepinephrine, and epinephrine. Therefore, the regulation of tyrosine hydroxylase enzyme number and intrinsic enzyme activity represents the central means for controlling the synthesis of these important biogenic amines. An intricate scheme has evolved whereby tyrosine hydroxylase activity is modulated by nearly every documented form of regulation. Beginning with the genomic DNA, evidence exists for the transcriptional regulation of tyrosine hydroxylase mRNA levels, alternative RNA processing, and the regulation of RNA stability. There is also experimental support for the role of both translational control and enzyme stability in establishing steady-state levels of active tyrosine hydroxylase protein. Finally, mechanisms have been proposed for feedback inhibition of the enzyme by catecholamine products, allosteric modulation of enzyme activity, and phosphorylation-dependent activation of the enzyme by various different kinase systems. Given the growing literature suggesting that different tissues regulate tyrosine hydroxylase mRNA levels and activity in different ways, regulatory mechanisms provide not only redundancy but also diversity in the control of catecholamine biosynthesis.

  3. Endogenous sialylation of the lipooligosaccharides of Neisseria meningitidis.

    PubMed Central

    Mandrell, R E; Kim, J J; John, C M; Gibson, B W; Sugai, J V; Apicella, M A; Griffiss, J M; Yamasaki, R

    1991-01-01

    Monoclonal antibodies (MAb) 3F11 and 06B4 recognize epitopes that are conserved on gonococcal lipooligosaccharides (LOS), present on some meningococcal LOS, and conserved on human erythrocytes. LOS of some group B and C prototype meningococcal LOS strains (LOS serotypes L1 to L8) treated with neuraminidase showed increased expression of the 3F11 and 06B4 MAb-defined epitopes. Neuraminidase-treated LOS separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stained showed a shift in migration from a component with a mass of approximately 4.8 kDa to a component with a mass of between 4.5 and 4.6 kDa. The same strains grown in medium with excess CMP-N-acetylneuraminic acid had LOS that shifted in migration to a slightly higher component (mass, approximately 4.8 kDa). Chemical analysis of the neuraminidase-digested products from one LOS indicated it contained approximately 1.5% sialic acid. Covalent linkage between sialic acid and the LOS was confirmed by analysis of de-O-acylated and dephosphorylated LOS by liquid secondary ion mass spectrometry. Three studies show that some meningococci contain sialic acid in their LOS, that the sialic acid is cleaved and lost in conventional acetic acid hydrolysis, and that the sialic acid alters the expression of MAb-defined epitopes. Images PMID:1708379

  4. Isolation and molecular characterisation of flavonoid 3'-hydroxylase and flavonoid 3', 5'-hydroxylase genes from a traditional Chinese medicinal plant, Epimedium sagittatum.

    PubMed

    Huang, Wenjun; Sun, Wei; Wang, Ying

    2012-04-10

    The epimedii herb, a traditional Chinese medicinal plant, has significant pharmacological effects on human health. The bioactive components in the herb (Epimedium sagittatum (Sieb. et Zucc.) Maxim) are mainly prenylated flavonol glycosides, which are end-products of the flavonoid biosynthetic pathway. This has not been clearly elucidated until recently. The genes encoding flavonoid 3'-hydroxylase (F3'H) and flavonoid 3', 5'-hydroxylase (F3'5'H) involved in the flavonoid biosynthetic pathway, designated as EsF3'H and EsF3'5'H, were isolated from E. sagittatum using a homology-based cloning method and deposited in the GenBank databases (GenBank ID: HM011054 and HM011055), respectively. EsF3'H and EsF3'5'H proteins shared high homology with other plant-specific flavonoid hydroxylases and were clustered into the CYP75B and CYP75A group, respectively. In addition, four conserved cytochrome P450-featured motifs were found in the amino acid sequences of both genes. Transcription levels of both genes were detected in all tissues tested and were high in most of the pigmented tissues. Moreover, the expression levels of both EsF3'H and EsF3'5'H correlated positively with the anthocyanin accumulation pattern in leaves from E. sagittatum. The cloning and molecular characterisation of EsF3'H and EsF3'5'H genes will accelerate progress in the study of the flavonoid biosynthetic pathway to elucidate the molecular mechanisms of the biosynthesis of the bioactive components in E. sagittatum.

  5. Phenylalanine hydroxylase: function, structure, and regulation.

    PubMed

    Flydal, Marte I; Martinez, Aurora

    2013-04-01

    Mammalian phenylalanine hydroxylase (PAH) catalyzes the rate-limiting step in the phenylalanine catabolism, consuming about 75% of the phenylalanine input from the diet and protein catabolism under physiological conditions. In humans, mutations in the PAH gene lead to phenylketonuria (PKU), and most mutations are mainly associated with PAH misfolding and instability. The established treatment for PKU is a phenylalanine-restricted diet and, recently, supplementation with preparations of the natural tetrahydrobiopterin cofactor also shows effectiveness for some patients. Since 1997 there has been a significant increase in the understanding of the structure, catalytic mechanism, and regulation of PAH by its substrate and cofactor, in addition to improved correlations between genotype and phenotype in PKU. Importantly, there has also been an increased number of studies on the structure and function of PAH from bacteria and lower eukaryote organisms, revealing an additional anabolic role of the enzyme in the synthesis of melanin-like pigments. In this review, we discuss these recent studies, which contribute to define the evolutionary adaptation of the PAH structure and function leading to sophisticated regulation for effective catabolic processing of phenylalanine in mammalian organisms.

  6. Tetrahydrobiopterin shows chaperone activity for tyrosine hydroxylase.

    PubMed

    Thöny, Beat; Calvo, Ana C; Scherer, Tanja; Svebak, Randi M; Haavik, Jan; Blau, Nenad; Martinez, Aurora

    2008-07-01

    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamine neurotransmitters. Primary inherited defects in TH have been associated with l-DOPA responsive and non-responsive dystonia and infantile parkinsonism. In this study, we show that both the cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and the feedback inhibitor and catecholamine product dopamine increase the kinetic stability of human TH isoform 1 in vitro. Activity measurements and synthesis of the enzyme by in vitro transcription-translation revealed a complex regulation by the cofactor including both enzyme inactivation and conformational stabilization. Oral BH(4) supplementation to mice increased TH activity and protein levels in brain extracts, while the Th-mRNA level was not affected. All together our results indicate that the molecular mechanisms for the stabilization are a primary folding-aid effect of BH(4) and a secondary effect by increased synthesis and binding of catecholamine ligands. Our results also establish that orally administered BH(4) crosses the blood-brain barrier and therapeutic regimes based on BH(4) supplementation should thus consider the effect on TH. Furthermore, BH(4) supplementation arises as a putative therapeutic agent in the treatment of brain disorders associated with TH misfolding, such as for the human TH isoform 1 mutation L205P.

  7. Tryptophan hydroxylase expression in human skin cells.

    PubMed

    Slominski, Andrzej; Pisarchik, Alexander; Johansson, Olle; Jing, Chen; Semak, Igor; Slugocki, George; Wortsman, Jacobo

    2003-10-15

    We attempted to further characterize cutaneous serotoninergic and melatoninergic pathways evaluating the key biosynthetic enzyme tryptophan hydroxylase (TPH). There was wide expression of TPH mRNA in whole human skin, cultured melanocytes and melanoma cells, dermal fibroblasts, squamous cell carcinoma cells and keratinocytes. Gene expression was associated with detection of TPH immunoreactive species by Western blotting. Characterization of the TPH immunoreactive species performed with two different antibodies showed expression of the expected protein (55-60 kDa), and of forms with higher and lower molecular weights. This pattern of broad spectrum of TPH expression including presumed degradation products suggests rapid turnover of the enzyme, as previously reported in mastocytoma cells. RP-HPLC of skin extracts showed fluorescent species with the retention time of serotonin and N-acetylserotonin. Immunocytochemistry performed in skin biopsies localized TPH immunoreactivity to normal and malignant melanocytes. We conclude that while the TPH mRNA and protein are widely expressed in cultured normal and pathological epidermal and dermal skin cells, in vivo TPH expression is predominantly restricted to cells of melanocytic origin.

  8. Biochemical and genetic characterization of three molybdenum cofactor hydroxylases in Arabidopsis thaliana.

    PubMed

    Hoff, T; Frandsen, G I; Rocher, A; Mundy, J

    1998-07-09

    Aldehyde oxidases and xanthine dehydrogenases/oxidases belong to the molybdenum cofactor dependent hydroxylase class of enzymes. Zymograms show that Arabidopsis thaliana has at least three different aldehyde oxidases and one xanthine oxidase. Three different cDNA clones encoding putative aldehyde oxidases (AtAO1, 2, 3) were isolated. An aldehyde oxidase is the last step in abscisic acid (ABA) biosynthesis. AtAO1 is mainly expressed in seeds and roots which might reflect that it is involved in ABA biosynthesis.

  9. Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase.

    PubMed

    Glieder, Anton; Farinas, Edgardo T; Arnold, Frances H

    2002-11-01

    We have converted cytochrome P450 BM-3 from Bacillus megaterium (P450 BM-3), a medium-chain (C12-C18) fatty acid monooxygenase, into a highly efficient catalyst for the conversion of alkanes to alcohols. The evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for the selective oxidation of hydrocarbons of small to medium chain length (C3-C8). Unlike naturally occurring alkane hydroxylases, the best known of which are the large complexes of methane monooxygenase (MMO) and membrane-associated non-heme iron alkane monooxygenase (AlkB), the evolved enzyme is monomeric, soluble, and requires no additional proteins for catalysis. The evolved alkane hydroxylase was found to be even more active on fatty acids than wild-type BM-3, which was already one of the most efficient fatty acid monooxgenases known. A broad range of substrates including the gaseous alkane propane induces the low to high spin shift that activates the enzyme. This catalyst for alkane hydroxylation at room temperature opens new opportunities for clean, selective hydrocarbon activation for chemical synthesis and bioremediation.

  10. Differential hepatocellular zonation pattern of cholesterol 7alpha-hydroxylase (Cyp7a1) and sterol 12alpha-hydroxylase (Cyp8b1) in the mouse.

    PubMed

    Wang, Jin; Olin, Maria; Rozell, Björn; Björkhem, Ingemar; Einarsson, Curt; Eggertsen, Gösta; Gåfvels, Mats

    2007-03-01

    The synthesis of primary bile acids is confined to the hepatocytes. This study aimed to evaluate the expression pattern within the liver architecture of the rate-limiting enzyme of the neutral pathway, cholesterol 7alpha-hydroxylase (Cyp7a1), and sterol 12alpha-hydroxylase (Cyp8b1), the enzyme necessary for the synthesis of cholic acid. Specific Cyp8b1 and Cyp7a1 peptide antiserums were used for immunohistochemical staining of livers from wild type and Cyp8b1 null mice, the latter instead expressing beta-galactosidase (beta-Gal) as a replacement reporter gene. Cyp8b1 was mainly expressed in the hepatocytes in a zonal pattern surrounding the central vein while the areas surrounding the portal zones showed much lower levels. The zonation was maintained in cholic acid-depleted mice using beta-Gal as a reporter protein. Cyp7a1 expression in wild type mice also showed a zonal distribution pattern, although less distinct, with a maximal expression within a 1-2 cell thick layer of hepatocytes surrounding the central vein. In Cyp8b1 null mice, a more intense staining was obtained, in accordance with the higher expression level of Cyp7a1, although the overall expression pattern was maintained. Our results in mice indicate possible differences in the regulation of the cellular zonation of Cyp7a1 and Cyp8b1. Also, cholic acid affects the set-point of Cyp7a1 expression but not its zonal distribution.

  11. Unexpected inhibition of cholesterol 7 alpha-hydroxylase by cholesterol in New Zealand white and Watanabe heritable hyperlipidemic rabbits.

    PubMed Central

    Xu, G; Salen, G; Shefer, S; Ness, G C; Nguyen, L B; Parker, T S; Chen, T S; Zhao, Z; Donnelly, T M; Tint, G S

    1995-01-01

    We investigated the effect of cholesterol feeding on plasma cholesterol concentrations, hepatic activities and mRNA levels of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase and hepatic LDL receptor function and mRNA levels in 23 New Zealand White (NZW) and 17 Watanabe heritable hyperlipidemic (WHHL) rabbits. Plasma cholesterol concentrations were 9.9 times greater in WHHL than NZW rabbits and rose significantly in both groups when cholesterol was fed. Baseline liver cholesterol levels were 50% higher but rose only 26% in WHHL as compared with 3.6-fold increase with the cholesterol diet in NZW rabbits. In both rabbit groups, hepatic total HMG-CoA reductase activity was similar and declined > 60% without changing enzyme mRNA levels after cholesterol was fed. In NZW rabbits, cholesterol feeding inhibited LDL receptor function but not mRNA levels. As expected, receptor-mediated LDL binding was reduced in WHHL rabbits. Hepatic cholesterol 7 alpha-hydroxylase activity and mRNA levels were 2.8 and 10.4 times greater in NZW than WHHL rabbits. Unexpectedly, cholesterol 7 alpha-hydroxylase activity was reduced 53% and mRNA levels were reduced 79% in NZW rabbits with 2% cholesterol feeding. These results demonstrate that WHHL as compared with NZW rabbits have markedly elevated plasma and higher liver cholesterol concentrations, less hepatic LDL receptor function, and very low hepatic cholesterol 7 alpha-hydroxylase activity and mRNA levels. Feeding cholesterol to NZW rabbits increased plasma and hepatic concentrations greatly, inhibited LDL receptor-mediated binding, and unexpectedly suppressed cholesterol 7 alpha-hydroxylase activity and mRNA to minimum levels similar to WHHL rabbits. Dietary cholesterol accumulates in the plasma of NZW rabbits, and WHHL rabbits are hypercholesterolemic because reduced LDL receptor function is combined with decreased catabolism of cholesterol to bile acids. Images PMID:7706454

  12. Dopamine beta-hydroxylase immunoreactivity in human cerebrospinal fluid: properties, relationship to central noradrenergic neuronal activity and variation in Parkinson's disease and congenital dopamine beta-hydroxylase deficiency.

    PubMed

    O'Connor, D T; Cervenka, J H; Stone, R A; Levine, G L; Parmer, R J; Franco-Bourland, R E; Madrazo, I; Langlais, P J; Robertson, D; Biaggioni, I

    1994-02-01

    1. Dopamine beta-hydroxylase is stored and released with catecholamines by exocytosis from secretory vesicles in noradrenergic neurons and chromaffin cells. Although dopamine beta-hydroxylase enzymic activity is measurable in cerebrospinal fluid, such activity is unstable, and its relationship to central noradrenergic neuronal activity in humans is not clearly established. To explore the significance of cerebrospinal fluid dopamine beta-hydroxylase, we applied a homologous human dopamine beta-hydroxylase radioimmunoassay to cerebrospinal fluid, in order to characterize the properties and stability of cerebrospinal fluid dopamine beta-hydroxylase, as well as its relationship to central noradrenergic neuronal activity and its variation in disease states such as hypertension, renal failure, Parkinsonism and congenital dopamine beta-hydroxylase deficiency. 2. Authentic, physically stable dopamine beta-hydroxylase immunoreactivity was present in normal human cerebrospinal fluid at a concentration of 31.3 +/- 1.4 ng/ml (range: 18.5-52.5 ng/ml), but at a 283 +/- 27-fold lower concentration than that found in plasma. Cerebrospinal fluid and plasma dopamine beta-hydroxylase concentrations were correlated (r = 0.67, P = 0.001). Some degree of local central nervous system control of cerebrospinal fluid dopamine beta-hydroxylase was suggested by incomplete correlation with plasma dopamine beta-hydroxylase (with an especially marked dissociation in renal disease) as well as the lack of a ventricular/lumbar cerebrospinal dopamine beta-hydroxylase concentration gradient. 3. Cerebrospinal fluid dopamine beta-hydroxylase was not changed by the central alpha 2-agonist clonidine at a dose that diminished cerebrospinal fluid noradrenaline, nor did cerebrospinal fluid dopamine beta-hydroxylase correspond between subjects to cerebrospinal fluid concentrations of noradrenaline or methoxyhydroxyphenylglycol; thus, cerebrospinal fluid dopamine beta-hydroxylase concentration was not closely

  13. Structural features of the regulatory ACT domain of phenylalanine hydroxylase.

    PubMed

    Carluccio, Carla; Fraternali, Franca; Salvatore, Francesco; Fornili, Arianna; Zagari, Adriana

    2013-01-01

    Phenylalanine hydroxylase (PAH) catalyzes the conversion of L-Phe to L-Tyr. Defects in PAH activity, caused by mutations in the human gene, result in the autosomal recessively inherited disease hyperphenylalaninemia. PAH activity is regulated by multiple factors, including phosphorylation and ligand binding. In particular, PAH displays positive cooperativity for L-Phe, which is proposed to bind the enzyme on an allosteric site in the N-terminal regulatory domain (RD), also classified as an ACT domain. This domain is found in several proteins and is able to bind amino acids. We used molecular dynamics simulations to obtain dynamical and structural insights into the isolated RD of PAH. Here we show that the principal motions involve conformational changes leading from an initial open to a final closed domain structure. The global intrinsic motions of the RD are correlated with exposure to solvent of a hydrophobic surface, which corresponds to the ligand binding-site of the ACT domain. Our results strongly suggest a relationship between the Phe-binding function and the overall dynamic behaviour of the enzyme. This relationship may be affected by structure-disturbing mutations. To elucidate the functional implications of the mutations, we investigated the structural effects on the dynamics of the human RD PAH induced by six missense hyperphenylalaninemia-causing mutations, namely p.G46S, p.F39C, p.F39L, p.I65S, p.I65T and p.I65V. These studies showed that the alterations in RD hydrophobic interactions induced by missense mutations could affect the functionality of the whole enzyme.

  14. Ovarian intratumoral 21-hydroxylase deficiency in a postmenopausal hirsute woman.

    PubMed

    Souto, Selma B; Baptista, Pedro V; Barreto, Filomena; Sousa, Pedro F; Braga, Daniel C; Carvalho, Davide

    2012-12-01

    Virilising ovarian tumours are a rare cause of hyperandrogenism in women, accounting for less than 5% of all ovarian neoplasms. It occurs most often in - and postmenopausal women. We report a case of a 64 year-old woman with signs of virilisation that had started 3 years before. Blood hormone analysis revealed increased levels of testosterone, and 17-hydroxyprogesterone. The tetracosactin test revealed 21-hydroxylase deficiency. Radiological imaging demonstrated a nodule in her left ovary. The patient was submitted to bilateral laparoscopic oophorectomy, and histopathological examination revealed a luteoma of the left ovary. Postoperative serum testosterone level and 17-hydroxyprogesterone returned to normal levels in one month. Virilism regressed within six months. Our patient also showed an elevation in 17-OHP serum levels. Normalization of 17-OHP after oophorectomy suggests a case of intratumoral 21-hydroxylase deficiency. To our knowledge, this is the first description of ovarian intratumoral 21-hydroxylase deficiency in a postmenopausal woman.

  15. Cloning, functional expression, and characterization of a chalcone 3-hydroxylase from Cosmos sulphureus.

    PubMed

    Schlangen, Karin; Miosic, Silvija; Thill, Jana; Halbwirth, Heidi

    2010-07-01

    A chalcone 3-hydroxylase (CH3H) cDNA clone was isolated and characterized from Cosmos sulphureus petals accumulating butein (2',3,4,4'-tetrahydroxychalcone) derivatives as yellow flower pigments. The recombinant protein catalyses the introduction of an additional hydroxyl group in the B-ring of chalcones, a reaction with high similarity to the hydroxylation of flavonoids catalysed by the well-studied flavonoid 3'-hydroxylase (F3'H). CH3H shows high specificity for chalcones, but a low F3'H activity was also detected. By contrast, the common F3'H from C. sulphureus does not accept chalcones as substrates and is therefore unlikely to be involved in the creation of the B-ring hydroxylation pattern of the yellow flower pigments. CH3H was primarily expressed in young buds, the main tissue for chalcone pigment formation. Expression levels in open flowers and 3-d-old seedlings were lower and almost no CH3H expression was observed in leaves. F3'H, in contrast, showed the highest expression also in buds, but comparable expression rates in all other tissues tested. Recombinant hybrid proteins constructed from CH3H and F3'H fragments demonstrated that amino acid residues at a substrate recognition site and an insertion of four amino acid residues in a putative loop region have an impact on chalcone acceptance. This is the first identification of a CH3H cDNA from any plant species.

  16. Proteasomal inhibition causes loss of nigral tyrosine hydroxylase neurons.

    PubMed

    Schapira, Anthony H V; Cleeter, Michael W J; Muddle, John R; Workman, Jane M; Cooper, J Mark; King, Rosalind H M

    2006-08-01

    Dysfunction of the ubiquitin-proteasomal system (UPS) has been implicated in the pathogenesis of Parkinson's disease. The systemic administration of UPS inhibitors has been reported to induce nigrostriatal cell death and model Parkinson's disease pathology in rodents. We administered a synthetic, specific UPS inhibitor (PSI) subcutaneously to rats and quantified substantia nigral tyrosine hydroxylase-positive dopaminergic neurons by stereology. PSI caused a 15% decrease in UPS activity at 2 weeks and a 42% reduction in substantia nigra pars compacta tyrosine hydroxylase-positive neurons at 8 weeks. Systemic inhibition of the UPS warrants further evaluation as a means to model Parkinson's disease.

  17. Structural insights into substrate specificity of Feruloyl-CoA 6’-Hydroxylase from Arabidopsis thaliana

    DOE PAGES

    Sun, Xinxiao; Zhou, Dayong; Kandavelu, Palani; ...

    2015-05-20

    Coumarins belong to an important class of plant secondary metabolites. Feruloyl-CoA 6’-hydroxylase (F6’H), a 2-oxoglutarate dependent dioxygenase (2OGD), catalyzes a pivotal step in the biosynthesis of a simple coumarin scopoletin. In this study, we determined the 3-dimensional structure of the F6’H1 apo enzyme by X-ray crystallography. It is the first reported structure of a 2OGD enzyme involved in coumarin biosynthesis and closely resembles the structure of Arabidopsis thaliana anthocyanidin synthase. To better understand the mechanism of enzyme catalysis and substrate specificity, we also generated a homology model of a related ortho-hydroxylase (C2’H) from sweet potato. By comparing these two structures,more » we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis.« less

  18. Regulation of phenylalanine hydroxylase: conformational changes upon phenylalanine binding detected by hydrogen/deuterium exchange and mass spectrometry.

    PubMed

    Li, Jun; Dangott, Lawrence J; Fitzpatrick, Paul F

    2010-04-20

    Phenylalanine acts as an allosteric activator of the tetrahydropterin-dependent enzyme phenylalanine hydroxylase. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into local conformational changes accompanying activation of rat phenylalanine hydroxylase by phenylalanine. Peptides in the regulatory and catalytic domains that lie in the interface between these two domains show large increases in the extent of deuterium incorporation from solvent in the presence of phenylalanine. In contrast, the effects of phenylalanine on the exchange kinetics of a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site for the amino acid substrate. These results support a model in which the N-terminus of the protein acts as an inhibitory peptide, with phenylalanine binding causing a conformational change in the regulatory domain that alters the interaction between the catalytic and regulatory domains.

  19. Structural Insights into Substrate Specificity of Feruloyl-CoA 6’-Hydroxylase from Arabidopsis thaliana

    PubMed Central

    Sun, Xinxiao; Zhou, Dayong; Kandavelu, Palani; Zhang, Hua; Yuan, Qipeng; Wang, Bi-Cheng; Rose, John; Yan, Yajun

    2015-01-01

    Coumarins belong to an important class of plant secondary metabolites. Feruloyl-CoA 6’-hydroxylase (F6’H), a 2-oxoglutarate dependent dioxygenase (2OGD), catalyzes a pivotal step in the biosynthesis of a simple coumarin scopoletin. In this study, we determined the 3-dimensional structure of the F6’H1 apo enzyme by X-ray crystallography. It is the first reported structure of a 2OGD enzyme involved in coumarin biosynthesis and closely resembles the structure of Arabidopsis thaliana anthocyanidin synthase. To better understand the mechanism of enzyme catalysis and substrate specificity, we also generated a homology model of a related ortho-hydroxylase (C2’H) from sweet potato. By comparing these two structures, we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis. PMID:25993561

  20. Molecular basis of nonclassical steroid 21-hydroxylase deficiency detected by neonatal mass screening in Japan.

    PubMed

    Tajima, T; Fujieda, K; Nakae, J; Toyoura, T; Shimozawa, K; Kusuda, S; Goji, K; Nagashima, T; Cutler, G B

    1997-07-01

    Since 1989, neonatal mass screening for congenital adrenal hyperplasia (CAH) has been performed in Japan, and the frequency of the classical form of 21-hydroxylase deficiency was found to be nearly identical to that in other countries. However, it has not yet been determined whether our mass screening program can detect the nonclassical (NC) form. From 1991 to 1994, about 4,500,000 infants underwent CAH mass screening in Japan. During this period, we identified by screening 2 siblings and 2 unrelated patients who had mild elevation of serum 17-hydroxyprogesterone levels at 5 days of age, but who revealed no symptoms of CAH. They were diagnosed as having probable NC steroid 21-hydroxylase deficiency. To clarify the molecular basis of NC CAH detectable by neonatal screening in Japan, the steroid 21-hydroxylase (CYP21) genes from these cases were analyzed. The 2 siblings (patients 1 and 2) had I172N and R356W mutations in 1 allele and in the other allele had local gene conversion, including the P30L mutation in exon 1. Patient 3, who was unrelated, had gene conversion encoding the same P30L mutation in 1 allele and in the other allele had an intron 2 mutation (668-12 A-->G), causing aberrant ribonucleic acid splicing, and the R356W mutation. Patient 4, also a compound heterozygote, had the R356W and 707del8 mutations. The estimated rate of detection of the NC form by mass screening (1:1,100,000) seemed low compare to the established detection rate for the classical form (1:18,000). As all of our 4 patients were compound heterozygotes with at least 1 allele bearing 1 or more mutations associated with classic CAH, it may be difficult to detect NC cases carrying only NC-associated alleles using our current neonatal mass screening methods.

  1. Therapeutic treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334) ameliorates murine colitis

    PubMed Central

    Gupta, Ram; Chaudhary, Anita R; Shah, Binita N; Jadhav, Avinash V; Zambad, Shitalkumar P; Gupta, Ramesh Chandra; Deshpande, Shailesh; Chauthaiwale, Vijay; Dutt, Chaitanya

    2014-01-01

    Background and aim Mucosal healing in inflammatory bowel disease (IBD) can be achieved by improvement of intestinal barrier protection. Activation of hypoxia-inducible factor (HIF) has been identified as a critical factor for barrier protection during mucosal insult and is linked with improvement in symptoms of colitis. Although prophylactic efficacy of HIF hydroxylase inhibitors in murine colitis have been established, its therapeutic efficacy in clinically relevant therapeutic settings have not been established. In the present study we aim to establish therapeutic efficacy of TRC160334, a novel HIF hydroxylase inhibitor, in animal models of colitis. Methods The efficacy of TRC160334 was evaluated in two different mouse models of colitis by oral route. A prophylactic efficacy study was performed in a 2,4,6-trinitrobenzene sulfonic acid-induced mouse model of colitis representing human Crohn’s disease pathology. Additionally, a therapeutic efficacy study was performed in a dextran sulfate sodium-induced mouse model of colitis, a model simulating human ulcerative colitis. Results TRC160334 treatment resulted in significant improvement in disease end points in both models of colitis. TRC160334 treatment resulted into cytoprotective heatshock protein 70 induction in inflamed colon. TRC160334 successfully attenuated the rate of fall in body weight, disease activity index, and macroscopic and microscopic scores of colonic damage leading to overall improvement in study outcome. Conclusion Our findings are the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor ameliorates IBD in disease models. These findings highlight the potential of TRC160334 for its clinical application in the treatment of IBD. PMID:24493931

  2. Amygdala responsiveness is modulated by tryptophan hydroxylase-2 gene variation.

    PubMed

    Canli, T; Congdon, E; Gutknecht, L; Constable, R T; Lesch, K P

    2005-11-01

    The tryptophan hydroxylase-2 gene (TPH2) codes for the enzyme of serotonin (5-HT) synthesis in the brain and variation of TPH2 has been implicated in disorders of emotion regulation. Here, we used functional magnetic resonance imaging (fMRI) to demonstrate that a potentially functional variant of TPH2 modulates amygdala responsiveness to emotional stimuli of both negative and positive valence.

  3. Recommendations for the nutrition management of phenylalanine hydroxylase deficiency.

    PubMed

    Singh, Rani H; Rohr, Fran; Frazier, Dianne; Cunningham, Amy; Mofidi, Shideh; Ogata, Beth; Splett, Patricia L; Moseley, Kathryn; Huntington, Kathleen; Acosta, Phyllis B; Vockley, Jerry; Van Calcar, Sandra C

    2014-02-01

    The effectiveness of a phenylalanine-restricted diet to improve the outcome of individuals with phenylalanine hydroxylase deficiency (OMIM no. 261600) has been recognized since the first patients were treated 60 years ago. However, the treatment regime is complex, costly, and often difficult to maintain for the long term. Improvements and refinements in the diet for phenylalanine hydroxylase deficiency have been made over the years, and adjunctive therapies have proven to be successful for certain patients. Yet evidence-based guidelines for managing phenylalanine hydroxylase deficiency, optimizing outcomes, and addressing all available therapies are lacking. Thus, recommendations for nutrition management were developed using evidence from peer-reviewed publications, gray literature, and consensus surveys. The areas investigated included choice of appropriate medical foods, integration of adjunctive therapies, treatment during pregnancy, monitoring of nutritional and clinical markers, prevention of nutrient deficiencies, providing of access to care, and compliance strategies. This process has not only provided assessment and refinement of current nutrition management and monitoring recommendations but also charted a direction for future studies. This document serves as a companion to the concurrently published American College of Medical Genetics and Genomics guideline for the medical treatment of phenylalanine hydroxylase deficiency.

  4. Levodopa-induced dyskinesias in tyrosine hydroxylase deficiency.

    PubMed

    Pons, Roser; Syrengelas, Dimitris; Youroukos, Sotiris; Orfanou, Irene; Dinopoulos, Arqirios; Cormand, Bru; Ormazabal, Aida; Garzía-Cazorla, Angels; Serrano, Mercedes; Artuch, Rafael

    2013-07-01

    The objective of this study was to characterize levodopa (l-dopa)-induced dyskinesias in patients with tyrosine hydroxylase deficiency. Clinical observation was carried out on 6 patients who were diagnosed with tyrosine hydroxylase deficiency and were treated with escalating doses of l-dopa. All 6 patients showed l-dopa-induced dyskinesias of variable intensity early in the course of treatment and regardless of the age of initiation. l-Dopa-induced dyskinesias were precipitated by increases in the dose of l-dopa and also by febrile illnesses and stress. They caused dysfunction and distress in 2 patients. The dyskinesias were improved by decreasing the l-dopa dose or by slowing its titration upward. Increasing the dose frequency was helpful in 2 patients, and introducing amantadine was helpful in another 2 patients. l-Dopa-induced dyskinesias are a common phenomenon in tyrosine hydroxylase deficiency. The current observations show that l-dopa-induced dyskinesias are frequent in a dopamine-deficient state in the absence of nigrostriatal degeneration. Although l-dopa-induced dyskinesias in tyrosine hydroxylase deficiency are phenomenologically similar to those that occur in Parkinson's disease, they are different in a number of other respects, suggesting intrinsic differences in the pathophysiologic basis of l-dopa-induced dyskinesias in the 2 conditions. © 2013 Movement Disorder Society.

  5. Association between Tryptophan Hydroxylase 2 Gene Polymorphism and Completed Suicide

    ERIC Educational Resources Information Center

    Fudalej, Sylwia; Ilgen, Mark; Fudalej, Marcin; Kostrzewa, Grazyna; Barry, Kristen; Wojnar, Marcin; Krajewski, Pawel; Blow, Frederic; Ploski, Rafal

    2010-01-01

    The association between suicide and a single nucleotide polymorphism (rs1386483) was examined in the recently identified tryptophan hydroxylase 2 (TPH2) gene. Blood samples of 143 suicide victims and 162 age- and sex-matched controls were examined. The frequency of the TT genotype in the TPH2 polymorphism was higher in suicide victims than in…

  6. Functional characterization of myrcene hydroxylases from two geographically distinct Ips pini populations.

    PubMed

    Song, Minmin; Kim, Amy C; Gorzalski, Andrew J; MacLean, Marina; Young, Sharon; Ginzel, Matthew D; Blomquist, Gary J; Tittiger, Claus

    2013-04-01

    Ips pini bark beetles use myrcene hydroxylases to produce the aggregation pheromone component, ipsdienol, from myrcene. The enantiomeric ratio of pheromonal ipsdienol is an important prezygotic mating isolation mechanism of I. pini and differs among geographically distinct populations. We explored the substrate and product ranges of myrcene hydroxylases (CYP9T2 and CYP9T3) from reproductively-isolated western and eastern I. pini. The two cytochromes P450 share 94% amino acid identity. CYP9T2 mRNA levels were not induced in adults exposed to myrcene-saturated atmosphere. Functional assays of recombinant enzymes showed both hydroxylated myrcene, (+)- and (-)-α-pinene, 3-carene, and R-(+)-limonene, but not α-phellandrene, (-)-β-pinene, γ-terpinene, or terpinolene, with evidence that CYP9T2 strongly preferred myrcene over other substrates. They differed in the enantiomeric ratios of ipsdienol produced from myrcene, and in the products resulting from different α-pinene enantiomers. These data provide new information regarding bark beetle pheromone evolution and factors affecting cytochrome P450 structure-function relationships.

  7. Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

    PubMed

    Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine

    2005-08-01

    Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

  8. Cytochrome P450 ω-hydroxylase promotes angiogenesis and metastasis by upregulation of VEGF and MMP-9 in non-small cell lung cancer

    PubMed Central

    Yu, Wei; Chen, Li; Yang, Yu-Qing; Falck, John R.; Guo, Austin M.; Li, Ying

    2013-01-01

    Purpose Cytochrome P450 (CYP) ω-hydroxylase, mainly consisting of CYP4A and CYP4F, converts arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) that induces angiogenic responses in vivo and in vitro. The present study examined the role of CYP ω-hydroxylase in angiogenesis and metastasis of human non-small cell lung cancer (NSCLC). Methods The effect of WIT003, a stable 20-HETE analog, on invasion was evaluated using a modified Boyden chamber in three NSCLC cell lines. A549 cells were transfected with CYP4A11 expression vector or exposed to CYP ω-hydroxylase inhibitor (HET0016) or 20-HETE antagonist (WIT002), and then ω-hydroxylation activity toward arachidonic acid and the levels of matrix metalloproteinases (MMPs) and VEGF were detected. The in vivo effects of CYP ω-hydroxylase were tested in established tumor xenografts and an experimental metastasis model in athymic mice. Results Addition of WIT003 or overexpression of CYP4A11 with an associated increase in 20-HETE production significantly induced invasion and expression of VEGF and MMP-9. Treatment of A549 cells with HET0016 or WIT002 inhibited invasion with reduction in VEGF and MMP-9. The PI3 K or ERK inhibitors also attenuated expression of VEGF and MMP-9. Compared with control, CYP4A11 transfection significantly increased tumor weight, microvessel density (MVD), and lung metastasis by 2.5-fold, 2-fold, and 3-fold, respectively. In contrast, WIT002 or HET0016 decreased tumor volume, MVD, and spontaneous pulmonary metastasis occurrences. Conclusion CYP ω-hydroxylase promotes tumor angiogenesis and metastasis by upregulation of VEGF and MMP-9 via PI3 K and ERK1/2 signaling in human NSCLC cells. PMID:21120482

  9. Biotransformation of Cholesterol and 16α,17α-Epoxypregnenolone and Isolation of Hydroxylase in Burkholderia cepacia SE-1.

    PubMed

    Zhu, XiangDong; Pang, CuiPing; Cao, Yuting; Fan, Dan

    2016-01-01

    The metabolism of cholesterol is critical in eukaryotes as a precursor for vitamins, steroid hormones, and bile acids. Some steroid compounds can be transformed into precursors of steroid medicine by some microorganisms. In this study, the biotransformation products of cholesterol and 16α,17α-epoxypregnenolone produced by Burkholderia cepacia SE-1 were investigated, and a correlative enzyme, hydroxylase, was also studied. The biotransformation products, 7β-hydroxycholesterol, 7-oxocholesterol, and 20-droxyl-16α,17α-epoxypregn-1,4-dien-3-one, were purified by silica gel and Sephadex LH-20 column chromatography and identified by nuclear magnetic resonance and mass spectroscopy. The hydroxylase was isolated from the bacterium and the partial sequences of the hydroxylase, which belong to the catalases/peroxidase family, were analyzed using MS/MS analyses. The enzyme showed activity toward cholesterol and had a specific activity of 37.2 U/mg of protein at 30°C and pH 7.0.

  10. Biotransformation of Cholesterol and 16α,17α-Epoxypregnenolone and Isolation of Hydroxylase in Burkholderia cepacia SE-1

    PubMed Central

    Zhu, XiangDong; Pang, CuiPing; Cao, Yuting; Fan, Dan

    2016-01-01

    The metabolism of cholesterol is critical in eukaryotes as a precursor for vitamins, steroid hormones, and bile acids. Some steroid compounds can be transformed into precursors of steroid medicine by some microorganisms. In this study, the biotransformation products of cholesterol and 16α,17α-epoxypregnenolone produced by Burkholderia cepacia SE-1 were investigated, and a correlative enzyme, hydroxylase, was also studied. The biotransformation products, 7β-hydroxycholesterol, 7-oxocholesterol, and 20-droxyl-16α,17α-epoxypregn-1,4-dien-3-one, were purified by silica gel and Sephadex LH-20 column chromatography and identified by nuclear magnetic resonance and mass spectroscopy. The hydroxylase was isolated from the bacterium and the partial sequences of the hydroxylase, which belong to the catalases/peroxidase family, were analyzed using MS/MS analyses. The enzyme showed activity toward cholesterol and had a specific activity of 37.2 U/mg of protein at 30°C and pH 7.0. PMID:27340662

  11. Evidence for a high-spin Fe(IV) species in the catalytic cycle of a bacterial phenylalanine hydroxylase.

    PubMed

    Panay, Aram Joel; Lee, Michael; Krebs, Carsten; Bollinger, J Martin; Fitzpatrick, Paul F

    2011-03-22

    Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mössbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme-(57)Fe(II)-phenylalanine-6-methyltetrahydropterin complex with O(2) reveal the accumulation of an intermediate at short reaction times (20-100 ms). The Mössbauer parameters of the intermediate (δ = 0.28 mm/s, and |ΔE(Q)| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme-substrate complex reacts with oxygen to form an Fe(IV)═O intermediate with a rate constant of 19 mM(-1) s(-1), the Fe(IV)═O intermediate hydroxylates phenylalanine with a rate constant of 42 s(-1), and rate-limiting product release occurs with a rate constant of 6 s(-1) at 5 °C.

  12. Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis.

    PubMed

    Laitala, Anu; Aro, Ellinoora; Walkinshaw, Gail; Mäki, Joni M; Rossi, Maarit; Heikkilä, Minna; Savolainen, Eeva-Riitta; Arend, Michael; Kivirikko, Kari I; Koivunen, Peppi; Myllyharju, Johanna

    2012-10-18

    An endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm(-/-) mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm(-/-) mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497-treated Hif-p4h-2 hypomorphic (Hif-p4h-2(gt/gt)) and Hif-p4h-3(-/-) mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm(-/-) and wild-type mice, but caused higher increases in both values in the Hif-p4h-2(gt/gt) mice and in hematocrit value in the Hif-p4h-3(-/-) mice than in the wild-type. Hif-p4h-2(gt/gt)/P4h-tm(-/-) double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2(gt/gt) or P4h-tm(-/-) mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

  13. A large duplication in the gene for lysyl hydroxylase accounts for the type VI variant of Ehlers-Danlos syndrome in two siblings

    SciTech Connect

    Hautala, T.; Heikkinen, J.; Kivirikko, K.I.; Myllylae, R. )

    1993-02-01

    Ehlers-Danlos syndrome is a deterogeneous disorder characterized by joint hypermobility, skin hyperextensibility, fragility, and other sign of connective tissue involvement. In addition to these, the type VI variant of the disease has some special characteristics such as kyphoscoliosis and ocular abnormalities. The biochemical abnormality in most patients with this autosomal recessively inherited type IV variant is a deficiency in the activity of lysyl hydroxylase (EC 1.14,11.4), the enzyme catalyzing the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. The type VI variant of Ehlers-Danlos syndrome was first identified in two sisters with a reduced amount of lysyl hydroxylase activity in their skin fibroblasts (S.R. Pinnell, S.M. Krane, J.E. Kenzora, and M.J. Glimcher (1972) N. Engl. J. Med. 286; 1013-1020). Our recent molecular cloning of lysyl hydroxylase has now made it possible to study the mutations leading to the deficiency in lysyl dydroxylase activity in these cells. Our data indicate that the mRNA for lysyl hydroxylase produced in the affected cells is about 4 kb in size, whereas it is 3.2 kb in the control cells. The sequencing of the cDNA for lysyl hydroxylase from the affected cells revealed an apparently homozygous duplication rearrangement of nucleotides 1176 to 1955, corresponding to amino acids 326 to 585 in the normal sequence. From Southern blotting data, the duplicated area in the gene equals about 6-9 kb and corresponds to seven exons. 35 refs., 4 figs.

  14. HIF hydroxylase pathways in cardiovascular physiology and medicine

    PubMed Central

    Bishop, Tammie; Ratcliffe, Peter J.

    2015-01-01

    Hypoxia inducible factors (HIFs) are alpha/beta heterodimeric transcription factors that direct multiple cellular and systemic responses in response to changes in oxygen availability. The oxygen sensitive signal is generated by a series of iron and 2-oxoglutarate dependent dioxygenases that catalyse post-translational hydroxylation of specific prolyl and asparaginyl residues in HIFalpha subunits and thereby promote their destruction and inactivation in the presence of oxygen. In hypoxia, these processes are suppressed allowing HIF to activate a massive transcriptional cascade. Elucidation of these pathways has opened several new fields of cardiovascular research. Here we review the role of HIF hydroxylase pathways in cardiac development and in cardiovascular control. We also consider the current status, opportunities and challenges of therapeutic modulation of HIF hydroxylases in the therapy of cardiovascular disease. PMID:26089364

  15. Ablation of cytochrome P450 omega-hydroxylase 4A14 gene attenuates hepatic steatosis and fibrosis.

    PubMed

    Zhang, Xiaoyan; Li, Sha; Zhou, Yunfeng; Su, Wen; Ruan, Xiongzhong; Wang, Bing; Zheng, Feng; Warner, Margaret; Gustafsson, Jan-Åke; Guan, Youfei

    2017-03-21

    Nonalcoholic fatty liver disease (NAFLD) is characterized by simple hepatic steatosis (SS), nonalcoholic steatohepatitis (NASH), hepatic fibrosis, and cirrhosis. Dysregulated fatty acid metabolism in the liver plays a critical role in the pathogenesis of NAFLD. Cytochrome P450 omega-hydroxylase 4A14 (CYP4A14) is a homolog of human CYP4A hydroxylase that catalyzes omega-hydroxylation of medium-chain fatty acids and arachidonic acid in mice. The goal of this study was to determine the role of CYP4A14 in the development and the progression of NAFLD. Here, we showed that hepatic CYP4A expression was up-regulated in the livers of patients and three murine models of NAFLD. Adenovirus-mediated overexpression of CYP4A14 in the livers of C57BL/6 mice resulted in a fatty liver phenotype with a significant increase in hepatic fatty acid translocase (FAT/CD36) expression. In contrast, CYP4A14 gene-deficient mice fed a high-fat diet or a methionine and choline-deficient (MCD) diet exhibited attenuated liver lipid accumulation and reduced hepatic FAT/CD36 expression. In addition, hepatic inflammation and fibrosis was markedly ameliorated in MCD diet-fed CYP4A14-deficient mice. Collectively, CYP4A14 plays an important role in the pathogenesis of both SS and NASH and may represent a potential therapeutic target for the treatment of NAFLD.

  16. Rice CYP703A3, a cytochrome P450 hydroxylase, is essential for development of anther cuticle and pollen exine.

    PubMed

    Yang, Xijia; Wu, Di; Shi, Jianxin; He, Yi; Pinot, Franck; Grausem, Bernard; Yin, Changsong; Zhu, Lu; Chen, Mingjiao; Luo, Zhijing; Liang, Wanqi; Zhang, Dabing

    2014-10-01

    Anther cuticle and pollen exine act as protective envelopes for the male gametophyte or pollen grain, but the mechanism underlying the synthesis of these lipidic polymers remains unclear. Previously, a tapetum-expressed CYP703A3, a putative cytochrome P450 fatty acid hydroxylase, was shown to be essential for male fertility in rice (Oryza sativa L.). However, the biochemical and biological roles of CYP703A3 has not been characterized. Here, we observed that cyp703a3-2 caused by one base insertion in CYP703A3 displays defective pollen exine and anther epicuticular layer, which differs from Arabidopsis cyp703a2 in which only defective pollen exine occurs. Consistently, chemical composition assay showed that levels of cutin monomers and wax components were dramatically reduced in cyp703a3-2 anthers. Unlike the wide range of substrates of Arabidopsis CYP703A2, CYP703A3 functions as an in-chain hydroxylase only for a specific substrate, lauric acid, preferably generating 7-hydroxylated lauric acid. Moreover, chromatin immunoprecipitation and expression analyses revealed that the expression of CYP703A3 is directly regulated by Tapetum Degeneration Retardation, a known regulator of tapetum PCD and pollen exine formation. Collectively, our results suggest that CYP703A3 represents a conserved and diversified biochemical pathway for in-chain hydroxylation of lauric acid required for the development of male organ in higher plants.

  17. Human circulating dopamine-beta-hydroxylase and epilepsy.

    PubMed

    Warter, J M; Coquillat, G; Kurtz, D

    1975-01-01

    The activity of circulatory dopamine-beta-hydroxylase (DBH) in humans is shown to be lower in some epileptic subjects than in normal subjects. The activity of the enzymes was found to be dramatically low in subjects who experienced an epileptic seizure 24 hrs before DBH activity was determined. The activity varied through the course of epileptic seizures induced by a convulsant drugs and these variations might be due to the "en masse" changes of the sympathetic nervous system.

  18. Epigenetic Control of Prolyl and Asparaginyl Hydroxylases in Prostate Cancer

    DTIC Science & Technology

    2011-07-01

    containing proteins (PHD/EGLN/HPH) which utilize iron , oxygen and 2-oxoglutarate as co-factors to enzymatically catalyze hydroxylation on the oxygen-dependent...proteosome [6]. Under hypoxic conditions, HIF prolyl hydroxylase activity is decreased and HIF-1a protein accumulates . HIF-a subunits translocate to the...mechanism might be responsible for their silencing. Unlike genetic mutations that accumulate in cancer, epigenetic modifications are reversible [20]. We

  19. Giving anemia a boost with inhibitors of prolyl hydroxylase.

    PubMed

    Denny, William A

    2012-04-12

    There is much current interest in the development of inhibitors of the prolyl hydroxylase (PHD) enzymes that regulate the hypoxia-inducible transcription factor (HIF), which in turn stimulates the production of erythropoietin and ultimately red blood cells, as a treatment for anemia. A recent paper reports the synthesis and evaluation of a novel class of potent spirohydantoin-based pan-PHD inhibitors for this purpose. The paper is an exemplar of drug development from high-throughput screen to clinical candidate.

  20. Structure-Guided Reprogramming of a Hydroxylase To Halogenate Its Small Molecule Substrate.

    PubMed

    Mitchell, Andrew J; Dunham, Noah P; Bergman, Jonathan A; Wang, Bo; Zhu, Qin; Chang, Wei-Chen; Liu, Xinyu; Boal, Amie K

    2017-01-24

    Enzymatic installation of chlorine/bromine into unactivated carbon centers provides a versatile, selective, and environmentally friendly alternative to chemical halogenation. Iron(II) and 2-(oxo)-glutarate (Fe(II)/2OG)-dependent halogenases are powerful biocatalysts that are capable of cleaving aliphatic C-H bonds to introduce useful functional groups, including halogens. Using the structure of the Fe/2OG halogenase, WelO5, in complex with its small molecule substrate, we identified a similar N-acyl amino acid hydroxylase, SadA, and reprogrammed it to halogenate its substrate, thereby generating a new chiral haloalkyl center. The work highlights the potential of Fe(II)/2OG enzymes as platforms for development of novel stereospecific catalysts for late-stage C-H functionalization.

  1. Modulating carnitine levels by targeting its biosynthesis pathway – selective inhibition of γ-butyrobetaine hydroxylase

    PubMed Central

    Rydzik, Anna M.; Chowdhury, Rasheduzzaman; Kochan, Grazyna T.; Williams, Sophie T.; McDonough, Michael A.; Kawamura, Akane; Schofield, Christopher J.

    2015-01-01

    Carnitine is essential for fatty acid metabolism, but is associated with both health benefits and risks, especially heart diseases. We report the identification of potent, selective and cell active inhibitors of γ-butyrobetaine hydroxylase (BBOX), which catalyses the final step of carnitine biosynthesis in animals. A crystal structure of BBOX in complex with a lead inhibitor reveals that it binds in two modes, one of which adopts an unusual ‘U-shape’ conformation stabilised by inter- and intra-molecular π-stacking interactions. Conformational changes observed on binding of the inhibitor to BBOX likely reflect those occurring in catalysis; they also rationalise the inhibition of BBOX by high levels of its substrate γ-butyrobetaine (GBB), as observed both with isolated BBOX protein and in cellular studies. PMID:26682037

  2. Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics

    PubMed Central

    Calì, Francesco; Ruggeri, Giuseppa; Vinci, Mirella; Meli, Concetta; Carducci, Carla; Leuzzi, Vincenzo; Pozzessere, Simone; Schinocca, Pietro; Ragalmuto, Alda; Chiavetta, Valeria; Miccichè, Salvatore

    2010-01-01

    A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH) alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation- dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8 (systematic name c.353-?_912 + ?del) and exon 6 (systematic name c.510-?_706 + ?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics. PMID:19946181

  3. Cloning and characterization of a flavanone 3-hydroxylase gene from Saussurea medusa.

    PubMed

    Jin, Zhiping; Grotewold, Erich; Qu, Wenquan; Fu, Ghunxiang; Zhao, Dexiu

    2005-04-01

    Flavanone 3-hydroxylase (F3H) is a key enzyme in the flavonoid biosynthetic pathway, providing a branching point for the biosynthesis of different flavonoids, including the formation of 3-deoxy and 3-hydroxy flavonoids found in the silks of maize. Here, we report the cloning and characterization of a F3H gene (Smf3h) from a cDNA library derived from a red line callus of Saussurea medusa, a traditional Chinese medicinal plant. The cDNA contains a 1032 bp open reading frame (ORF) encoding a protein of 343 amino acid residues, a 149 bp long 5'untranslated regions (UTR) and a 163 bp long 3'UTR containing three putative polyadenylation signals (AATAAA) and an ATTTA element. The secondary structure of the mRNA predicted by MFOLD is very complex, suggesting a role in a post-transcriptional mechanism of regulation of Smf3h. The genomic structure of Smf3h includes four exons and three introns within the coding region, with all the splice donor/acceptor site sequences in accordance with the "GU-AG" consensus rule. The deduced SmF3H protein is 343 amino acid residues in length and has 40% and 39% identity and 60% and 58% similarity to the F3H of Arabidopsis and rice, respectively. Strikingly, the identity of SmF3H is higher to the H6H (hyoscyamine 3beta-hydroxylase, 45%) from Atropa belladonna. However, the analysis of the active center and the predicted protein secondary structure are more related to F3H than H6H. Together, our studies provide the first identification of a S. medusa flavonoid gene and its similarities to metabolic enzymes from other plants.

  4. The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13.

    PubMed

    Divari, Sara; Valetti, Francesca; Caposio, Patrizia; Pessione, Enrica; Cavaletto, Maria; Griva, Ersilia; Gribaudo, Giorgio; Gilardi, Gianfranco; Giunta, Carlo

    2003-05-01

    Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicomponent aromatic ring monooxygenase made up of three moieties: a reductase (PHR), an oxygenase (PHO) and a regulative component (PHI). The function of the oxygenase component (PHO), here characterized for the first time, is to bind molecular oxygen and catalyse the mono-hydroxylation of substrates (phenol, and with less efficiency, chloro- and methyl-phenol and naphthol). PHO was purified from extracts of A. radioresistens S13 cells and shown to be a dimer of 206 kDa. Each monomer is composed by three subunits: alpha (54 kDa), beta (38 kDa) and gamma (11 kDa). The gene encoding PHO alpha (named mopN) was cloned and sequenced and the corresponding amino acid sequence matched with that of functionally related oxygenases. By structural alignment with the catalytic subunits of methane monooxygenase (MMO) and alkene monooxygenase, we propose that PHO alpha contains the enzyme active site, harbouring a dinuclear iron centre Fe-O-Fe, as also suggested by spectral analysis. Conserved hydrophobic amino acids known to define the substrate recognition pocket, are also present in the alpha-subunit. The prevalence of alpha-helices (99.6%) as studied by CD confirmed the hypothized structural homologies between PHO and MMO. Three parameters (optimum ionic strength, temperature and pH) that affect kinetics of the overall phenol hydroxylase reaction were further analyzed with a fixed optimal PHR/PHI/PHO ratio of 2/1/1. The highest level of activity was evaluated between 0.075 and 0.1 m of ionic strength, the temperature dependence showed a maximum of activity at 24 degrees C and finally the pH for optimal activity was determined to be 7.5.

  5. The effectiveness of inhibitors of soluble prolyl hydroxylase against the enzyme in the cisternae of isolated bone microsomes.

    PubMed

    Tschank, G; Hanauske-Abel, H M; Peterkofsky, B

    1988-03-01

    Inhibitors of purified, soluble prolyl hydroxylase (K. Majamaa et al. (1984) Eur. J. Biochem. 138, 239-245; K. Majamaa et al. (1986) J. Biol. Chem. 261, 7819-7823) were tested against isolated chick embryo bone microsomes containing intracisternal prolyl hydroxylase and its radiolabeled, unhydroxylated procollagen substrate. Two groups of inhibitors were used which consisted of pyridine-2-carboxylate and 1,2-dihydroxybenzene (catechol) derivatives. The 2,4- and 2,5-pyridine dicarboxylic acids, which are potent inhibitors of the soluble enzyme (Ki values 2 and 0.8 microM, respectively), were effective in the same concentration range against intracisternal prolyl hydroxylase, although their relative affinities were reversed. Inhibition by pyridine-2,4-dicarboxylate in the microsomal system was reversed by increasing the concentration of 2-oxoglutarate. Pyridine-2,4-dicarboxylic acid did not inhibit the uptake of 2-[14C]oxoglutarate into microsomes, so it appears likely that the inhibitor must traverse the microsomal membrane and act directly at the enzyme level. Pyridine-2-carboxylic acid was ineffective in the microsomal system at 1 mM whereas it is a relatively potent inhibitor of the soluble enzyme with a Ki of 25 microM. This finding suggests that the second carboxyl group of the pyridine carboxylate derivatives may be required for their transport into the microsomal lumen. In the soluble system, 3,4-dihydroxybenzoic acid and 1,2-dihydroxybenzene had been found to be competitive inhibitors with relatively low Ki values of 5 and 25 microM, respectively. In the microsomal system, half-maximal inhibition was obtained at approximately 50-100 microM and inhibition was not reversed by increasing the concentrations of either 2-oxoglutarate or ascorbate, alone or together. These results imply that in situ these compounds do not inhibit prolyl hydroxylase directly. Thus, the microsomal system can assess the accessibility of the intracisternal enzyme to potential

  6. Insights into the catalytic mechanisms of phenylalanine and tryptophan hydroxylase from kinetic isotope effects on aromatic hydroxylation.

    PubMed

    Pavon, Jorge Alex; Fitzpatrick, Paul F

    2006-09-12

    Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-(2)H]-, [3,5-(2)H(2)]-, and (2)H(5)-phenylalanine as substrates. All (D)k(cat) values are normal with Delta117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12-1.41. In contrast, for Delta117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the (D)k(cat) value with [4-(2)H]-phenylalanine is 0.92 but is normal with [3,5-(2)H(2)]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Delta117PheH V379D shows a similar inverse isotope effect with [4-(2)H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-(2)H]-and [3,5(2)H(2)]-phenylalanine, are identical for Delta117PheH and Delta117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the (D)k(cat) value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight ( approximately 34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With (2)H(5)-indole-tryptophan as a substrate for Delta117PheH, the (D)k(cat) value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the (D)k(cat) values are within error of those for Delta117PheH V379D. Overall, these results

  7. Plasma Dopamine-Beta-Hydroxylase as an Index of Peripheral Noradrenergic Activity

    DTIC Science & Technology

    1981-08-17

    ABSTRACT Tit le of Dissertation: Plasma Dopamine-Beta- Hydroxylase as an Index of Peripheral Noradrenergic Act iv i ty John P. A f f ron t i...Pharmacology Dopamine-Beta- Hydroxylase (DBH) (E .C . I .14.17.1) is the biosynthetic enzyme for norepinephrine and is released with the neurotransmitter dur...lu id DBH as an index of central non- adrenergic act ivi ty is addressed. PLASMA DOPAMINE-BETA- HYDROXYLASE ACTIVITY AS AN INDEX OF PERIPHERAL

  8. Comparison of aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase induction by polycyclic aromatic compounds in human and mouse cell lines.

    PubMed

    Jaiswal, A K; Nebert, D W; Eisen, H W

    1985-08-01

    The human MCF-7 and the mouse Hepa-1 cell culture lines were compared for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]anthracene (BA) and TCDD- and BA-specific binding in the cytosol and nucleus. The effective concentration of BA in the growth medium required to induce either enzyme to 50% of its maximally inducible activity (EC50) was the same (5-11 microM) in both MCF-7 and Hepa-1 cells. On the other hand, the EC50 for TCDD in MCF-7 cells (5-25 nM) was more than 40-fold greater than that in Hepa-1 cells (0.4 to 0.6 nM). P1-450- and P3-450-specific mouse cDNA probes were used to quantitate mRNA induction in the Hepa-1 cell line. P1-450 mRNA was induced markedly by TCDD and benzo[a] anthracene, whereas P3-450 mRNA was induced negligibly. A P1-450-specific human cDNA probe was used to quantitate P1-450 mRNA induction in the MCF-7 cell line. Aryl hydrocarbon hydroxylase inducibility by TCDD or BA always paralleled P1-450 mRNA inducibility in either the mouse or human line. Although the cytosolic Ah receptor in Hepa-1 cells was easily detected by sucrose density gradient centrifugation, gel permeation chromatography, and anion-exchange high-performance liquid chromatography, the cytosolic receptor cannot be detected in MCF-7 cells. Following in vivo exposure of cultures to radiolabeled TCDD, the intranuclear concentration of inducer-receptor complex was at least fifty times greater in Hepa-1 than MCF-7 cultures. The complete lack of measurable cytosolic receptor and almost totally absent inducer-receptor complex in the nucleus of MCF-7 cells was, therefore, out of proportion to its capacity for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility. This MCF-7 line should provide an interesting model for a better understanding of the mechanisms of drug-metabolizing enzyme induction by polycyclic aromatic compounds, including the Ah receptor-mediated mechanism.

  9. Enhancement of L-tryptophan 5-hydroxylation activity by structure-based modification of L-phenylalanine 4-hydroxylase from Chromobacterium violaceum.

    PubMed

    Kino, Kuniki; Hara, Ryotaro; Nozawa, Ai

    2009-09-01

    The objective of this study was to enhance l-tryptophan hydroxylation activity of l-phenylalanine 4-hydroxylase. It had been known that l-phenylalanine 4-hydroxylase from Chromobacterium violaceum could convert l-tryptophan to 5-hydroxy-l-tryptophan and l-phenylalanine to l-tyrosine; however, the activity for l-tryptophan was extremely low compared to l-phenylalanine activity levels. We used the information on the crystal structures of aromatic amino acid hydroxylases to generate C. violaceuml-phenylalanine 4-hydroxylase with high l-tryptophan hydroxylating activity. In silico structural modeling analysis suggested that hydrophobic and/or stacking interactions with the substrate and cofactor at L101 and W180 in C. violaceuml-phenylalanine 4-hydroxylase would increase hydroxylation activity. Based on this hypothesis, we introduced a saturation mutagenesis towards these sites followed by the evaluation of 5-hydroxy-l-tryptophan productivity using a modified Gibbs assay. Three and nine positive mutants were obtained from the L101 and W180 mutant libraries, respectively. Among the mutants, L101Y and W180F showed the highest l-tryptophan hydroxylation activity at the respective residues. Steady-state kinetic analysis revealed that k(cat) values for l-tryptophan hydroxylation were increased from 0.40 (wild-type) to 1.02 (L101Y) and 0.51 s(-1) (W180F). In addition, the double mutant (L101Y-W180F) displayed higher l-tryptophan hydroxylation activity than the wild-type and the W180F and L101Y mutants. The k(cat) value of L101Y-W180F increased to 2.08 s(-1), showing a 5.2-fold increase compared to wild-type enzyme levels.

  10. Purification, characterization, and directed evolution study of a vitamin D{sub 3} hydroxylase from Pseudonocardia autotrophica

    SciTech Connect

    Fujii, Yoshikazu; Kabumoto, Hiroki; Nishimura, Kenji; Fujii, Tadashi; Yanai, Satoshi; Takeda, Koji; Tamura, Noriko; Arisawa, Akira; Tamura, Tomohiro

    2009-07-24

    Vitamin D{sub 3} (VD{sub 3}) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD{sub 3} into its physiologically active forms, namely, 25(OH)VD{sub 3} or 1{alpha},25(OH){sub 2}VD{sub 3}. In this study, we isolated and characterized Vdh (vitamin D{sub 3} hydroxylase), which hydroxylates VD{sub 3} from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368 Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V{sub max} values for VD{sub 3} 25-hydroxylation and 25(OH)VD{sub 3} 1{alpha}-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD{sub 3}. We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD{sub 3} 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD{sub 3} into 25(OH)VD{sub 3} was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD{sub 3} by a single cytochrome P450.

  11. Cholesterol 7α-hydroxylase protects the liver from inflammation and fibrosis by maintaining cholesterol homeostasis.

    PubMed

    Liu, Hailiang; Pathak, Preeti; Boehme, Shannon; Chiang, John Y L

    2016-10-01

    Cholesterol 7α-hydroxylase (CYP7A1) plays a critical role in control of bile acid and cholesterol homeostasis. Bile acids activate farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5) to regulate lipid, glucose, and energy metabolism. However, the role of bile acids in hepatic inflammation and fibrosis remains unclear. In this study, we showed that adenovirus-mediated overexpression of Cyp7a1 ameliorated lipopolysaccharide (LPS)-induced inflammatory cell infiltration and pro-inflammatory cytokine production in WT and TGR5-deficient (Tgr5(-/-)) mice, but not in FXR-deficient (Fxr(-/-)) mice, suggesting that bile acid signaling through FXR protects against hepatic inflammation. Nuclear factor κ light-chain enhancer of activated B cells (NF-κB)-luciferase reporter assay showed that FXR agonists significantly inhibited TNF-α-induced NF-κB activity. Furthermore, chromatin immunoprecipitation and mammalian two-hybrid assays showed that ligand-activated FXR interacted with NF-κB and blocked recruitment of steroid receptor coactivator-1 to cytokine promoter and resulted in inhibition of NF-κB activity. Methionine/choline-deficient (MCD) diet increased hepatic inflammation, free cholesterol, oxidative stress, apoptosis, and fibrosis in CYP7A1-deficient (Cyp7a1(-/-)) mice compared with WT mice. Remarkably, adenovirus-mediated overexpression of Cyp7a1 effectively reduced hepatic free cholesterol and oxidative stress and reversed hepatic inflammation and fibrosis in MCD diet-fed Cyp7a1(-/-) mice. Current studies suggest that increased Cyp7a1 expression and bile acid synthesis ameliorate hepatic inflammation through activation of FXR, whereas reduced bile acid synthesis aggravates MCD diet-induced hepatic inflammation and fibrosis. Maintaining bile acid and cholesterol homeostasis is important for protecting against liver injury and nonalcoholic fatty liver disease.

  12. Genetic Variations of Tyrosine Hydroxylase in the Pathogenesis of Hypertension

    PubMed Central

    Lee, Yu Ho; Kim, Yang Gyun; Moon, Ju-Young; Kim, Jin Sug; Jeong, Kyung-Hwan; Lee, Tae Won; Ihm, Chun-Gyoo

    2016-01-01

    One of the major pathophysiological features of primary hypertension is an inappropriate activation of the sympathetic nervous system, which is mediated by excessive synthesis and secretion of catecholamine into the blood. Tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of catecholamine, has been highlighted because genetic variations of TH could alter the activity of the sympathetic nervous system activity and subsequently contribute to the pathogenesis of hypertension. Here, we discuss the role of TH as a regulator of sympathetic activity and review several studies that investigated the relationship between genetic variations of TH and hypertension. PMID:28275384

  13. Molecular Characterization of Ferulate 5-Hydroxylase Gene from Kenaf (Hibiscus cannabinus L.)

    PubMed Central

    Park, Young-Hwan; Lim, Hyoun-Sub; Natarajan, Savithiry; Park, Sang-Un

    2013-01-01

    The purpose of this study is to clone and characterize the expression pattern of a F5H gene encoding ferulate 5-hydroxylase in the phenylpropanoid pathway from kenaf (Hibiscus cannabinus L.). Kenaf is a fast-growing dicotyledonous plant valued for its biomass. F5H, a cytochrome P450-dependent monooxygenase (CYP84), is a key enzyme for syringyl lignin biosynthesis. The full length of the F5H ortholog was cloned and characterized. The full-length F5H ortholog consists of a 1,557-bp open reading frame (ORF) encoding 518 amino acids (GenBank Accession number JX524278). The deduced amino acid sequence showed that kenaf F5H had the highest similarity (78%) with that of Populus trichocarpa. Transcriptional analysis of F5H ortholog was conducted using quantitative real-time PCR during the developmental stages of various tissues and in response to various abiotic stresses. The highest transcript level of the F5H ortholog was observed in immature flower tissues and in early stage (6 week-old) of stem tissues, with a certain level of expression in all tissues tested. The highest transcript level of F5H ortholog was observed at the late time points after treatments with NaCl (48 h), wounding (24 h), cold (24 h), abscisic acid (24 h), and methyl jasmonate (24 h). PMID:24204204

  14. Molecular characterization of ferulate 5-hydroxylase gene from kenaf (Hibiscus cannabinus L.).

    PubMed

    Kim, Jonggeun; Choi, Bosung; Park, Young-Hwan; Cho, Byoung-Kwan; Lim, Hyoun-Sub; Natarajan, Savithiry; Park, Sang-Un; Bae, Hanhong

    2013-01-01

    The purpose of this study is to clone and characterize the expression pattern of a F5H gene encoding ferulate 5-hydroxylase in the phenylpropanoid pathway from kenaf (Hibiscus cannabinus L.). Kenaf is a fast-growing dicotyledonous plant valued for its biomass. F5H, a cytochrome P450-dependent monooxygenase (CYP84), is a key enzyme for syringyl lignin biosynthesis. The full length of the F5H ortholog was cloned and characterized. The full-length F5H ortholog consists of a 1,557-bp open reading frame (ORF) encoding 518 amino acids (GenBank Accession number JX524278). The deduced amino acid sequence showed that kenaf F5H had the highest similarity (78%) with that of Populus trichocarpa. Transcriptional analysis of F5H ortholog was conducted using quantitative real-time PCR during the developmental stages of various tissues and in response to various abiotic stresses. The highest transcript level of the F5H ortholog was observed in immature flower tissues and in early stage (6 week-old) of stem tissues, with a certain level of expression in all tissues tested. The highest transcript level of F5H ortholog was observed at the late time points after treatments with NaCl (48 h), wounding (24 h), cold (24 h), abscisic acid (24 h), and methyl jasmonate (24 h).

  15. Nonflowering Plants Possess a Unique Folate-Dependent Phenylalanine Hydroxylase That Is Localized in Chloroplasts[W

    PubMed Central

    Pribat, Anne; Noiriel, Alexandre; Morse, Alison M.; Davis, John M.; Fouquet, Romain; Loizeau, Karen; Ravanel, Stéphane; Frank, Wolfgang; Haas, Richard; Reski, Ralf; Bedair, Mohamed; Sumner, Lloyd W.; Hanson, Andrew D.

    2010-01-01

    Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of full-length AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have Phe hydroxylase activity by functional complementation of an Escherichia coli Tyr auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for Phe, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate to any physiological tetrahydropterin as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli host strains. Targeting assays in Arabidopsis thaliana mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified Pisum sativum chloroplasts, indicated chloroplastic localization. Targeting assays further indicated that pterin-4a-carbinolamine dehydratase, which regenerates the AAH cofactor, is also chloroplastic. Ablating the single AAH gene in P. patens caused accumulation of Phe and caffeic acid esters. These data show that nonflowering plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism. PMID:20959559

  16. Plasma dopamine beta-hydroxylase in a noradrenalin-secreting pheochromocytoma.

    PubMed

    Aunis, D; Miras-Portugal, M T; Coquillat, G; Warter, J M; Mandel, P

    1976-08-02

    Circulating dopamine beta-hydroxylase activity was studied in a case of a hypertensive patient bearing a pheochromocytoma. The tumour secreted noradrenalin. The enzyme activity was found to decrease gradually after removal of the tumour, and to reach a stable value, in correlation with the decrease of urinary catecholamines. It is concluded that some pheochromocytoma tumours are able to secrete dopamine beta-hydroxylase.

  17. Gene Structures and Regulation of the Alkane Hydroxylase Complex in Acinetobacter sp. Strain M-1

    PubMed Central

    Tani, Akio; Ishige, Takeru; Sakai, Yasuyoshi; Kato, Nobuo

    2001-01-01

    In the long-chain n-alkane degrader Acinetobacter sp. strain M-1, two alkane hydroxylase complexes are switched by controlling the expression of two n-alkane hydroxylase-encoding genes in response to the chain length of n-alkanes, while rubredoxin and rubredoxin ruductase are encoded by a single gene and expressed constitutively. PMID:11160120

  18. Pentachlorophenol Hydroxylase: Analysis of Catalytic Abilities and Evolution of a Better Enzyme

    DTIC Science & Technology

    2004-01-19

    391, 288-291. 4 Xun, L.; Orser, C. S. "Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. Strain...pentachlorophenol by a Flavobacterium pentachlorophenol hydroxylase." J. Bacteriol. 1992, 174, 5745-5747. 6 Orser, C. S.; Lange, C. C. "Molecular analysis of

  19. Transcriptional regulation of steroid hydroxylase genes by corticotropin.

    PubMed Central

    John, M E; John, M C; Boggaram, V; Simpson, E R; Waterman, M R

    1986-01-01

    Maintenance of optimal steroidogenic capacity in the adrenal cortex is the result of a cAMP-dependent response to the peptide hormone corticotropin (ACTH). The molecular mechanism of this action of ACTH has been examined by using five recombinant DNA clones specific for enzymes of the steroidogenic pathway (P-450scc, P-45011 beta, P-450C21, P-45017 alpha, and adrenodoxin). The presence of nuclear precursors in steady-state RNA samples derived from cultured bovine adrenocortical cells and moderate increases in the number of RNA chain initiations, as determined by in vitro nuclear run-off assays, indicate that ACTH controls the expression of the gene(s) for each of these proteins at the transcriptional level. The ACTH-mediated increase in accumulation of transcripts specific for steroid hydroxylases in nuclear RNA can be specifically blocked by inhibiting protein synthesis in bovine adrenocortical cell cultures. The steady-state concentrations of nuclear RNA for control genes show no decrease upon cycloheximide treatment. These studies suggest that a primary action of ACTH in the adrenal cortex is to activate (via cAMP) the synthesis of rapidly turning over protein factors that in turn mediate increased initiation of transcription of steroid hydroxylase genes. We propose that these protein factors impart specificity of induction to genes encoding components of this pathway in steroidogenic tissues. Images PMID:3014507

  20. Spectrum of mutations in Lebanese patients with phenylalanine hydroxylase deficiency.

    PubMed

    Karam, Pascale E; Alhamra, Rasha Shahabeddeen; Nemer, Georges; Usta, Julnar

    2013-02-15

    Phenylketonuria is an autosomal recessive inborn error of metabolism resulting from phenylalanine hydroxylase deficiency. Genetic basis of phenylalanine hydroxylase deficiency has been reported in various European and Asian countries with few reports available in Arab populations of the Mediterranean region. This is the first pilot study describing phenotype and genotype of 23 Lebanese patients with phenylketonuria. 48% of the patients presented mainly with neurological signs at a mean age of 2 years 9 months, as newborn screening is not yet a nationwide policy. 56.5% of the patients had classical phenylketonuria. Thirteen different mutations were identified: splice site 52%, frameshift 31%, and missense 17% with no nonsense mutations. IVS10-11G>A was found mainly in Christians at high relative frequency whereas Muslims carried the G352fs and R261Q mutations. A rare splice mutation IVS7+1G>T, not described before, was identified in the homozygous state in one family with moderate phenylketonuria phenotype. Genotype-phenotype correlation using Guldberg arbitrary value method showed high consistency between predicted and observed phenotypes. Calculated homozygosity rate was 0.07 indicating the genetic heterogeneity in our patients. Our findings underline the admixture of different ethnicities and religions in Lebanon that might help tracing back the PAH gene flux history across the Mediterranean region.

  1. Dynamic regulation of phenylalanine hydroxylase by simulated redox manipulation.

    PubMed

    Fuchs, Julian E; Huber, Roland G; von Grafenstein, Susanne; Wallnoefer, Hannes G; Spitzer, Gudrun M; Fuchs, Dietmar; Liedl, Klaus R

    2012-01-01

    Recent clinical studies revealed increased phenylalanine levels and phenylalanine to tyrosine ratios in patients suffering from infection, inflammation and general immune activity. These data implicated down-regulation of activity of phenylalanine hydroxylase by oxidative stress upon in vivo immune activation. Though the structural damage of oxidative stress is expected to be comparably small, a structural rationale for this experimental finding was lacking. Hence, we investigated the impact of side chain oxidation at two vicinal cysteine residues on local conformational flexibility in the protein by comparative molecular dynamics simulations. Analysis of backbone dynamics revealed a highly flexible loop region (Tyr138-loop) in proximity to the active center of phenylalanine hydroxylase. We observed elevated loop dynamics in connection with a loop movement towards the active site in the oxidized state, thereby partially blocking access for the substrate phenylalanine. These findings were confirmed by extensive replica exchange molecular dynamics simulations and serve as a first structural explanation for decreased enzyme turnover in situations of oxidative stress.

  2. Adult human liver mesenchymal progenitor cells express phenylalanine hydroxylase.

    PubMed

    Baruteau, Julien; Nyabi, Omar; Najimi, Mustapha; Fauvart, Maarten; Sokal, Etienne

    2014-09-01

    Phenylketonuria (PKU) is one of the most prevalent inherited metabolic diseases and is accountable for a severe encephalopathy by progressive intoxication of the brain by phenylalanine. This results from an ineffective L-phenylalanine hydroxylase enzyme (PAH) due to a mutated phenylalanine hydroxylase (PAH) gene. Neonatal screening programs allow an early dietetic treatment with restrictive phenylalanine intake. This diet prevents most of the neuropsychological disabilities but remains challenging for lifelong compliance. Adult-derived human liver progenitor cells (ADHLPC) are a pool of precursors that can differentiate into hepatocytes. We aim to study PAH expression and PAH activity in a differenciated ADHLPC. ADHLPC were isolated from human hepatocyte primary culture of two different donors and differenciated under specific culture conditions. We demonstrated the high expression of PAH and a large increase of PAH activity in differenciated LPC. The age of the donor, the cellular viability after liver digestion and cryopreservation affects PAH activity. ADHLPC might therefore be considered as a suitable source for cell therapy in PKU.

  3. Glucocorticoid-Induced Osteoporosis in Children with 21-Hydroxylase Deficiency

    PubMed Central

    Ventura, Annamaria; Brunetti, Giacomina; Colucci, Silvia; Oranger, Angela; Ladisa, Filomena; Cavallo, Luciano; Grano, Maria; Faienza, Maria Felicia

    2013-01-01

    21-Hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH), resulting from deletions or mutations of the P450 21-hydroxylase gene (CYP21A2). Children with 21-OHD need chronic glucocorticoid (cGC) therapy, both to replace congenital deficit in cortisol synthesis and to reduce androgen secretion by adrenal cortex. GC-induced osteoporosis (GIO) is the most common form of secondary osteoporosis that results in an early, transient increase in bone resorption accompanied by a decrease in bone formation, maintained for the duration of GC therapy. Despite the conflicting results in the literature about the bone status on GC-treated patients with 21-OHD, many reports consider these subjects to be at risk for osteoporosis and fractures. In bone cells, at the molecular level, GCs regulate various functions including osteoblastogenesis, osteoclastogenesis, and the apoptosis of osteoblasts and osteocytes. In this paper, we focus on the physiology and biosynthesis of endogenous steroid hormones as well as on the effects of GCs on bone cells, highlighting the pathogenetic mechanism of GIO in children with 21-OHD. PMID:23484098

  4. Partial purification of benzene hydroxylase activity from rat liver mitoplasts

    SciTech Connect

    Karaszkiewicz, J.W.; Snyder, R.; Kalf, G.F.

    1986-05-01

    Benzene is a hemopoietic toxin and a carcinogen which causes aplastic anemia and leukemia in humans and leukemia and certain solid tumors in rodents. Bioactivation of benzene is required for toxicity and, presumably, carcinogenicity. The authors have demonstrated that rat liver mitochondria, stripped of outer membrane to avoid microsomal contamination (mitoplasts), metabolize benzene in an NADPH-dependent reaction to compounds capable of covalently binding to mitochondrial DNA. They report here on the partial purification of a benzene hydroxylase activity from rat liver mitoplasts which converts benzene to phenol and which appears to be a cytochrome P-450. The activity is solubilized with 0.4% sodium cholate and further purified by a 5-15% polyethylene glycol (PEG) fractionation. The production of (/sup 3/H)phenol from (/sup 3/H)benzene by the 5-15% PEG fraction requires an NADPH-generating system and is completely dependent on the addition of exogenous bovine adrenodoxin. Although benzene hydroxylase activity has been demonstrated in rat liver microsomes, adrenodoxin has no effect on the activity of microsomal cytochromes P-450, providing evidence that our activity is indeed of mitochondrial origin.

  5. Glucocorticoid-induced osteoporosis in children with 21-hydroxylase deficiency.

    PubMed

    Ventura, Annamaria; Brunetti, Giacomina; Colucci, Silvia; Oranger, Angela; Ladisa, Filomena; Cavallo, Luciano; Grano, Maria; Faienza, Maria Felicia

    2013-01-01

    21-Hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH), resulting from deletions or mutations of the P450 21-hydroxylase gene (CYP21A2). Children with 21-OHD need chronic glucocorticoid (cGC) therapy, both to replace congenital deficit in cortisol synthesis and to reduce androgen secretion by adrenal cortex. GC-induced osteoporosis (GIO) is the most common form of secondary osteoporosis that results in an early, transient increase in bone resorption accompanied by a decrease in bone formation, maintained for the duration of GC therapy. Despite the conflicting results in the literature about the bone status on GC-treated patients with 21-OHD, many reports consider these subjects to be at risk for osteoporosis and fractures. In bone cells, at the molecular level, GCs regulate various functions including osteoblastogenesis, osteoclastogenesis, and the apoptosis of osteoblasts and osteocytes. In this paper, we focus on the physiology and biosynthesis of endogenous steroid hormones as well as on the effects of GCs on bone cells, highlighting the pathogenetic mechanism of GIO in children with 21-OHD.

  6. Characterization of two carnation petal prolyl 4 hydroxylases.

    PubMed

    Vlad, Florina; Tiainen, Päivi; Owen, Carolyn; Spano, Thodhoraq; Daher, Firas Bou; Oualid, Fatiha; Senol, Namik Ozer; Vlad, Daniela; Myllyharju, Johanna; Kalaitzis, Panagiotis

    2010-10-01

    Prolyl 4-hydroxylases (P4Hs) catalyze the proline hydroxylation, a major post-translational modification, of hydroxyproline-rich glycoproteins. Two carnation petal P4H cDNAs, (Dianthus caryophyllus prolyl 4-hydroxylase) DcP4H1 and DcP4H2, were identified and characterized at the gene expression and biochemical level in order to investigate their role in flower senescence. Both mRNAs showed similar patterns of expression with stable transcript abundance during senescence progression and differential tissue-specific expression with DcP4H1 and DcP4H2 strongly expressed in ovaries and stems, respectively. Recombinant DcP4H1 and DcP4H2 proteins were produced and their catalytic properties were determined. Pyridine 2,4-dicarboxylate (PDCA) was identified as a potent inhibitor of the in vitro enzyme activity of both P4Hs and used to determine whether inhibition of proline hydroxylation in petals is involved in senescence progression of cut carnation flowers. PDCA suppressed the climacteric ethylene production indicating a strong correlation between the inhibition of DcP4H1 and DcP4H2 activity in vitro by PDCA and the suppression of climacteric ethylene production in cut carnation flowers.

  7. Structural insights into substrate specificity of Feruloyl-CoA 6’-Hydroxylase from Arabidopsis thaliana

    SciTech Connect

    Sun, Xinxiao; Zhou, Dayong; Kandavelu, Palani; Zhang, Hua; Yuan, Qipeng; Wang, Bi -Cheng; Rose, John; Yan, Yajun

    2015-05-20

    Coumarins belong to an important class of plant secondary metabolites. Feruloyl-CoA 6’-hydroxylase (F6’H), a 2-oxoglutarate dependent dioxygenase (2OGD), catalyzes a pivotal step in the biosynthesis of a simple coumarin scopoletin. In this study, we determined the 3-dimensional structure of the F6’H1 apo enzyme by X-ray crystallography. It is the first reported structure of a 2OGD enzyme involved in coumarin biosynthesis and closely resembles the structure of Arabidopsis thaliana anthocyanidin synthase. To better understand the mechanism of enzyme catalysis and substrate specificity, we also generated a homology model of a related ortho-hydroxylase (C2’H) from sweet potato. By comparing these two structures, we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis.

  8. A novel NADPH-dependent reductase of Sulfobacillus acidophilus TPY phenol hydroxylase: expression, characterization, and functional analysis.

    PubMed

    Li, Meng; Guo, Wenbin; Chen, Xinhua

    2016-12-01

    The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X 4 -Cys-X 2 -Cys-X 29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.

  9. Immunolocalization of phenylalanine ammonia-lyase and cinnamate-4-hydroxylase in differentiating xylem of poplar.

    PubMed

    Sato, Takahiko; Takabe, Keiji; Fujita, Minoru

    2004-01-01

    Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and cinnamate-4-hydroxylase (C4H; EC 1.14.13.11) are pivotal enzymes involved in lignification. We synthesized peptides as the epitopes according to the amino acid sequences of these enzymes, coupled them with hemocyanin, and injected them into mice. The antiserums against peptides of PAL and C4H specifically detected PAL and C4H in the crude enzymes extracted from differentiating xylem of poplar, respectively. PAL and C4H were localized in differentiating xylem of poplar. PAL labeling was mainly localized in the cytosol, and somewhat localized on the rough-endoplasmic reticulum (r-ER) and the Golgi apparatus. In contrast, C4H was mainly observed on r-ER and the Golgi apparatus. These findings suggest that conversion of phenylalanine to cinnamic acid occurs in the cytosol and the following reaction occurs near the membrane of r-ER and the Golgi apparatus. The possibility of coordinated localization of PAL and C4H is discussed.

  10. Erythrocytes encapsulated with phenylalanine hydroxylase exhibit improved pharmacokinetics and lowered plasma phenylalanine levels in normal mice.

    PubMed

    Yew, Nelson S; Dufour, Emmanuelle; Przybylska, Malgorzata; Putelat, Julie; Crawley, Cristin; Foster, Meta; Gentry, Sarah; Reczek, David; Kloss, Alla; Meyzaud, Aurélien; Horand, Françoise; Cheng, Seng H; Godfrin, Yann

    2013-08-01

    Enzyme replacement therapy is often hampered by the rapid clearance and degradation of the administered enzyme, limiting its efficacy and requiring frequent dosing. Encapsulation of therapeutic molecules into red blood cells (RBCs) is a clinically proven approach to improve the pharmacokinetics and efficacy of biologics and small molecule drugs. Here we evaluated the ability of RBCs encapsulated with phenylalanine hydroxylase (PAH) to metabolize phenylalanine (Phe) from the blood and confer sustained enzymatic activity in the circulation. Significant quantities of PAH were successfully encapsulated within murine RBCs (PAH-RBCs) with minimal loss of endogenous hemoglobin. While intravenously administered free PAH enzyme was rapidly eliminated from the blood within a few hours, PAH-RBCs persisted in the circulation for at least 10days. A single injection of PAH-RBCs was able to decrease Phe levels by nearly 80% in normal mice. These results demonstrate the ability of enzyme-loaded RBCs to metabolize circulating amino acids and highlight the potential to treat disorders of amino acid metabolism.

  11. Levodopa-responsive infantile parkinsonism due to a novel mutation in the tyrosine hydroxylase gene and exacerbation by viral infections.

    PubMed

    Diepold, Katharina; Schütz, Barbara; Rostasy, Kevin; Wilken, Bernd; Hougaard, Pia; Güttler, Flemming; Romstad, Anne; Birk Møller, Lisbeth

    2005-06-01

    Autosomal recessive forms of infantile dystonia due to mutations in the tyrosine hydroxylase (TH) gene have been described recently. The main clinical manifestations are Segawa's disease, or infantile hypokinetic rigid Parkinsonism. Here, we report on a patient with hyperrigidity, psychomotor developmental delay, and dystonic posturing of the hands, symptoms that appeared after a viral infection at the age of 14 months. Low homovanillic acid/5-hydroxyindolacetic acid (HVA/5HIAA) ratio in cerebrospinal fluid suggested a TH deficiency. Molecular analysis revealed a novel (H246Y) and a known (D498G) compound heterozygote mutation in the TH gene. The patient showed a remarkable response to treatment with levodopa. The new mutation and the association of viral infections with the onset and worsening of symptoms are discussed.

  12. Scallop phenylalanine hydroxylase implicates in immune response and can be induced by human TNF-α.

    PubMed

    Zhou, Zhi; Wang, Lingling; Wang, Mengqiang; Zhang, Huan; Wu, Tiantian; Qiu, Limei; Song, Linsheng

    2011-12-01

    Phenylalanine hydroxylase (PAH) is an important metabolic enzyme of aromatic amino acids, which is responsible for the irreversible oxidation of phenylalanine to tyrosine. In the present study, the full-length cDNA encoding PAH from Chlamys farreri (designated CfPAH) was cloned by using rapid amplification of cDNA ends (RACE) approaches and expression sequence tag (EST) analysis. The open reading frame of CfPAH encoded a polypeptide of 460 amino acids, and its sequence shared 64.4-74.2% similarity with those of PAHs from other animals. There were an N-terminal regulatory ACT domain and a C-terminal catalytic Biopterin_H domain in the deduced CfPAH protein. The mRNA transcripts of CfPAH could be detected in all the tested tissues, including adductor muscle, mantle, gill, gonad, haemocytes and hepatopancreas. And its expression level in haemocytes was increased significantly during 3-48 h after bacteria Vibrio anguillarum challenge with the highest level (9.1-fold, P < 0.05) at 24 h. Furthermore, the mRNA expression of CfPAH in haemocytes also increased significantly to 2.6-fold (P < 0.05) at 4 h and 3.3-fold (P < 0.05) at 6 h after the stimulation of 50.0 ng mL(-1) human TNF-α. The cDNA fragment encoding the mature peptide of CfPAH was recombined and expressed in the prokaryotic expression system, and 1 mg recombinant CfPAH protein (rCfPAH) could catalyze the conversion of 192.23 ± 32.35 nmol phenylalanine to tyrosine within 1 min (nmol min(-1) mg(-1) protein) in vitro. These results indicated collectively that CfPAH, as a homologue of phenylalanine hydroxylase in scallop C. farreri, could be induced by cytokine and involved in the immunomodulation of scallops by supplying the starting material tyrosine for the synthesis of melanin and catecholamines.

  13. 2,4-Dichlorophenol hydroxylase for chlorophenol removal: Substrate specificity and catalytic activity.

    PubMed

    Ren, Hejun; Li, Qingchao; Zhan, Yang; Fang, Xuexun; Yu, Dahai

    2016-01-01

    Chlorophenols (CPs) are common environmental pollutants. As such, different treatments have been assessed to facilitate their removal. In this study, 2,4-dichlorophenol (2,4-DCP) hydroxylase was used to systematically investigate the activity and removal ability of 19CP congeners at 25 and 0 °C. Results demonstrated that 2,4-DCP hydroxylase exhibited a broad substrate specificity to CPs. The activities of 2,4-DCP hydroxylase against specific CP congeners, including 3-CP, 2,3,6-trichlorophenol, 2-CP, and 2,3-DCP, were higher than those against 2,4-DCP, which is the preferred substrate of previously reported 2,4-DCP hydroxylase. To verify whether cofactors are necessary to promote hydroxylase activity against CP congeners, we added FAD and found that the added FAD induced a 1.33-fold to 5.13-fold significant increase in hydroxylase activity against different CP congeners. The metabolic pathways of the CP degradation in the enzymatic hydroxylation step were preliminarily proposed on the basis of the analyses of the enzymatic activities against 19CP congeners. We found that the high activity and removal rate of 2,4-DCP hydroxylase against CPs at 0 °C enhance the low-temperature-adaptability of this enzyme to the CP congeners; as such, the proposed removal process may be applied to biochemical, bioremediation, and industrial processes, particularly in cold environments.

  14. Quantitation of tyrosine hydroxylase, protein levels: Spot immunolabeling with an affinity-purified antibody

    SciTech Connect

    Haycock, J.W. )

    1989-09-01

    Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and {sup 125}I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background {sup 125}I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

  15. Current status of tyrosine hydroxylase in management of Parkinson's disease.

    PubMed

    Feve, Annaik Petithomme

    2012-06-01

    Tyrosine hydroxylase (TH) is the rate limiting enzyme responsible for converting tyrosine to L-DOPA in the dopamine synthesis pathway. The pathophysiology of Parkinson's disease (PD) is largely due to the nigrostriatal dopaminergic system, with a decrease in TH activity, TH synthesis and TH mRNA in the striatum of PD and animal experimental models. TH is thus one of the main targets for gene therapy in PD. TH activity variations during L-DOPA and new antiparkinsonian treatments have been extensively studied. Pharmacological trials with neuroprotective treatments could modify these variations, suggesting a direct involvement of TH cells in the neurodegenerative process. α- Synuclein, the main component of Lewy bodies regulates the production of dopamine through its interaction with TH. Over-expression of α-synuclein reduces the levels of TH mRNA and protein in the brain and in this way links the histological description of PD and its pathological biochemistry.

  16. Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine.

    PubMed

    Kim, Hye-Lim; Park, Mi-Bee; Kim, Yumin; Yang, Yun Gyeong; Lee, Soo-Woong; Zhuang, Ningning; Lee, Kon Ho; Park, Young Shik

    2012-10-19

    We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

  17. Bioavailable affinity label for collagen prolyl 4-hydroxylase

    PubMed Central

    Vasta, James D.; Higgin, Joshua J.; Kersteen, Elizabeth A.

    2013-01-01

    Collagen is the most abundant protein in animals. Its prevalent 4-hydroxyproline residues contribute greatly to its conformational stability. The hydroxyl groups arise from a post-translational modification catalyzed by the non-heme iron-dependent enzyme, collagen prolyl 4-hydroxylase (P4H). Here, we report that 4-oxo-5,6-epoxyhexanoate, a mimic of the α-ketoglutarate co-substrate, inactivates human P4H. The inactivation installs a ketone functionality in P4H, providing a handle for proteomic experiments. Caenorhabditis elegans exposed to the esterified epoxy ketone displays the phenotype of a worm lacking P4H. Thus, this affinity label can be used to mediate collagen stability in an animal, as is desirable in the treatment of a variety of fibrotic diseases. PMID:23702396

  18. Organ specificity of aryl hydrocarbon hydroxylase induction by cigarette smoke

    SciTech Connect

    Yoshikawa, M.; Arashidani, K.; Kawamoto, T.; Kodama, Y. )

    1990-06-01

    Biotransformation of many chemicals found in cigarette smoke, such as PAHs and nitrosamines, is generally considered essential for the mutagenic, carcinogenic effects of these xenobiotics. In fact, the genotic action of these premutagens or precarcinogens is dependent on metabolic activation catalyzed by microsomal monooxygenases. The first enzymatic reaction of the PAHs metabolic pathway is catalyzed by a cytochrome P-450-dependent monooxygenase, the aryl hydrocarbon hydroxylase (AHH). AHH leads to the formation of reactive arene oxides, which are further metabolized by enzymatic and non-enzymatic reaction into many metabolites. AHH induction in laboratory animals exposed to cigarette smoke has also been reported, and the data show that this response is highly dependent on species and tissues. Exposure of small laboratory animals to cigarette smoke generally induces AHH in the kidney and lung, while the effect of cigarette smoke on the hepatic AHH activity appears variable.

  19. Bone matrix hypermineralization in prolyl-3 hydroxylase 1 deficient mice.

    PubMed

    Fratzl-Zelman, Nadja; Bächinger, Hans-Peter; Vranka, Janice A; Roschger, Paul; Klaushofer, Klaus; Rauch, Frank

    2016-04-01

    Lack of prolyl 3-hydroxylase 1 (P3H1) due to mutations in P3H1 results in severe forms of recessive osteogenesis imperfecta. In the present study, we investigated the bone tissue characteristics of P3H1 null mice. Histomorphometric analyses of cancellous bone in the proximal tibia and lumbar vertebra in 1-month and 3-month old mice demonstrated that P3H1 deficient mice had low trabecular bone volume and low mineral apposition rate, but normal osteoid maturation time and normal osteoblast and osteoclast surfaces. Quantitative backscattered electron imaging revealed that the bone mineralization density distribution was shifted towards higher values, indicating hypermineralization of bone matrix. It thus appears that P3H1 deficiency leads to decreased deposition of extracellular matrix by osteoblasts and increased incorporation of mineral into the matrix.

  20. Delayed puberty from partial 17-alpha hydroxylase enzyme deficiency.

    PubMed

    Croxson, Michael; Ogilvie, C Megan; Milsom, Stella; Lewis, John; Davidson, James; Rumsby, Gill

    2012-05-25

    An 18-year-old woman with primary amenorrhoea and pubertal delay was investigated for mild labile hypertension and secondary hypogonadism. Low renin and normal aldosterone levels combined with evidence of primary adrenal insufficiency suggested partial 17-alpha hydroxylase enzyme deficiency. The diagnosis was confirmed by measurement of 24-hour urine steroid metabolites and whole gene sequencing of CYP17A1 that demonstrated c.160_162delTTC (p.Phe54del) homozygous mutation. Ultrasound showed bilateral small ovaries with multiple cysts. The serum anti-mullerian hormone concentration was unremarkable at 6.6 (normal <12.6 ng/ml) but the outlook for her future ovulatory potential is uncertain. Dexamethasone 0.25 mg pre-bed and hydrocortisone 5 mg on waking normalised her hormonal profile and her blood pressure without side-effects.

  1. Origin and evolution of peptide-modifying dioxygenases and identification of the wybutosine hydroxylase/hydroperoxidase

    PubMed Central

    Iyer, Lakshminarayan M.; Abhiman, Saraswathi; de Souza, Robson F.; Aravind, L.

    2010-01-01

    Unlike classical 2-oxoglutarate and iron-dependent dioxygenases, which include several nucleic acid modifiers, the structurally similar jumonji-related dioxygenase superfamily was only known to catalyze peptide modifications. Using comparative genomics methods, we predict that a family of jumonji-related enzymes catalyzes wybutosine hydroxylation/peroxidation at position 37 of eukaryotic tRNAPhe. Identification of this enzyme raised questions regarding the emergence of protein- and nucleic acid-modifying activities among jumonji-related domains. We addressed these with a natural classification of DSBH domains and reconstructed the precursor of the dioxygenases as a sugar-binding domain. This precursor gave rise to sugar epimerases and metal-binding sugar isomerases. The sugar isomerase active site was exapted for catalysis of oxygenation, with a radiation of these enzymes in bacteria, probably due to impetus from the primary oxygenation event in Earth’s history. 2-Oxoglutarate-dependent versions appear to have further expanded with rise of the tricarboxylic acid cycle. We identify previously under-appreciated aspects of their active site and multiple independent innovations of 2-oxoacid-binding basic residues among these superfamilies. We show that double-stranded β-helix dioxygenases diversified extensively in biosynthesis and modification of halogenated siderophores, antibiotics, peptide secondary metabolites and glycine-rich collagen-like proteins in bacteria. Jumonji-related domains diversified into three distinct lineages in bacterial secondary metabolism systems and these were precursors of the three major clades of eukaryotic enzymes. The specificity of wybutosine hydroxylase/peroxidase probably relates to the structural similarity of the modified moiety to the ancestral amino acid substrate of this superfamily. PMID:20423905

  2. Body temperature effect on methylenedioxymethamphetamine-induced acute decrease in tryptophan hydroxylase activity.

    PubMed

    Che, S; Johnson, M; Hanson, G R; Gibb, J W

    1995-12-07

    Brain tryptophan hydroxylase activity decreases within 15 min after a single administration of 3,4-methylenedioxymethamphetamine. In the present study, the effect of body temperature on this acute decrease of tryptophan hydroxylase activity was examined. 2 h after a single dose of 3,4-methylenedioxymethamphetamine (20 mg/kg, s.c.), rats exhibited hyperthermia (38.7 degrees C) or hypothermia (35.8 degrees C) when maintained at 25 degrees C or 6 degrees C, respectively. The rectal temperature of control animals maintained at 6 degrees C was not altered. Tryptophan hydroxylase activity measured in the hippocampus, striatum and frontal cortex of hyperthermic rats treated with 3,4-methylenedioxymethamphetamine was decreased to 61%, 65%, and 71% of control levels, respectively, 2 h after drug treatment. However, in hypothermic rats, 3,4-methylenedioxymethamphetamine had no effect on tryptophan hydroxylase activity in the hippocampus, striatum or frontal cortex. Non-drug-induced hyperthermia or hypothermia did not affect tryptophan hydroxylase activity. Since hypothermia may prevent the 3,4-methylenedioxymethamphetamine-induced decrease in tryptophan hydroxylase activity by reducing the formation of free radicals, the effect of a free radical scavenging agent, N-tert-butyl-alpha-phenylnitrone, was examined. N-tert-butyl-alpha-phenylnitrone (200 mg/kg, i.p.) alone caused hypothermia but had no direct effect on tryptophan hydroxylase activity. Preadministration of N-tert-butyl-alpha-phenylnitrone prevented 3,4-methylenedioxymethamphetamine from raising the temperature above normal and attenuated the drug-induced decrease in tryptophan hydroxylase activity in hippocampus, striatum and frontal cortex. However, when the rats treated with a combination of N-tert-butyl-alpha-phenylnitrone and 3,4-methylenedioxymethamphetamine were maintained at hyperthermic conditions, N-tert-butyl-alpha-phenylnitrone had no protective effect. These results suggest that body temperature plays a

  3. Molecular cloning and bioinformatic analysis of the Streptococcus agalactiae neuA gene isolated from tilapia.

    PubMed

    Wang, E L; Wang, K Y; Chen, D F; Geng, Y; Huang, L Y; Wang, J; He, Y

    2015-06-01

    Cytidine monophosphate (CMP) N-acetylneuraminic acid (NeuNAc) synthetase, which is encoded by the neuA gene, can catalyze the activation of sialic acid with CMP, and plays an important role in Streptococcus agalactiae infection pathogenesis. To study the structure and function of the S. agalactiae neuA gene, we isolated it from diseased tilapia, amplified it using polymerase chain reaction (PCR) with specific primers, and cloned it into a pMD19-T vector. The recombinant plasmid was confirmed by PCR and restriction enzyme digestion, and identified by sequencing. Molecular characterization analyses of the neuA nucleotide amino acid sequence were performed using bioinformatic tools and an online server. The results showed that the neuA nucleotide sequence contained a complete coding region, which comprised 1242 bp, encoding 413 amino acids (aa). The aa sequence was highly conserved and contained a Glyco_tranf_GTA_type superfamily and an SGNH_hydrolase superfamily conserved domain, which are related to sialic acid activation catalysis. The NeuA protein possessed many important sites related to post-translational modification, including 28 potential phosphorylation sites and 2 potential N-glycosylation sites, had no signal peptides or transmembrane regions, and was predicted to reside in the cytoplasm. Moreover, the protein had some B-cell epitopes, which suggests its potential in development of a vaccine against S. agalactiae infection. The codon usage frequency of neuA differed greatly in Escherichia coli and Homo sapiens genes, and neuA may be more efficiently expressed in eukaryotes (yeast). S. agalactiae neuA from tilapia maintains high structural homology and sequence identity with CMP-NeuNAc synthetases from other bacteria.

  4. Functional Characterization and Subcellular Localization of Poplar (Populus trichocarpa × Populus deltoides) Cinnamate 4-Hydroxylase1

    PubMed Central

    Ro, Dae Kyun; Mah, Nancy; Ellis, Brian E.; Douglas, Carl J.

    2001-01-01

    Cinnamic acid 4-hydroxylase (C4H), a member of the cytochrome P450 monooxygenase superfamily, plays a central role in phenylpropanoid metabolism and lignin biosynthesis and possibly anchors a phenylpropanoid enzyme complex to the endoplasmic reticulum (ER). A full-length cDNA encoding C4H was isolated from a hybrid poplar (Populus trichocarpa × P. deltoides) young leaf cDNA library. RNA-blot analysis detected C4H transcripts in all organs tested, but the gene was most highly expressed in developing xylem. C4H expression was also strongly induced by elicitor-treatment in poplar cell cultures. To verify the catalytic activity of the putative C4H cDNA, two constructs, C4H and C4H fused to the FLAG epitope (C4H::FLAG), were expressed in yeast. Immunoblot analysis showed that C4H was present in the microsomal fraction and microsomal preparations from strains expressing both enzymes efficiently converted cinnamic acid to p-coumaric acid with high specific activities. To investigate the subcellular localization of C4H in vivo, a chimeric C4H-green fluorescent protein (GFP) gene was engineered and stably expressed in Arabidopsis. Confocal laser microscopy analysis clearly showed that in Arabidopsis the C4H::GFP chimeric enzyme was localized to the ER. When expressed in yeast, the C4H::GFP fusion enzyme was also active but displayed significantly lower specific activity than either C4H or C4H::FLAG in in vitro and in vivo enzyme assays. These data definitively show that C4H is localized to the ER in planta. PMID:11351095

  5. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    SciTech Connect

    Sun, Zhichao; Yu, Xuemei; Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin; Li, Yintao; Yang, Lili; Ruan, Yuanyuan; Gu, Jianxin; Ren, Shifang; Zhang, Songwen

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

  6. Cinnamate 4-Hydroxylase (C4H) genes from Leucaena leucocephala: a pulp yielding leguminous tree.

    PubMed

    Kumar, Santosh; Omer, Sumita; Patel, Krunal; Khan, Bashir M

    2013-02-01

    Leucaena leucocephala is a leguminous tree species accounting for one-fourth of raw material supplied to paper and pulp industry in India. Cinnamate 4-Hydroxylase (C4H, EC 1.14.13.11) is the second gene of phenylpropanoid pathway and a member of cytochrome P450 family. There is currently intense interest to alter or modify lignin content of L. leucocephala. Three highly similar C4H alleles of LlC4H1 gene were isolated and characterized. The alleles shared more than 98 % sequence identity at amino acid level to each other. Binding of partial promoter of another C4H gene LlC4H2, to varying amounts of crude nuclear proteins isolated from leaf and stem tissues of L. leucocephala formed two loose and one strong complex, respectively, suggesting that the abundance of proteins that bind with the partial C4H promoter is higher in stem tissue than in leaf tissue. Quantitative Real Time PCR study suggested that among tissues of same age, root tissues had highest level of C4H transcripts. Maximum transcript level was observed in 30 day old root tissue. Among the tissues investigated, C4H activity was highest in 60 day old root tissues. Tissue specific quantitative comparison of lignin from developing seedling stage to 1 year old tree stage indicated that Klason lignin increased in tissues with age.

  7. Kinetic mechanism of phenylalanine hydroxylase: intrinsic binding and rate constants from single-turnover experiments.

    PubMed

    Roberts, Kenneth M; Pavon, Jorge Alex; Fitzpatrick, Paul F

    2013-02-12

    Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH(4)) and O(2). A complete kinetic mechanism for PheH was determined by global analysis of single-turnover data in the reaction of PheHΔ117, a truncated form of the enzyme lacking the N-terminal regulatory domain. Formation of the productive PheHΔ117-BH(4)-phenylalanine complex begins with the rapid binding of BH(4) (K(d) = 65 μM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (K(d) = 130 μM) is approximately 10-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O(2) rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, which can be detected as a decrease in absorbance at 340 nm, with a rate constant of 140 s(-1). Formation of the 4a-hydroxypterin and Fe(IV)O intermediates is 10-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines k(cat). Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation.

  8. Purification and characterization of a benzene hydroxylase: A cytochrome P-450 from rat liver mitochondria

    SciTech Connect

    Karaszkiewicz, J.W.

    1989-01-01

    This laboratory previously demonstrated that incubation of ({sup 14}C)benzene with isolated mitochondria resulted in the formation of mtDNA adducts. Since benzene is incapable of spontaneously covalently binding to nuclei acids, it was hypothesized that enzyme(s) present in the organelle metabolized benzene to reactive derivatives. We have purified, to electrophoretic homogeneity, a 52 kDa cytochrome P-450 from liver mitoplasts which metabolizes benzene to phenol. The enzyme has a K{sub M} for benzene of 0.012 mM, and a V{sub MAX} of 22.6 nmol phenol/nmol P-450/10 min, and requires NADPH, adrenodoxin, and adrenodoxin reductase for activity. Activity also can be reconstituted with microsomal cytochrome P-450 reductase. Benzene hydroxylase activity could be inhibited by carbon monoxide and SKF-525A, and by specific inhibitors of microsomal benzene metabolism. The purified enzyme oxidized phenol, forming catechol; aminopyrine N-demethylase activity was also demonstrated. These data confirm that a cytochrome P-450 of mitochondrial origin is involved in benzene metabolism, and indicate a role for the mitochondrion in xenobiotic activation.

  9. Lipidomic profiling of tryptophan hydroxylase 2 knockout mice reveals novel lipid biomarkers associated with serotonin deficiency.

    PubMed

    Weng, Rui; Shen, Sensen; Burton, Casey; Yang, Li; Nie, Honggang; Tian, Yonglu; Bai, Yu; Liu, Huwei

    2016-04-01

    Serotonin is an important neurotransmitter that regulates a wide range of physiological, neuropsychological, and behavioral processes. Consequently, serotonin deficiency is involved in a wide variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, schizophrenia, and depression. The pathophysiological mechanisms underlying serotonin deficiency, particularly from a lipidomics perspective, remain poorly understood. This study therefore aimed to identify novel lipid biomarkers associated with serotonin deficiency by lipidomic profiling of tryptophan hydroxylase 2 knockout (Tph2-/-) mice. Using a high-throughput normal-/reversed-phase two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry (NP/RP 2D LC-QToF-MS) method, 59 lipid biomarkers encompassing glycerophospholipids (glycerophosphocholines, lysoglycerophosphocholines, glycerophosphoethanolamines, lysoglycerophosphoethanolamines glycerophosphoinositols, and lysoglycerophosphoinositols), sphingolipids (sphingomyelins, ceramides, galactosylceramides, glucosylceramides, and lactosylceramides) and free fatty acids were identified. Systemic oxidative stress in the Tph2-/- mice was significantly elevated, and a corresponding mechanism that relates the lipidomic findings has been proposed. In summary, this work provides preliminary findings that lipid metabolism is implicated in serotonin deficiency.

  10. Overexpression of Tyrosine hydroxylase and Dopa decarboxylase associated with pupal melanization in Spodoptera exigua

    PubMed Central

    Liu, Sisi; Wang, Mo; Li, Xianchun

    2015-01-01

    Melanism has been found in a wide range of species, but the molecular mechanisms involved remain largely elusive. In this study, we studied the molecular mechanisms of the pupal melanism in Spodoptera exigua. The full length cDNA sequences of tyrosine hydroxylase (TH) and dopa decarboxylase (DDC), two key enzymes in the biosynthesis pathway of melanin, were cloned, and their temporal expression patterns in the integument were compared during the larval-pupal metamorphosis process of the S. exigua wild type (SEW) and melanic mutant (SEM) strains. No amino acid change in the protein sequence of TH and DDC was found between the two strains. Both DDC and TH were significantly over-expressed in the integument of the SEM strain at late-prepupa and 0 h pupa, respectively, compared with those of the SEW strain. Feeding 5th instar larvae of SEM with diets incorporated with 1 mg/g of the DDC inhibitor L-α-Methyl-DOPA and 0.75 mg/g of the TH inhibitor 3-iodo-tyrosine (3-IT) resulted in 20% pupae with partially-rescued phenotype and 68.2% of pupae with partially- or fully-rescued phenotype, respectively. These results indicate that overexpressions of TH and DDC are involved in the pupal melanization of S. exigua. PMID:26084938

  11. Carboxyl-terminal domain characterization of polyene-specific P450 hydroxylase in Pseudonocardia autotrophica.

    PubMed

    Kim, Min-Kyung; Won, Hyung-Jin; Kim, Hye-Jin; Choi, Si-Sun; Lee, Heung-Shick; Kim, Pil; Kim, Eung-Soo

    2016-11-01

    A polyene compound NPP identified in Pseudonocardia autotrophica was shown to contain an aglycone identical to nystatin, but to harbor a unique disaccharide moiety that led to higher solubility and reduced hemolytic activity. Recently, it was revealed that the final step of NPP (nystatin-like polyene) biosynthesis is C10 regio-specific hydroxylation by the cytochrome P450 hydroxylase (CYP) NppL (Kim et al. [7]). Through mutation and cross-complementation, here we found that NppL preferred a polyene substrate containing a disaccharide moiety for C10 hydroxylation, while its orthologue NysL involved in nystatin biosynthesis showed no substrate preference toward mono- and disaccharide moieties, suggesting that two homologous polyene CYPs, NppL and NysL might possess a unique domain recognizing a sugar moiety. Two hybrid NppL constructs containing the C-terminal domain of NysL exhibited no substrate preference toward 10-deoxy NPP and 10-deoxy nystatin-like NysL, implying that the C-terminal domain plays a major role in differentiating the sugar moiety responsible for substrate specificity. Further C-terminal domain dissection of NppL revealed that the last fifty amino acids play a critical role in determining substrate specificity of polyene-specific hydroxylation, setting the stage for the biotechnological application of hydroxyl diversification for novel polyene biosynthesis in actinomycetes.

  12. Signaling hypoxia by hypoxia-inducible factor protein hydroxylases: a historical overview and future perspectives

    PubMed Central

    Bishop, Tammie; Ratcliffe, Peter J

    2014-01-01

    By the early 1900s, the close matching of oxygen supply with demand was recognized to be a fundamental requirement for physiological function, and multiple adaptive responses to environment hypoxia had been described. Nevertheless, the widespread operation of mechanisms that directly sense and respond to levels of oxygen in animal cells was not appreciated for most of the twentieth century with investigators generally stressing the regulatory importance of metabolic products. Work over the last 25 years has overturned that paradigm. It has revealed the existence of a set of “oxygen-sensing” 2-oxoglutarate dependent dioxygenases that catalyze the hydroxylation of specific amino acid residues and thereby control the stability and activity of hypoxia-inducible factor. The hypoxia-inducible factor hydroxylase pathway regulates a massive transcriptional cascade that is operative in essentially all animal cells. It transduces a wide range of responses to hypoxia, extending well beyond the classical boundaries of hypoxia physiology. Here we review the discovery and elucidation of these pathways, and consider the opportunities and challenges that have been brought into focus by the findings, including new implications for the integrated physiology of hypoxia and therapeutic approaches to ischemic/hypoxic disease. PMID:27774477

  13. Insights into the proline hydroxylase (PHD) family, molecular evolution and its impact on human health.

    PubMed

    Minervini, Giovanni; Quaglia, Federica; Tosatto, Silvio C E

    2015-09-01

    PHDs (proline hydroxylases) are a small protein family found in all organisms, considered the central regulator of the molecular hypoxia response due to PHDs being completely inactivated under low oxygen concentration. At physiological oxygen concentration, PHDs drive the degradation of the HIF-1α (hypoxia-inducible factor 1-α), which is responsible for upregulating the expression of genes involved in the cellular response to hypoxia. Hypoxia is a common feature of most tumors, in particular during metastasis development. Indeed, cancer reacts by activating pathways promoting new blood vessel formation and activating strategies aimed to improve survival. In this scenario, the PHD family regulates the activation of HIF-1α and cell-cycle regulation. Several PHD mutations were found in cancer patients, underlining their importance for human health. Here, we propose a Bayesian model able to predict the pathological effect of human PHD mutations and their correlation with cancer outcome. The model was developed through an integrative in silico approach, where data collected from the literature has been coupled with sequence evolution and structural analysis. The model was used to assess 135 human PHD variants. Finally, bioinformatics characterization was used to demonstrate how few amino acid changes are able to explain the functional specialization of PHD family members and their physiological role in human health.

  14. Structural consequences of effector protein complex formation in a diiron hydroxylase

    SciTech Connect

    Bailey, Lucas J.; McCoy, Jason G.; Phillips, Jr., George N.; Fox, Brian G.

    2009-06-12

    Carboxylate-bridged diiron hydroxylases are multicomponent enzyme complexes responsible for the catabolism of a wide range of hydrocarbons and as such have drawn attention for their mechanism of action and potential uses in bioremediation and enzymatic synthesis. These enzyme complexes use a small molecular weight effector protein to modulate the function of the hydroxylase. However, the origin of these functional changes is poorly understood. Here, we report the structures of the biologically relevant effector protein-hydroxylase complex of toluene 4-monooxygenase in 2 redox states. The structures reveal a number of coordinated changes that occur up to 25 {angstrom} from the active site and poise the diiron center for catalysis. The results provide a structural basis for the changes observed in a number of the measurable properties associated with effector protein binding. This description provides insight into the functional role of effector protein binding in all carboxylate-bridged diiron hydroxylases.

  15. Changes of human plasma dopamine-beta-hydroxylase activity after intravenous administration of theophylline.

    PubMed

    Aunis, D; Mandel, P; Miras-Portugal, M T; Coquillat, G; Rohmer, F; Warter, J M

    1975-03-01

    The intravenous administration of theophylline to ten healthy human subjects produced either an increase of circulating plasma dopamine-beta-hydroxylase or no change. The rise of plasma enzyme activity may reflect the increased peripheral catecholamine release induced by theophylline.

  16. Genetics Home Reference: congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency

    MedlinePlus

    ... collectively called congenital adrenal hyperplasia) that affect the adrenal glands . The adrenal glands are located on top of the kidneys and ... CAH due to 11-beta-hydroxylase deficiency, the adrenal glands produce excess androgens, which are male sex hormones. ...

  17. Phytoecdysteroid C2-hydroxylase is microsomal in spinach, Spinacia oleracea L.

    PubMed

    Bakrim, Ahmed; Guittard, Emilie; Maria, Annick; De Virville, Jacques Davy; Lafont, René; Takvorian, Najat

    2009-12-01

    An enzyme involved in the biosynthesis of phytoecdysteroids, the C2-hydroxylase, has been investigated in spinach, Spinacia oleracea. This enzyme is microsomal and its K(m) has been determined using 2-deoxy-20-hydroxyecdysone as substrate (K(m)=3.72 microM). It is much more efficient with 2-deoxy-20-hydroxyecdysone than with 2-deoxyecdysone and, conversely, the C20-hydroxylase is more active on 2-deoxyecdysone than on ecdysone. These data support the conclusion that C20-hydroxylation precedes C2-hydroxylation. The C2-hydroxylase is inhibited by high concentrations of 20E. Substrate specificity and subcellular localization of C2-hydroxylase differ between plants and insects, and these data, as well as those previously reported on other biosynthetic steps, show the great difference between plant and insect ecdysteroid biosynthetic pathways and suggest an independent origin for the pathways in both kingdoms.

  18. Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

    SciTech Connect

    Arnold, Frances H.

    2012-02-27

    The goals of this project are to investigate (1) the kinetics and stabilities of engineered cytochrome P450 (P450) small alkane hydroxylases and their evolutionary intermediates, (2) the structural basis for catalytic proficiency on small alkanes of these engineered P450s, and (3) the changes in redox control resulting from protein engineering. To reach these goals, we have established new methods for determining the kinetics and stabilities of multicomponent P450s such as CYP153A6. Using these, we were able to determine that CYP153A6 is proficient for hydroxylation of alkanes as small as ethane, an activity that has never been observed previously in any natural P450. To elucidate the structures of the engineered P450s, we obtained x-ray diffraction data for two variants in the P450PMO (propane monooxygenase) lineage and a preliminary structure for the most evolved variant. This structure shows changes in the substrate binding regions of the enzyme and a reduction in active site volume that are consistent with the observed changes in substrate specificity from fatty acids in the native enzyme to small alkanes in P450PMO. We also constructed semi-rational designed libraries mutating only residues in the enzyme active site that in one round of mutagenesis and screening produced variants that achieved nearly half of the activity of the most evolved enzymes of the P450PMO lineage. Finally, we found that changes in redox properties of the laboratory-evolved P450 alkane hydroxylases did not reflect the improvement in their electron transfer efficiency. The heme redox potential remained constant throughout evolution, while activity increased and coupling efficiency improved from 10% to 90%. The lack of correlation between heme redox potential and enzyme activity and coupling efficiency led us to search for other enzyme properties that could be better predictors for activity towards small alkanes, specifically methane. We investigated the oxidation potential of the radical

  19. The hypoxia-inducible-factor hydroxylases bring fresh air into hypoxia signalling

    PubMed Central

    Berra, Edurne; Ginouvès, Amandine; Pouysségur, Jacques

    2006-01-01

    Metazoans rapidly respond to changes in oxygen availability by regulating gene expression. The transcription factor hypoxia-inducible-factor (HIF), which controls the expression of several genes, ‘senses' the oxygen concentration indirectly through the hydroxylation of two proline residues that earmarks the HIF-α subunits for proteasomal degradation. We review the expression, regulation and function of the HIF prolyl hydroxylases or prolyl hydroxylases domain proteins, which are genuine oxygen sensors. PMID:16391536

  20. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes.

    PubMed Central

    Peng, H; Armentano, D; MacKenzie-Graham, L; Shen, R F; Darlington, G; Ledley, F D; Woo, S L

    1988-01-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. We report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human alpha 1-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating form the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the alpha 1-antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes. Images PMID:3186716

  1. Verbascoside promotes the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra

    PubMed Central

    Liang, Jian-qing; Wang, Li; He, Jian-cheng; Hua, Xian-dong

    2016-01-01

    Tyrosine hydroxylase is a key enzyme in dopamine biosynthesis. Change in tyrosine hydroxylase expression in the nigrostriatal system is closely related to the occurrence and development of Parkinson's disease. Verbascoside, an extract from Radix Rehmanniae Praeparata has been shown to be clinically effective in treating Parkinson's disease. However, the underlying mechanisms remain unclear. It is hypothesized that the effects of verbascoside on Parkinson's disease are related to tyrosine hydroxylase expression change in the nigrostriatal system. Rat models of Parkinson's disease were established and verbascoside (60 mg/kg) was administered intraperitoneally once a day. After 6 weeks of verbascoside treatment, rat rotational behavior was alleviated; tyrosine hydroxylase mRNA and protein expression and the number of tyrosine hydroxylase-immunoreactive neurons in the rat right substantia nigra were significantly higher than the Parkinson's model group. These findings suggest that the mechanism by which verbascoside treats Parkinson's disease is related to the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra. PMID:26981096

  2. Synthetic glycosylation of proteins using N-(beta-saccharide) iodoacetamides: applications in site-specific glycosylation and solid-phase enzymic oligosaccharide synthesis.

    PubMed Central

    Wong, S Y; Guile, G R; Dwek, R A; Arsequell, G

    1994-01-01

    A simple and efficient synthetic glycosylation method suitable for use in solid-phase enzymic oligosaccharide synthesis and site-specific glycosylation of recombinant proteins to produce defined glycoforms is described. This strategy utilizes N-(beta-saccharide) haloacetamides for attaching oligosaccharides specifically to cysteine residues of proteins in solution to form neoglycoproteins. The alkylation reaction was tested using N-(beta-chitotriose) bromoacetamide and an unprotected synthetic hexapeptide containing a single cysteine residue. The glycosylated product was confirmed by amino acid and hexosamine analyses as well as laser desorption mass spectrometry. Similarly N-(beta-chitotriose) iodoacetamide was covalently linked to non-reduced BSA to produce a defined glycoform of this protein. The specific attachment of chitotriose at the single cysteine residue in non-reduced serum albumin was suggested by Ellman's assay for free thiols. This was verified by amino acid sequencing of tryptic glycopeptide derived from this neoglycoprotein. Multiple sugar attachment was accomplished using fully reduced serum albumin as demonstrated by the formation of two neoglycoproteins using iodoacetamide derivatives of galactose beta 1-3-N-acetylgalactosamine (Gal beta 1-3GalNAc) and the major xylose/fucose-class plant-type oligosaccharide of horseradish peroxidase. These two neoglycoproteins with an average of 18-21 sugar residues attached were assayed positively for binding to peanut agglutinin and a sugar-specific anti-(horseradish peroxidase) monoclonal antibody YZ1/2.23 respectively. Sialylation of the neoglycoprotein containing Gal beta 1-3GalNAc was accomplished using alpha-2,3-sialyltransferase and radiolabelled CMP-N-acetylneuraminic acid. Significantly, glycan attachment using this conjugation method is reversible as demonstrated by the release of oligosaccharides from these two neoglycoproteins using hydrazinolysis. Therefore this method could provide invaluable

  3. Loss of FERULATE 5-HYDROXYLASE Leads to Mediator-Dependent Inhibition of Soluble Phenylpropanoid Biosynthesis in Arabidopsis1[OPEN

    PubMed Central

    Anderson, Nickolas A.; Bonawitz, Nicholas D.; Nyffeler, Kayleigh; Chapple, Clint

    2015-01-01

    Phenylpropanoids are phenylalanine-derived specialized metabolites and include important structural components of plant cell walls, such as lignin and hydroxycinnamic acids, as well as ultraviolet and visible light-absorbing pigments, such as hydroxycinnamate esters (HCEs) and anthocyanins. Previous work has revealed a remarkable degree of plasticity in HCE biosynthesis, such that most Arabidopsis (Arabidopsis thaliana) mutants with blockages in the pathway simply redirect carbon flux to atypical HCEs. In contrast, the ferulic acid hydroxylase1 (fah1) mutant accumulates greatly reduced levels of HCEs, suggesting that phenylpropanoid biosynthesis may be repressed in response to the loss of FERULATE 5-HYDROXYLASE (F5H) activity. Here, we show that in fah1 mutant plants, the activity of HCE biosynthetic enzymes is not limiting for HCE accumulation, nor is phenylpropanoid flux diverted to the synthesis of cell wall components or flavonol glycosides. We further show that anthocyanin accumulation is also repressed in fah1 mutants and that this repression is specific to tissues in which F5H is normally expressed. Finally, we show that repression of both HCE and anthocyanin biosynthesis in fah1 mutants is dependent on the MED5a/5b subunits of the transcriptional coregulatory complex Mediator, which are similarly required for the repression of lignin biosynthesis and the stunted growth of the phenylpropanoid pathway mutant reduced epidermal fluorescence8. Taken together, these observations show that the synthesis of HCEs and anthocyanins is actively repressed in a MEDIATOR-dependent manner in Arabidopsis fah1 mutants and support an emerging model in which MED5a/5b act as central players in the homeostatic repression of phenylpropanoid metabolism. PMID:26048881

  4. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  5. Prolyl hydroxylase 3 inhibited the tumorigenecity of gastric cancer cells.

    PubMed

    Cui, Lei; Qu, Jianguo; Dang, Shengchun; Mao, Zhengfa; Wang, Xuqing; Fan, Xin; Sun, Kang; Zhang, Jianxin

    2014-09-01

    Gastric cancer is one of the most common malignancies and the second leading cause of cancer-related death in the world, and it is very urgent to develop novel therapeutic strategies. Although HIF-1α is the most highly characterized target of prolyl hydroxylase 3 (PHD3), PHD3 has been shown to regulate several signal pathways independent of HIF-1α. Here, we found that the expression of PHD3 was decreased in the clinical gastric cancer samples and reversely correlated with tumor size and tumor stage. Over-expression of PHD3 in the gastric cancer cells significantly inhibited cell growth in vitro and in vivo, while knockdown the expression of PHD3 promoted the tumorigenecity of gastric cancer cells. Mechanistically, it showed that PHD3 downregulated the expression of beta-catenin and inhibited beta-catenin/T-cell factor (TCF) signaling. Taken together, our findings demonstrate that PHD3 inhibits gastric cancer by suppressing the beta-catenin/TCF signaling and PHD3 might be an important therapeutic target in gastric cancer.

  6. Substrate recognition by "password" in p-hydroxybenzoate hydroxylase.

    PubMed

    Palfey, B A; Moran, G R; Entsch, B; Ballou, D P; Massey, V

    1999-01-26

    The flavin of p-hydroxybenzoate hydroxylase (PHBH) adopts two conformations [Gatti, D. L., Palfey, B. A., Lah, M.-S., Entsch, B., Massey, V., Ballou, D. P., and Ludwig, M. L. (1994) Science 266, 110-114; Schreuder, H. A., Mattevi, A., Obmolova, G., Kalk, K. H., Hol, W. G. J., van der Bolt, F. J. T., and van Berkel, W. J. H. (1994) Biochemistry 33, 10161-10170]. Kinetic studies detected the movement of the flavin from the buried conformation to the exposed conformation caused by the binding of NADPH prior to its reaction with the flavin. The pH dependence of the rate constant for flavin reduction in wild-type PHBH and the His72Asn mutant indicates that the deprotonation of bound p-hydroxybenzoate is also required for flavin movement, and is accomplished by the same internal proton transport network previously found to be involved in substrate oxidation. The linkage of substrate deprotonation to flavin movement constitutes a novel mode of molecular recognition in which the enzyme tests the suitability of aromatic substrates before committing to the catalytic cycle.

  7. Complex regulation of prolyl-4-hydroxylases impacts root hair expansion.

    PubMed

    Velasquez, Silvia M; Ricardi, Martiniano M; Poulsen, Christian Peter; Oikawa, Ai; Dilokpimol, Adiphol; Halim, Adnan; Mangano, Silvina; Denita Juarez, Silvina Paola; Marzol, Eliana; Salgado Salter, Juan D; Dorosz, Javier Gloazzo; Borassi, Cecilia; Möller, Svenning Rune; Buono, Rafael; Ohsawa, Yukiko; Matsuoka, Ken; Otegui, Marisa S; Scheller, Henrik V; Geshi, Naomi; Petersen, Bent Larsen; Iusem, Norberto D; Estevez, José M

    2015-05-01

    Root hairs are single cells that develop by tip growth, a process shared with pollen tubes, axons, and fungal hyphae. However, structural plant cell walls impose constraints to accomplish tip growth. In addition to polysaccharides, plant cell walls are composed of hydroxyproline-rich glycoproteins (HRGPs), which include several groups of O-glycoproteins, including extensins (EXTs). Proline hydroxylation, an early post-translational modification (PTM) of HRGPs catalyzed by prolyl 4-hydroxylases (P4Hs), defines their subsequent O-glycosylation sites. In this work, our genetic analyses prove that P4H5, and to a lesser extent P4H2 and P4H13, are pivotal for root hair tip growth. Second, we demonstrate that P4H5 has in vitro preferred specificity for EXT substrates rather than for other HRGPs. Third, by P4H promoter and protein swapping approaches, we show that P4H2 and P4H13 have interchangeable functions but cannot replace P4H5. These three P4Hs are shown to be targeted to the secretory pathway, where P4H5 forms dimers with P4H2 and P4H13. Finally, we explore the impact of deficient proline hydroxylation on the cell wall architecture. Taken together, our results support a model in which correct peptidyl-proline hydroxylation on EXTs, and possibly in other HRGPs, is required for proper cell wall self-assembly and hence root hair elongation in Arabidopsis thaliana.

  8. Diverse alkane hydroxylase genes in microorganisms and environments

    PubMed Central

    Nie, Yong; Chi, Chang-Qiao; Fang, Hui; Liang, Jie-Liang; Lu, She-Lian; Lai, Guo-Li; Tang, Yue-Qin; Wu, Xiao-Lei

    2014-01-01

    AlkB and CYP153 are important alkane hydroxylases responsible for aerobic alkane degradation in bioremediation of oil-polluted environments and microbial enhanced oil recovery. Since their distribution in nature is not clear, we made the investigation among thus-far sequenced 3,979 microbial genomes and 137 metagenomes from terrestrial, freshwater, and marine environments. Hundreds of diverse alkB and CYP153 genes including many novel ones were found in bacterial genomes, whereas none were found in archaeal genomes. Moreover, these genes were detected with different distributional patterns in the terrestrial, freshwater, and marine metagenomes. Hints for horizontal gene transfer, gene duplication, and gene fusion were found, which together are likely responsible for diversifying the alkB and CYP153 genes adapt to the ubiquitous distribution of different alkanes in nature. In addition, different distributions of these genes between bacterial genomes and metagenomes suggested the potentially important roles of unknown or less common alkane degraders in nature. PMID:24829093

  9. Prolyl hydroxylase domain enzymes: important regulators of cancer metabolism

    PubMed Central

    Yang, Ming; Su, Huizhong; Soga, Tomoyoshi; Kranc, Kamil R; Pollard, Patrick J

    2014-01-01

    The hypoxia-inducible factor (HIF) prolyl hydroxylase domain enzymes (PHDs) regulate the stability of HIF protein by post-translational hydroxylation of two conserved prolyl residues in its α subunit in an oxygen-dependent manner. Trans-4-prolyl hydroxylation of HIFα under normal oxygen (O2) availability enables its association with the von Hippel-Lindau (VHL) tumor suppressor pVHL E3 ligase complex, leading to the degradation of HIFα via the ubiquitin-proteasome pathway. Due to the obligatory requirement of molecular O2 as a co-substrate, the activity of PHDs is inhibited under hypoxic conditions, resulting in stabilized HIFα, which dimerizes with HIFβ and, together with transcriptional co-activators CBP/p300, activates the transcription of its target genes. As a key molecular regulator of adaptive response to hypoxia, HIF plays important roles in multiple cellular processes and its overexpression has been detected in various cancers. The HIF1α isoform in particular has a strong impact on cellular metabolism, most notably by promoting anaerobic, whilst inhibiting O2-dependent, metabolism of glucose. The PHD enzymes also seem to have HIF-independent functions and are subject to regulation by factors other than O2, such as by metabolic status, oxidative stress, and abnormal levels of endogenous metabolites (oncometabolites) that have been observed in some types of cancers. In this review, we aim to summarize current understandings of the function and regulation of PHDs in cancer with an emphasis on their roles in metabolism. PMID:27774472

  10. Regulation of hepatic 7 alpha-hydroxylase expression by dietary psyllium in the hamster.

    PubMed Central

    Horton, J D; Cuthbert, J A; Spady, D K

    1994-01-01

    Soluble fiber consistently lowers plasma total and low density lipoprotein (LDL)-cholesterol concentrations in humans and various animal models including the hamster; however, the mechanism of this effect remains incompletely defined. We performed studies to determine the activity of dietary psyllium on hepatic 7 alpha-hydroxylase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and LDL receptor expression in the hamster. In animals fed a cholesterol-free semisynthetic diet containing 7.5% cellulose (avicel) as a fiber source, substitution of psyllium for avicel increased hepatic 7 alpha-hydroxylase activity and mRNA levels by 3-4-fold. Comparable effects on 7 alpha-hydroxylase expression were observed with 1% cholestyramine. Psyllium also increased hepatic 7 alpha-hydroxylase activity and mRNA in animals fed a diet enriched with cholesterol and triglyceride. Activation of 7 alpha-hydroxylase was associated with an increase in hepatic cholesterol synthesis that was apparently not fully compensatory since the cholesterol content of the liver declined. Although dietary psyllium did not increase hepatic LDL receptor expression in animals fed the cholesterol-free, very-low-fat diet, it did increase (or at least restore) receptor expression that had been downregulated by dietary cholesterol and triglyceride. Thus, 7.5% dietary psyllium produced effects on hepatic 7 alpha-hydroxylase and LDL metabolism that were similar to those of 1% cholestyramine. Induction of hepatic 7 alpha-hydroxylase activity by dietary psyllium may account, in large part, for the hypocholesterolemic effect of this soluble fiber. Images PMID:8182140

  11. Infarction-induced cytokines cause local depletion of tyrosine hydroxylase in cardiac sympathetic nerves

    PubMed Central

    Parrish, Diana C.; Alston, Eric N.; Rohrer, Hermann; Nkadi, Paul; Woodward, William R.; Schütz, Günther; Habecker, Beth A.

    2010-01-01

    Myocardial infarction causes heterogeneity of noradrenergic transmission that contributes to the development of ventricular arrhythmias and sudden cardiac death. Ischemia-induced alterations in sympathetic transmission include regional variations in cardiac norepinephrine (NE) and in tyrosine hydroxylase, the rate-limiting enzyme in NE synthesis. Inflammatory cytokines that act through gp130 are elevated in the heart after myocardial infarction. These cytokines decrease expression of tyrosine hydroxylase in sympathetic neurons, and indirect evidence suggests they contribute to the local depletion of tyrosine hydroxylase in the damaged left ventricle. However, gp130 cytokines are also important for the survival of cardiac myocytes following damage to the heart. To examine the effect of cytokines on tyrosine hydroxylase and NE content in cardiac nerves we used gp130DBH-Cre/lox mice, which have a deletion of the gp130 receptor in neurons expressing dopamine beta hydroxylase. The absence of neuronal gp130 prevented the loss of tyrosine hydroxylase in cardiac sympathetic nerves innervating the left ventricle one week after ischemia-reperfusion. Surprisingly, restoring tyrosine hydroxylase in the damaged ventricle did not return neuronal NE content to normal levels. NE uptake into cardiac nerves was significantly lower in gp130 KO mice, contributing to the lack of neuronal NE stores. There were no significant differences in left ventricular peak systolic pressure, dP/dtMAX, or dP/dtMIN between the two genotypes after myocardial infarction, but ganglionic blockade revealed differences in autonomic tone between the genotypes. Stimulating the heart with dobutamine or releasing endogenous NE with tyramine generated similar responses in both genotypes. Thus, the removal of gp130 from sympathetic neurons prevents the post-infarct depletion of TH in the left ventricle, but does not alter NE content or cardiac function. PMID:19880537

  12. Regulation of diurnal variation of cholesterol 7alpha-hydroxylase (CYP7A1) activity in healthy subjects.

    PubMed

    Kovár, J; Lenícek, M; Zimolová, M; Vítek, L; Jirsa, M; Pitha, J

    2010-01-01

    Cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory enzyme of bile acid synthesis, displays a pronounced diurnal variation. To better understand the regulation of CYP7A1 activity, three day-long examinations were carried out in 12 healthy men. The concentrations of 7alpha-hydroxycholest-4-en-3-one (C4), a surrogate marker of CYP7A1 activity, bile acids (BA), insulin, glucose, nonesterified fatty acids, triglycerides, and cholesterol were measured in serum in 90-min intervals from 7 AM till 10 PM. To lower and to increase BA concentration during the study, the subjects received cholestyramine and chenodeoxycholic acid (CDCA), respectively, in two examinations. No drug was used in the control examination. There was a pronounced diurnal variation of C4 concentration with a peak around 1 PM in most of the subjects. The area under the curve (AUC) of C4 concentration was five times higher and three times lower when subjects were treated with cholestyramine and CDCA, respectively. No relationship was found between AUC of C4 and AUC of BA concentration, but AUC of C4 correlated positively with that of insulin. Moreover, short-term treatment with cholestyramine resulted in about 10 % suppression of glycemia throughout the day. Our results suggest that insulin is involved in the regulation of diurnal variation of CYP7A1 activity in humans.

  13. Enhanced Bacterial α(2,6)-Sialyltransferase Reaction through an Inhibition of Its Inherent Sialidase Activity by Dephosphorylation of Cytidine-5'-Monophosphate.

    PubMed

    Kang, Ji-Yeon; Lim, Se-Jong; Kwon, Ohsuk; Lee, Seung-Goo; Kim, Ha Hyung; Oh, Doo-Byoung

    2015-01-01

    Bacterial α(2,6)-sialyltransferases (STs) from Photobacterium damsela, Photobacterium sp. JT-ISH-224, and P. leiognathi JT-SHIZ-145 were recombinantly expressed in Escherichia coli and their ST activities were compared directly using a galactosylated bi-antennary N-glycan as an acceptor substrate. In all ST reactions, there was an increase of sialylated glycans at shorter reaction times and later a decrease in prolonged reactions, which is related with the inherent sialidase activities of bacterial STs. These sialidase activities are greatly increased by free cytidine monophosphate (CMP) generated from a donor substrate CMP-N-acetylneuraminic acid (CMP-Neu5Ac) during the ST reactions. The decrease of sialylated glycans in prolonged ST reaction was prevented through an inhibition of sialidase activity by simple treatment of alkaline phosphatase (AP), which dephosphorylates CMP to cytidine. Through supplemental additions of AP and CMP-Neu5Ac to the reaction using the recombinant α(2,6)-ST from P. leiognathi JT-SHIZ-145 (P145-ST), the content of bi-sialylated N-glycan increased up to ~98% without any decrease in prolonged reactions. This optimized P145-ST reaction was applied successfully for α(2,6)-sialylation of asialofetuin, and this resulted in a large increase in the populations of multi-sialylated N-glycans compared with the reaction without addition of AP and CMP-Neu5Ac. These results suggest that the optimized reaction using the recombinant P145-ST readily expressed from E. coli has a promise for economic glycan synthesis and glyco-conjugate remodeling.

  14. Enhanced Bacterial α(2,6)-Sialyltransferase Reaction through an Inhibition of Its Inherent Sialidase Activity by Dephosphorylation of Cytidine-5'-Monophosphate

    PubMed Central

    Kang, Ji-Yeon; Lim, Se-Jong; Kwon, Ohsuk; Lee, Seung-Goo; Kim, Ha Hyung; Oh, Doo-Byoung

    2015-01-01

    Bacterial α(2,6)-sialyltransferases (STs) from Photobacterium damsela, Photobacterium sp. JT-ISH-224, and P. leiognathi JT-SHIZ-145 were recombinantly expressed in Escherichia coli and their ST activities were compared directly using a galactosylated bi-antennary N-glycan as an acceptor substrate. In all ST reactions, there was an increase of sialylated glycans at shorter reaction times and later a decrease in prolonged reactions, which is related with the inherent sialidase activities of bacterial STs. These sialidase activities are greatly increased by free cytidine monophosphate (CMP) generated from a donor substrate CMP-N-acetylneuraminic acid (CMP-Neu5Ac) during the ST reactions. The decrease of sialylated glycans in prolonged ST reaction was prevented through an inhibition of sialidase activity by simple treatment of alkaline phosphatase (AP), which dephosphorylates CMP to cytidine. Through supplemental additions of AP and CMP-Neu5Ac to the reaction using the recombinant α(2,6)-ST from P. leiognathi JT-SHIZ-145 (P145-ST), the content of bi-sialylated N-glycan increased up to ~98% without any decrease in prolonged reactions. This optimized P145-ST reaction was applied successfully for α(2,6)-sialylation of asialofetuin, and this resulted in a large increase in the populations of multi-sialylated N-glycans compared with the reaction without addition of AP and CMP-Neu5Ac. These results suggest that the optimized reaction using the recombinant P145-ST readily expressed from E. coli has a promise for economic glycan synthesis and glyco-conjugate remodeling. PMID:26231036

  15. Associations of tryptophan hydroxylase gene polymorphisms with IBS

    PubMed Central

    Jun, Sang-Eun; Kohen, Ruth; Cain, Kevin C.; Jarrett, Monica E.; Heitkemper, Margaret M.

    2010-01-01

    Background Alterations in serotonin (5-HT) are suspected in the pathophysiology of irritable bowel syndrome (IBS). Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of serotonin and has two isoforms, TPH1 and TPH2. Genetic variants in both genes have been studied in various disorders related to serotonin dysregulation. The aim of this study was to examine whether TPH gene variants were associated with IBS and IBS-related gastrointestinal (GI) symptoms. Methods Five single nucleotide polymorphisms (SNPs) from the TPH1 and one SNP from the TPH2 were genotyped in 199 IBS patients and 79 healthy controls. All subjects were Caucasian women of European origin. IBS patients filled in a daily diary with five GI symptoms and stool characteristics for 28 days. Key Results The TPH1 SNPs showed no association with the diagnosis of IBS. However among IBS patients, all five TPH1 SNPs showed some association with diarrhea and loose type of stool consistency, with p-values rating from 0.01 to 0.20. The TPH2 SNP showed a trend towards a reduced risk of IBS as well as possible associations with stool characteristics, both hard and loose stools. However, no p-values were less than the conservative multiple-comparison-adjusted threshold of 0.001 and hence these results must be interpreted cautiously. Conclusions & Inferences This study is the first to assess associations of TPH gene variants with IBS-related GI symptoms and stool characteristics. The possible association of TPH gene variants with diarrhea needs to be verified in an independent sample. PMID:21073637

  16. Computational study of missense mutations in phenylalanine hydroxylase.

    PubMed

    Réblová, Kamila; Kulhánek, Petr; Fajkusová, Lenka

    2015-04-01

    Hyperphenylalaninemia (HPA) is one of the most common metabolic disorders. HPA, which is transmitted by an autosomal recessive mode of inheritance, is caused by mutations of the phenylalanine hydroxylase gene. Most mutations are missense and lead to reduced protein stability and/or impaired catalytic function. The impact of such mutations varies, ranging from classical phenylketonuria (PKU), mild PKU, to non-PKU HPA phenotypes. Despite the fact that HPA is a monogenic disease, clinical data show that one PKU genotype can be associated with more in vivo phenotypes, which indicates the role of other (still unknown) factors. To better understand the phenotype-genotype relationships, we analyzed computationally the impact of missense mutations in homozygotes stored in the BIOPKU database. A total of 34 selected homozygous genotypes was divided into two main groups according to their phenotypes: (A) genotypes leading to non-PKU HPA or combined phenotype non-PKU HPA/mild PKU and (B) genotypes leading to classical PKU, mild PKU or combined phenotype mild PKU/classical PKU. Combining in silico analysis and molecular dynamics simulations (in total 3 μs) we described the structural impact of the mutations, which allowed us to separate 32 out of 34 mutations between groups A and B. Testing the simulation conditions revealed that the outcome of mutant simulations can be modulated by the ionic strength. We also employed programs SNPs3D, Polyphen-2, and SIFT but based on the predictions performed we were not able to discriminate mutations with mild and severe PKU phenotypes.

  17. Human DJ-1-specific Transcriptional Activation of Tyrosine Hydroxylase Gene*

    PubMed Central

    Ishikawa, Shizuma; Taira, Takahiro; Takahashi-Niki, Kazuko; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M. M.

    2010-01-01

    Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-l-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice. PMID:20938049

  18. Human DJ-1-specific transcriptional activation of tyrosine hydroxylase gene.

    PubMed

    Ishikawa, Shizuma; Taira, Takahiro; Takahashi-Niki, Kazuko; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M M

    2010-12-17

    Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-L-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice.

  19. Tyrosine Hydroxylase Expression in Type II Cochlear Afferents in Mice.

    PubMed

    Vyas, Pankhuri; Wu, Jingjing Sherry; Zimmerman, Amanda; Fuchs, Paul; Glowatzki, Elisabeth

    2017-02-01

    Acoustic information propagates from the ear to the brain via spiral ganglion neurons that innervate hair cells in the cochlea. These afferents include unmyelinated type II fibers that constitute 5 % of the total, the majority being myelinated type I neurons. Lack of specific genetic markers of type II afferents in the cochlea has been a roadblock in studying their functional role. Unexpectedly, type II afferents were visualized by reporter proteins induced by tyrosine hydroxylase (TH)-driven Cre recombinase. The present study was designed to determine whether TH-driven Cre recombinase (TH-2A-CreER) provides a selective and reliable tool for identification and genetic manipulation of type II rather than type I cochlear afferents. The "TH-2A-CreER neurons" radiated from the spiral lamina, crossed the tunnel of Corti, turned towards the base of the cochlea, and traveled beneath the rows of outer hair cells. Neither the processes nor the somata of TH-2A-CreER neurons were labeled by antibodies that specifically labeled type I afferents and medial efferents. TH-2A-CreER-positive processes partially co-labeled with antibodies to peripherin, a known marker of type II afferents. Individual TH-2A-CreER neurons gave off short branches contacting 7-25 outer hair cells (OHCs). Only a fraction of TH-2A-CreER boutons were associated with CtBP2-immunopositive ribbons. These results show that TH-2A-CreER provides a selective marker for type II versus type I afferents and can be used to describe the morphology and arborization pattern of type II cochlear afferents in the mouse cochlea.

  20. Phenylalanine hydroxylase activity and expression in chicks subjected to phenylalanine imbalance or phenylalanine toxicity.

    PubMed

    Lartey, F M; Austic, R E

    2009-04-01

    Experiments were performed to investigate the activity of hepatic Phe hydroxylase (PAH) and plasma amino acid concentrations under conditions of Phe imbalance or toxicity in chicks fed on experimental diets from 7 to 14 or 16 d of age. In experiment 1, Phe imbalance was created by adding 10% of a mixture of indispensable amino acids lacking Phe (IAA - Phe) to a basal diet containing 0.46% Phe. The activity of PAH was not significantly affected by the imbalance. Correcting the imbalance by adding 1.12% Phe to the diet prevented the growth impairment and increased the activity of PAH. In experiment 2, growth was reduced by the addition of excess (2%) Phe to the basal diet. Correcting the excess by adding the IAA - Phe to the diet prevented the growth reduction. The activity of PAH was not significantly affected by 2% Phe, but it increased in chicks fed the corrected diet. The levels of PAH mRNA were not affected by the dietary treatments. A factorial arrangement of treatments with 3 dietary levels of Phe (0.46, 1.58, and 2.46%) with or without the IAA - Phe was used in experiment 3. The effects on growth were similar to those of the same treatments in experiments 1 and 2. The addition of Phe significantly increased hepatic PAH activity, but there was no detectable main effect of the IAA - Phe and no interaction. Plasma Phe concentration was increased by dietary Phe and decreased by the IAA - Phe mixture. We conclude that hepatic PAH activity in chicks variably increases in response to Phe or a 10% dietary supplement of indispensable amino acids including Phe but does not increase in response to IAA - Phe when the amino acids are added to a diet that is marginally adequate in Phe. The increased activity does not involve changes in PAH mRNA. The effects of IAA - Phe on plasma Phe concentrations appear to be independent of hepatic PAH activity as measured in vitro.

  1. Monoamine Oxidase and Dopamine β-Hydroxylase Inhibitors from the Fruits of Gardenia jasminoides

    PubMed Central

    Kim, Ji Ho; Kim, Gun Hee; Hwang, Keum Hee

    2012-01-01

    This research was designed to determine what components of Gardenia jasminoides play a major role in inhibiting the enzymes related antidepressant activity of this plant. In our previous research, the ethyl acetate fraction of G. jasminosides fruits inhibited the activities of both monoamine oxidase-A (MAO-A) and monoamine oxidase-B (MAO-B), and oral administration of the ethanolic extract slightly increased serotonin concentrations in the brain tissues of rats and decreased MAO-B activity. In addition, we found through in vitro screening test that the ethyl acetate fraction showed modest inhibitory activity on dopamine-β hydroxylase (DBH). The bioassay-guided fractionation led to the isolation of five bio-active compounds, protocatechuic acid (1), geniposide (2), 6'-O-trans-p-coumaroylgeniposide (3), 3,5-d-ihydroxy-1,7-bis (4-hydroxyphenyl) heptanes (4), and ursolic acid (5), from the ethyl acetate fraction of G. jasminoides fruits. The isolated compounds showed different inhibitory potentials against MAO-A, -B, and DBH. Protocatechuic acid showed potent inhibition against MAO-B (IC50 300 μmol/L) and DBH (334 μmol/L), exhibiting weak MAO-A inhibition (2.41 mmol/L). Two iridoid glycosides, geniposide (223 μmol/L) and 6'-O-trans-p-coumaroylgeniposide (127μmol/L), were selective MAO-B inhibitor. Especially, 6'-O-trans-p-coumaroylgeniposide exhibited more selective MAO-B inhibition than deprenyl, well-known MAO-B inhibitor for the treatment of early-stage Parkinson’s disease. The inhibitory activity of 3,5-di-hydroxy-1,7-bis (4-hydroxyphenyl) heptane was strong for MAO-B (196 μmol/L), modest for MAO-A (400 μmol/L), and weak for DBH (941 μmol/L). Ursolic acid exhibited significant inhibition of DBH (214 μmol/L), weak inhibition of MAO-B (780 μmol/L), and no inhibition against MAO-A. Consequently, G. jasminoides fruits are considerable for development of biofunctional food materials for the combination treatment of depression and neurodegenerative disorders

  2. Clinical, genetic, and structural basis of congenital adrenal hyperplasia due to 11β-hydroxylase deficiency.

    PubMed

    Khattab, Ahmed; Haider, Shozeb; Kumar, Ameet; Dhawan, Samarth; Alam, Dauood; Romero, Raquel; Burns, James; Li, Di; Estatico, Jessica; Rahi, Simran; Fatima, Saleel; Alzahrani, Ali; Hafez, Mona; Musa, Noha; Razzghy Azar, Maryam; Khaloul, Najoua; Gribaa, Moez; Saad, Ali; Charfeddine, Ilhem Ben; Bilharinho de Mendonça, Berenice; Belgorosky, Alicia; Dumic, Katja; Dumic, Miroslav; Aisenberg, Javier; Kandemir, Nurgun; Alikasifoglu, Ayfer; Ozon, Alev; Gonc, Nazli; Cheng, Tina; Kuhnle-Krahl, Ursula; Cappa, Marco; Holterhus, Paul-Martin; Nour, Munier A; Pacaud, Daniele; Holtzman, Assaf; Li, Sun; Zaidi, Mone; Yuen, Tony; New, Maria I

    2017-03-07

    Congenital adrenal hyperplasia (CAH), resulting from mutations in CYP11B1, a gene encoding 11β-hydroxylase, represents a rare autosomal recessive Mendelian disorder of aberrant sex steroid production. Unlike CAH caused by 21-hydroxylase deficiency, the disease is far more common in the Middle East and North Africa, where consanguinity is common often resulting in identical mutations. Clinically, affected female newborns are profoundly virilized (Prader score of 4/5), and both genders display significantly advanced bone ages and are oftentimes hypertensive. We find that 11-deoxycortisol, not frequently measured, is the most robust biochemical marker for diagnosing 11β-hydroxylase deficiency. Finally, computational modeling of 25 missense mutations of CYP11B1 revealed that specific modifications in the heme-binding (R374W and R448C) or substrate-binding (W116C) site of 11β-hydroxylase, or alterations in its stability (L299P and G267S), may predict severe disease. Thus, we report clinical, genetic, hormonal, and structural effects of CYP11B1 gene mutations in the largest international cohort of 108 patients with steroid 11β-hydroxylase deficiency CAH.

  3. Superstoichiometric binding of L-Phe to phenylalanine hydroxylase from Caenorhabditis elegans: evolutionary implications.

    PubMed

    Flydal, Marte I; Mohn, Tonje C; Pey, Angel L; Siltberg-Liberles, Jessica; Teigen, Knut; Martinez, Aurora

    2010-11-01

    Phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of L-Phe to L-Tyr. Dysfunctional PAH results in phenylketonuria and mammalian PAH is therefore highly regulated and displays positive cooperativity for L-Phe (Hill coefficient (h)=2). L-Phe does not bind to the regulatory ACT domain in full-length tetrameric human PAH and cooperativity is elicited by homotropic binding to the catalytic site (Thórólfsson et al. in Biochemistry 41:7573-7585, 2002). PAH from Caenorhabditis elegans (cePAH) is devoid of cooperativity for L-Phe (h=0.9), and, as shown in this work, structural analysis reveal an additional L-Phe binding site at the regulatory domain of full-length cePAH. This site involves the GA(S)L/ISRP motifs, which are also found in ACT domains of other L-Phe binding proteins, such as prephenate dehydratase. Isothermal titration calorimetry further demonstrated 2 binding sites per subunit for cePAH versus ~1 for hPAH. Steric occlusion of the regulatory site, notably by residues Lys215/Tyr216 from the adjacent catalytic domain, appears to hinder regulatory binding in full-length hPAH. Accordingly, the humanized mutant Q215K/N216Y of cePAH binds ~1.4 L-Phe/subunit. This mutant also displays high catalytic activity and certain positive cooperativity for L-Phe (h=1.4). Our results support that the acquisition of positive cooperativity in mammalian forms of PAH is accompanied by a closure of the regulatory L: -Phe binding site. Concomitantly, the function of the regulatory ACT domain appears to be adapted from amino acid binding to serving the communication of conformational changes among catalytic subunits.

  4. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.

    PubMed

    Arturo, Emilia C; Gupta, Kushol; Héroux, Annie; Stith, Linda; Cross, Penelope J; Parker, Emily J; Loll, Patrick J; Jaffe, Eileen K

    2016-03-01

    Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.

  5. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  6. Domain Characterization of Cyclosporin Regio-Specific Hydroxylases in Rare Actinomycetes.

    PubMed

    Woo, Min-Woo; Lee, Bo-Ram; Nah, Hee-Ju; Choi, Si-Sun; Li, Shengying; Kim, Eung-Soo

    2015-10-01

    Cytochrome P450 hydroxylase (CYP) in actinomycetes plays an important role in the biosynthesis and bioconversion of various secondary metabolites. Two unique CYPs named CYP-sb21 and CYP-pa1, which were identified from Sebekia benihana and Pseudonocardia autotrophica, respectively, were proven to transfer a hydroxyl group at the 4(th) or 9(th) N-methyl leucine position of immunosuppressive agent cyclosporin A (CsA). Interestingly, these two homologous CYPs showed different CsA regio-selectivities. CYP-sb21 exhibited preferential hydroxylation activity at the 4(th) position over the 9(th) position, whereas CYP-pa1 showed the opposite preference. To narrow down the CYP domain critical for CsA regio-selectivity, each CYP was divided into four domains, and each domain was swapped with its counterpart from the other CYP. A total of 18 hybrid CYPs were then individually tested for CsA regioselectivity. Although most of the hybrid CYPs failed to exhibit a significant change in regioselectivity in the context of CsA hydroxylation, hybrid CYP-pa1 swapped with the second domain of CYP-sb21 showed a higher preference for the 9th position. Moreover, hybrid CYPsb21 containing seven amino acids from the 2nd domain of CYP-pa1 showed higher preference for the 4(th) position. These results imply that the 2nd domain of CsA-specific CYP plays a critical role in CsA regio-selectivity, thereby setting the stage for biotechnological application of CsA regio-selective hydroxylation.

  7. Molecular cloning and characterization of a flavanone 3-Hydroxylase gene from Artemisia annua L.

    PubMed

    Xiong, Shuo; Tian, Na; Long, Jinhua; Chen, Yuhong; Qin, Yu; Feng, Jinyu; Xiao, Wenjun; Liu, Shuoqian

    2016-08-01

    Flavonoids were found to synergize anti-malaria and anti-cancer compounds in Artemisia annua, a very important economic crop in China. In order to discover the regulation mechanism of flavonoids in Artemisia annua, the full length cDNA of flavanone 3-hydroxylase (F3H) were isolated from Artemisia annua for the first time by using RACE (rapid amplification of cDNA ends). The completed open read frame of AaF3H was 1095 bp and it encoded a 364-amino acid protein with a predicted molecular mass of 41.18 kDa and a pI of 5.67. The recombinant protein of AaF3H was expressed in E. coli BL21(DE3) as His-tagged protein, purified by Ni-NTA agrose affinity chromatography, and functionally characterized in vitro. The results showed that the His-tagged protein (AaF3H) catalyzed naringenin to dihydrokaempferol in the present of Fe(2+). The Km for naringenin was 218.03 μM. The optimum pH for AaF3H reaction was determined to be pH 8.5, and the optimum temperature was determined to be 35 °C. The AaF3H transcripts were found to be accumulated in the cultivar with higher level of flavonoids than that with lower level of flavonoids, which implied that AaF3H was a potential target for regulation of flavonoids biosynthesis in Artemisia annua through metabolic engineering.

  8. Spatial distribution of synapses on tyrosine hydroxylase-expressing juxtaglomerular cells in the mouse olfactory glomerulus.

    PubMed

    Kiyokage, Emi; Kobayashi, Kazuto; Toida, Kazunori

    2017-04-01

    Olfactory sensory axons converge in specific glomeruli where they form excitatory synapses onto dendrites of mitral/tufted (M/T) and juxtaglomerular (JG) cells, including periglomerular (PG), external tufted (ET), and superficial-short axon cells. JG cells consist of heterogeneous subpopulations with different neurochemical, physiological, and morphological properties. Among JG cells, previous electron microscopic (EM) studies have shown that the majority of synaptic inputs to tyrosine hydroxylase (TH)-immunoreactive neurons were asymmetrical synapses from olfactory nerve (ON) terminals. However, recent physiological results revealed that 70% of dopaminergic/γ-aminobutyric acid (GABA)ergic neurons received polysynaptic inputs via ET cells, whereas the remaining 30% received monosynaptic ON inputs. To understand the discrepancies between EM and physiological data, we used serial EM analysis combined with confocal laser scanning microscope images to examine the spatial distribution of synapses on dendrites using mice expressing enhanced green fluorescent protein under the control of the TH promoter. The majority of synaptic inputs to TH-expressing JG cells were from ON terminals, and they preferentially targeted distal dendrites from the soma. On the other hand, the numbers of non-ON inputs were fewer and targeted proximal dendrites. Furthermore, individual TH-expressing JG cells formed serial synapses, such as M/T→TH→another presumed M/T or ON→TH→presumed M/T, but not reciprocal synapses. Serotonergic fibers also associated with somatic regions of TH neurons, displaying non-ON profiles. Thus, fewer proximal non-ON synapses provide more effective inputs than large numbers of distal ON synapses and may occur on the physiologically characterized population of dopaminergic-GABAergic neurons (70%) that receive their most effective inputs indirectly via an ON→ET→TH circuit. J. Comp. Neurol. 525:1059-1074, 2017. © 2017 Wiley Periodicals, Inc.

  9. Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding

    PubMed Central

    Lee, Chang Woo; Yu, Sang-Cheol; Lee, Joo-Ho; Park, Sun-Ha; Park, Hyun; Oh, Tae-Jin; Lee, Jun Hyuck

    2016-01-01

    Enzymatic alkane hydroxylation reactions are useful for producing pharmaceutical and agricultural chemical intermediates from hydrocarbons. Several cytochrome P450 enzymes catalyze the regio- and stereo-specific hydroxylation of alkanes. We evaluated the substrate binding of a putative CYP alkane hydroxylase (CYP153D17) from the bacterium Sphingomonas sp. PAMC 26605. Substrate affinities to C10–C12 n-alkanes and C10–C14 fatty acids with Kd values varied from 0.42 to 0.59 μM. A longer alkane (C12) bound more strongly than a shorter alkane (C10), while shorter fatty acids (C10, capric acid; C12, lauric acid) bound more strongly than a longer fatty acid (C14, myristic acid). These data displayed a broad substrate specificity of CYP153D17, hence it was named as a putative CYP alkane hydroxylase. Moreover, the crystal structure of CYP153D17 was determined at 3.1 Å resolution. This is the first study to provide structural information for the CYP153D family. Structural analysis showed that a co-purified alkane-like compound bound near the active-site heme group. The alkane-like substrate is in the hydrophobic pocket containing Thr74, Met90, Ala175, Ile240, Leu241, Val244, Leu292, Met295, and Phe393. Comparison with other CYP structures suggested that conformational changes in the β1–β2, α3–α4, and α6–α7 connecting loop are important for incorporating the long hydrophobic alkane-like substrate. These results improve the understanding of the catalytic mechanism of CYP153D17 and provide valuable information for future protein engineering studies. PMID:27941697

  10. Chemical inhibition of potato ABA-8'-hydroxylase activity alters in vitro and in vivo ABA metabolism and endogenous ABA levels but does not affect potato microtuber dormancy duration.

    PubMed

    Suttle, Jeffrey C; Abrams, Suzanne R; De Stefano-Beltrán, Luis; Huckle, Linda L

    2012-09-01

    The effects of azole-type P450 inhibitors and two metabolism-resistant abscisic acid (ABA) analogues on in vitro ABA-8'-hydroxylase activity, in planta ABA metabolism, endogenous ABA content, and tuber meristem dormancy duration were examined in potato (Solanum tuberosum L. cv. Russet Burbank). When functionally expressed in yeast, three potato CYP707A genes were demonstrated to encode enzymatically active ABA-8'-hydroxylases with micromolar affinities for (+)-ABA. The in vitro activity of the three enzymes was inhibited by the P450 azole-type inhibitors ancymidol, paclobutrazol, diniconazole, and tetcyclasis, and by the 8'-acetylene- and 8'-methylene-ABA analogues, with diniconazole and tetcyclasis being the most potent inhibitors. The in planta metabolism of [(3)H](±)-ABA to phaseic acid and dihydrophaseic acid in tuber meristems was inhibited by diniconazole, tetcyclasis, and to a lesser extent by 8'-acetylene- and 8'-methylene-ABA. Continuous exposure of in vitro generated microtubers to diniconazole resulted in a 2-fold increase in endogenous ABA content and a decline in dihydrophaseic acid content after 9 weeks of development. Similar treatment with 8'-acetylene-ABA had no effects on the endogenous contents of ABA or phaseic acid but reduced the content of dihydrophaseic acid. Tuber meristem dormancy progression was determined ex vitro in control, diniconazole-, and 8'-acetylene-ABA-treated microtubers following harvest. Continuous exposure to diniconazole during microtuber development had no effects on subsequent sprouting at any time point. Continuous exposure to 8'-acetylene-ABA significantly increased the rate of microtuber sprouting. The results indicate that, although a decrease in ABA content is a hallmark of tuber dormancy progression, the decline in ABA levels is not a prerequisite for dormancy exit and the onset of tuber sprouting.

  11. Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency, state of the art.

    PubMed

    Spaapen, Leo J M; Rubio-Gozalbo, M Estela

    2003-02-01

    Since 1999 an increasing number of patients with phenylalanine hydroxylase (PAH) deficiency are reported to be able to decrease their plasma phenylalanine (Phe) concentrations after a 6R-tetrahydrobiopterin (BH(4)) challenge. The majority of these patients have mild PKU or MHP (mild hyperphenylalaninemia) and harbour at least one missense mutation in the PAH gene associated with this phenotype. The rate of decrease and the lowest achieved Phe level vary between patients with different genotypes but appears to be similar in patients with the same genotype. A number of the mutations associated with BH(4)-responsiveness have been studied in an 'in vitro' eukaryotic cell expression system leading to biosynthesis of a mutant PAH enzyme with some residual activity. Patients bearing mutations that cause severe structural distortion in the expressed protein (loss of function mutations), leading to undetectable PAH activity, are not responsive to BH(4). These observations suggest that residual PAH activity (in vitro) is a prerequisite for BH(4)-responsiveness. However, an in vitro residual PAH activity is not a guarantee for in vivo BH(4)-responsiveness. Mechanisms behind this responsiveness could be relieve of decreased binding affinity for BH(4), BH(4)-mediated increase of PAH gene expression or stabilization of the mutant enzyme protein by BH(4). BH(4)-responsive PAH-deficient patients have only been reported since 1999. For the western countries this is explained by the fact that the manufacturer changed the diastereoisomeric purity of the BH4 preparation from 69% of the natural 6R-BH4 (31% of 6S-BH4) to 99.5% 6R-BH4. The new findings on BH(4)-responsiveness may be of clinical relevance because these patients can be treated with BH(4) with concomitant relief or withdrawal of the burdensome PKU diet. These observations warrant further clinical studies to assess efficacy, optimal dosage, and safety of BH(4) treatment in this group. The data strongly emphasize the

  12. A steady-state kinetic analysis of the prolyl-4-hydroxylase mechanism.

    PubMed

    Soskel, N T; Kuby, S A

    1981-01-01

    Published kinetic data by Kivirikko, et al. on the prolyl-4-hydroxylase reaction have been re-evaluated using the overall steady-state velocity equation in the forward and reverse directions for an ordered ter ter kinetic mechanism. Qualitatively, the published data for prolyl-4-hydroxylase appear to fit the predicted patterns for this kinetic mechanism. More kinetic data are needed to confirm these results and to quantitate the kinetic parameters but, tentatively, the order of substrate addition would appear to be alpha-ketoglutarate, oxygen, and peptide; and the order of product release would be hydroxylated peptide (or collagen), carbon dioxide, and succinate.

  13. Phenylalanine binding is linked to dimerization of the regulatory domain of phenylalanine hydroxylase.

    PubMed

    Zhang, Shengnan; Roberts, Kenneth M; Fitzpatrick, Paul F

    2014-10-28

    Analytical ultracentrifugation has been used to analyze the oligomeric structure of the isolated regulatory domain of phenylalanine hydroxylase. The protein exhibits a monomer-dimer equilibrium with a dissociation constant of ~46 μM; this value is unaffected by the removal of the 24 N-terminal residues or by phosphorylation of Ser16. In contrast, phenylalanine binding (Kd = 8 μM) stabilizes the dimer. These results suggest that dimerization of the regulatory domain of phenylalanine hydroxylase is linked to allosteric activation of the enzyme.

  14. [Dopamine beta hydroxylase. Value and limits of its study in neurology].

    PubMed

    Miras Portugal, M T; Aunis, D; Mandel, P; Warter, J M; Coquillat, G; Collard, M; Rohmer, F

    1976-06-01

    Plasma dopamine-beta-hydroxylase was studied in 96 subjects, 33 of them controls and 63 of them patients (Parkinson's disease, chronic chorea, torsion dystonia, postural tremor and epilepsy). Only the epileptics showed a significant decrease in the average level of dopamine-beta-hydroxylase activity in comparison with the controls. During the cold test, DBH did not vary except in one case. On the other hand, during epileptic attacks, DBH activity underwent considerable fluctuations. Therefore, except in special pathological conditions, such as epileptic attacks, measurement of plasma or serum DBH activity is of limited value for neurological pathology and is not a good indication of the activity of the sympathetic nervous system.

  15. Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly

    PubMed Central

    Bates, Philip D.; Johnson, Sean R.; Cao, Xia; Li, Jia; Nam, Jeong-Won; Jaworski, Jan G.; Ohlrogge, John B.; Browse, John

    2014-01-01

    Degradation of unusual fatty acids through β-oxidation within transgenic plants has long been hypothesized as a major factor limiting the production of industrially useful unusual fatty acids in seed oils. Arabidopsis seeds expressing the castor fatty acid hydroxylase accumulate hydroxylated fatty acids up to 17% of total fatty acids in seed triacylglycerols; however, total seed oil is also reduced up to 50%. Investigations into the cause of the reduced oil phenotype through in vivo [14C]acetate and [3H]2O metabolic labeling of developing seeds surprisingly revealed that the rate of de novo fatty acid synthesis within the transgenic seeds was approximately half that of control seeds. RNAseq analysis indicated no changes in expression of fatty acid synthesis genes in hydroxylase-expressing plants. However, differential [14C]acetate and [14C]malonate metabolic labeling of hydroxylase-expressing seeds indicated the in vivo acetyl–CoA carboxylase activity was reduced to approximately half that of control seeds. Therefore, the reduction of oil content in the transgenic seeds is consistent with reduced de novo fatty acid synthesis in the plastid rather than fatty acid degradation. Intriguingly, the coexpression of triacylglycerol synthesis isozymes from castor along with the fatty acid hydroxylase alleviated the reduced acetyl–CoA carboxylase activity, restored the rate of fatty acid synthesis, and the accumulation of seed oil was substantially recovered. Together these results suggest a previously unidentified mechanism that detects inefficient utilization of unusual fatty acids within the endoplasmic reticulum and activates an endogenous pathway for posttranslational reduction of fatty acid synthesis within the plastid. PMID:24398521

  16. Vascular Endothelial Over-Expression of Human Soluble Epoxide Hydrolase (Tie2-sEH Tr) Attenuates Coronary Reactive Hyperemia in Mice: Role of Oxylipins and ω-Hydroxylases

    PubMed Central

    Zeldin, Darryl C.; Morisseau, Christophe; Falck, John R.

    2017-01-01

    Cytochromes P450 metabolize arachidonic acid (AA) into two vasoactive oxylipins with opposing biologic effects: epoxyeicosatrienoic acids (EETs) and omega-(ω)-terminal hydroxyeicosatetraenoic acids (HETEs). EETs have numerous beneficial physiological effects, including vasodilation and protection against ischemia/reperfusion injury, whereas ω-terminal HETEs induce vasoconstriction and vascular dysfunction. We evaluated the effect of these oxylipins on post-ischemic vasodilation known as coronary reactive hyperemia (CRH). CRH prevents the potential harm associated with transient ischemia. The beneficial effects of EETs are reduced after their hydrolysis to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). ω-terminal HETEs are formed by ω-hydroxylase family members. The relationship among endothelial over-expression of sEH (Tie2-sEH Tr), the changes in oxylipins it may produce, the pharmacologic inhibition of ω-hydroxylases, activation of PPARγ, and CRH response to a brief ischemia is not known. We hypothesized that CRH is attenuated in isolated mouse hearts with endothelial sEH over-expression through modulation of oxylipin profiles, whereas both inhibition of ω-hydroxylases and activation of PPARγ enhance CRH. Compared to WT mice, Tie2-sEH Tr mice had decreased CRH, including repayment volume, repayment duration, and repayment/debt ratio (P < 0.05), whereas inhibition of ω-hydroxylases increased these same CRH parameters in Tie2-sEH Tr mice. Inhibition of sEH with t-AUCB reversed the decreased CRH in Tie2-sEH Tr mice. Endothelial over-expression of sEH significantly changed oxylipin profiles, including decreases in DHETs, mid-chain HETEs, and prostaglandins (P < 0.05). Treatment with rosiglitazone, PPARγ-agonist, enhanced CRH (P < 0.05) in both Tie2-sEH Tr and wild type (WT) mice. These data demonstrate that endothelial over-expression of sEH (through changing the oxylipin profiles) attenuates CRH, whereas inhibition of ω-hydroxylases

  17. Potent inhibition of retinoic acid metabolism enzyme(s) by novel azolyl retinoids.

    PubMed

    Njar, V C; Nnane, I P; Brodie, A M

    2000-09-04

    Novel (+/-)-4-azolyl retinoic acid analogues 4, 5, 7 and 8 have been designed and synthesized and have been shown to be powerful inhibitors of hamster microsomal all-trans-retinoic acid 4-hydroxylase enzyme(s). (+/-)-4-(1H-Imidazol-1-yl)retinoic acid (4) is the most potent inhibitor of this enzyme reported to date.

  18. Characteristics of hydrocarbon hydroxylase genes in a thermophilic aerobic biological system treating oily produced wastewater.

    PubMed

    Liu, Ruyin; Gao, Yingxin; Ji, Yifeng; Zhang, Yu; Yang, Min

    2015-01-01

    Alkane and aromatic hydroxylase genes in a full-scale aerobic system treating oily produced wastewater under thermophilic condition (45-50 °C) in the Jidong oilfield, China, were investigated using clone library and quantitative polymerase chain reaction methods. Rather than the normally encountered integral-membrane non-haem iron monooxygenase (alkB) genes, only CYP153-type P450 hydroxylase genes were detected for the alkane activation, indicating that the terminal oxidation of alkanes might be mainly mediated by the CYP153-type alkane hydroxylases in the thermophilic aerobic process. Most of the obtained CYP153 gene clones showed distant homology with the reference sequences, which might represent novel alkane hydroxylases. For the aromatic activation, the polycyclic aromatic hydrocarbon-ring hydroxylating dioxygenase (PAH-RHD) gene was derived from Gram-negative PAH-degraders belonging to the Burkholderiales order, with a 0.72% relative abundance of PAH-RHD gene to 16S rRNA gene. This was consistent with the result of 16S rRNA gene analysis, indicating that Burkholderiales bacteria might play a key role in the full-scale process of thermophilic hydrocarbon degradation.

  19. 17α-hydroxylase/17,20-lyase deficiency in congenital adrenal hyperplasia: A case report

    PubMed Central

    Xu, Simiao; Hu, Shuhong; Yu, Xuefeng; Zhang, Muxun; Yang, Yan

    2016-01-01

    Congenital adrenal hyperplasia (CAH) is a rare autosomal recessive disorder caused by mutations in the cytochrome P450 family 17 subfamily A member 1 (CYP17A1) gene located on chromosome 10q24.3, which leads to a deficiency in 17α-hydroxylase/17,20-lyase. The disorder is characterized by low blood levels of estrogens, androgens and cortisol, which leads to a compensatory increase in adrenocorticotropic hormone levels that stimulate the production of mineralocorticoid precursors. This subsequently leads to hypertension, hypokalemia, primary amenorrhea and sexual infantilism. Over 90 distinct genetic lesions have been identified in patients with this disorder. The prevalence of common mutation of CYP17A1 gene differs among ethnic groups. Treatment of this disorder involves replacement of glucocorticoids and sex steroids. Estrogen alone is prescribed for patients who are biologically male with 17α-hydroxylase deficiencies that identify as female. However, genetically female patients may receive estrogen and progesterone supplementation. In the present study, a 17-year-old female with 17α-hydroxylase/17,20-lyase deficiency that presented with primary amenorrhea and sexual infantilism and no hypertension, was examined. The karyotype of the patient was 46, XX, and genetic analysis revealed the presence of a compound heterozygous mutation in exons 6 and 8, leading to the complete absence of 17α-hydroxylase/17,20-lyase activity. The patient was treated with prednisolone and ethinyl estradiol. In addition, a summary of the recent literature regarding CAH is presented. PMID:27959413

  20. Mutant torsinA interacts with Tyrosine Hydroxylase in cultured cells

    PubMed Central

    O'Farrell, Casey A.; Martin, Kirstee; Hutton, Michael; Delatycki, Martin B.; Cookson, Mark R.; Lockhart, Paul J.

    2010-01-01

    A specific mutation (ΔE302/303) in the torsinA gene underlies most cases of dominantly inherited early-onset torsion dystonia. This mutation causes the protein to aggregate and form intracellular inclusion bodies in cultured cells and animal models. Co-expression of the wildtype and mutant proteins resulted in the redistribution of the wildtype protein from the endoplasmic reticulum to inclusion bodies in cultured HEK293 cells, and this was associated with increased interaction between the two proteins. Expression of ΔE302/303 but not wildtype torsinA in primary postnatal midbrain neurons resulted in the formation of intracellular inclusion bodies, predominantly in dopaminergic neurons. Tyrosine hydroxylase was sequestered in these inclusions and this process was mediated by increased protein-protein interaction between mutant torsinA and tyrosine hydroxylase. Analysis in an inducible neuroblastoma cell culture model demonstrated altered tyrosine hydroxylase activity in the presence of the mutant but not wildtype torsinA protein. Our results suggest that the interaction of tyrosine hydroxylase and mutant torsinA may contribute to the phenotype and reported dopaminergic dysfunction in torsinA-mediated dystonia. PMID:19761814

  1. Changes of human plasma dopamine-beta-hydroxylase activity after intravenous administration of theophylline.

    PubMed Central

    Aunis, D; Mandel, P; Miras-Portugal, M T; Coquillat, G; Rohmer, F; Warter, J M

    1975-01-01

    The intravenous administration of theophylline to ten healthy human subjects produced either an increase of circulating plasma dopamine-beta-hydroxylase or no change. The rise of plasma enzyme activity may reflect the increased peripheral catecholamine release induced by theophylline. PMID:1137731

  2. Tryptophan hydroxylase is modulated by L-type calcium channels in the rat pineal gland.

    PubMed

    Barbosa, Roseli; Scialfa, Julieta Helena; Terra, Ilza Mingarini; Cipolla-Neto, José; Simonneaux, Valérie; Afeche, Solange Castro

    2008-02-27

    Calcium is an important second messenger in the rat pineal gland, as well as cAMP. They both contribute to melatonin synthesis mediated by the three main enzymes of the melatonin synthesis pathway: tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. The cytosolic calcium is elevated in pinealocytes following alpha(1)-adrenergic stimulation, through IP(3)-and membrane calcium channels activation. Nifedipine, an L-type calcium channel blocker, reduces melatonin synthesis in rat pineal glands in vitro. With the purpose of investigating the mechanisms involved in melatonin synthesis regulation by the L-type calcium channel, we studied the effects of nifedipine on noradrenergic stimulated cultured rat pineal glands. Tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase activities were quantified by radiometric assays and 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin contents were quantified by HPLC with electrochemical detection. The data showed that calcium influx blockaded by nifedipine caused a decrease in tryptophan hydroxylase activity, but did not change either arylalkylamine N-acetyltransferase or hydroxyindole-O-methyltransferase activities. Moreover, there was a reduction of 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin intracellular content, as well as a reduction of serotonin and melatonin secretion. Thus, it seems that the calcium influx through L-type high voltage-activated calcium channels is essential for the full activation of tryptophan hydroxylase leading to melatonin synthesis in the pineal gland.

  3. Adrenal scan in 17-alpha-hydroxylase deficiency: false indication of adrenal adenoma

    SciTech Connect

    Shore, R.M.; Lieberman, L.M.; Newman, T.J.; Friedman, A.; Bargman, G.J.

    1981-07-01

    A patient who was thought to have testicular feminization syndrome and primary aldosteronism had an adrenal scan that suggested an adrenal adenoma. After later diagnosis of 17-alpha-hydroxylase deficiency, she was treated with glucocorticoids rather than surgery. Her clinical course and a repeat adrenal scan confirmed she did not have a tumor.

  4. Elicitor-induced prolyl hydroxylase from French bean (Phaseolus vulgaris). Localization, purification and properties.

    PubMed

    Bolwell, G P; Robbins, M P; Dixon, R A

    1985-08-01

    The enzyme prolyl hydroxylase (proline: 2-oxoglutarate dioxygenase, EC 1.14.11.12), induced in suspension-cultured cells of Phaseolus vulgaris L. (French bean) by treatment with an elicitor preparation from the phytopathogenic fungus Colletotrichum lindemuthianum, has been investigated. The enzyme, which catalyses the hydroxylation of poly-L-proline with the stoichiometric decarboxylation of 2-oxoglutarate, has been shown to be localized mainly in smooth endoplasmic reticulum. After solubilization from microsomal membranes, the hydroxylase was purified by ion-exchange chromatography and affinity chromatography on poly-L-proline-Sepharose 4B. The subunit Mr, as assessed by sodium dodecyl sulphate/poly-acrylamide-gel electrophoresis, was 65 000, the subunit apparently being recovered as a doublet: the subunits associate under non-denaturing conditions to give at least a tetramer. The bean hydroxylase has kinetic properties and cofactor requirements similar to those previously reported for the enzyme from other plants. Elicitor treatment of suspension-cultured bean cells leads to a rapid induction of prolyl hydroxylase activity concomitant with induction of a protein: arabinosyl-transferase and increased levels of an arabinosylated hydroxyproline-rich protein.

  5. An Unusual Case: The Comorbidity of Mood Disorder and 17-α-Hydroxylase Deficiency.

    PubMed

    Tunç, Serhat; Yiğiter, Sera; Altinbaş, Kürşat; Kurt, Erhan; Oral, Timuçin

    2013-09-01

    17-α-hydroxylase enzyme has a crucial role in the steroid biosynthesis and, deficiency of this enzyme is an autosomal recessive monogenic disorder which is one of the two hypertensive form of congenital adrenal hyperplasia. It is characterized with the deficiency in glucocorticoid, adrenal androgen, and sex steroid synthesis with concomitant mineralocorticoid excess due to genetic defect in steroid biosynthesis. The relationship of hormone system physiology with psychiatric signs and syndromes are complex. Any problem in the hypothalamo-pituitary axis may cause psychiatric syndromes. On the other hand, many psychiatric disorders, such as mood-anxiety symptoms, depression, mania, psychosis, and delirium can be seen secondary to the treatment of hormone deficiency. We present the case of a male patient with pseudohermaphroditism who has been followed and treated in Raşit Tahsin Mood Clinic with the diagnosis of mood disorder not otherwise specified and was diagnosed with 46, XY karyotype and 17-α-hydroxylase deficiency after referring to a hospital with delayed puberty. Considering the medical literature, 17-α-hydroxylase deficiency has been evaluated from the aspects of gender-related behavioral disorders, psychological developmental and anxiety disorders. To the best of our knowledge, in the medical literature, this is the first case of 17-α-hydroxylase deficiency associated with mood disorder. Here, the relationship between mood disorders and hypothalamo-pituitary axis is discussed in the light of the literature.

  6. An Unusual Case: The Comorbidity of Mood Disorder and 17-α-Hydroxylase Deficiency

    PubMed Central

    TUNÇ, Serhat; YİĞİTER, Sera; ALTINBAŞ, Kürşat; KURT, Erhan; ORAL, Timuçin

    2013-01-01

    17-α-hydroxylase enzyme has a crucial role in the steroid biosynthesis and, deficiency of this enzyme is an autosomal recessive monogenic disorder which is one of the two hypertensive form of congenital adrenal hyperplasia. It is characterized with the deficiency in glucocorticoid, adrenal androgen, and sex steroid synthesis with concomitant mineralocorticoid excess due to genetic defect in steroid biosynthesis. The relationship of hormone system physiology with psychiatric signs and syndromes are complex. Any problem in the hypothalamo-pituitary axis may cause psychiatric syndromes. On the other hand, many psychiatric disorders, such as mood-anxiety symptoms, depression, mania, psychosis, and delirium can be seen secondary to the treatment of hormone deficiency. We present the case of a male patient with pseudohermaphroditism who has been followed and treated in Raşit Tahsin Mood Clinic with the diagnosis of mood disorder not otherwise specified and was diagnosed with 46, XY karyotype and 17-α-hydroxylase deficiency after referring to a hospital with delayed puberty. Considering the medical literature, 17-α-hydroxylase deficiency has been evaluated from the aspects of gender-related behavioral disorders, psychological developmental and anxiety disorders. To the best of our knowledge, in the medical literature, this is the first case of 17-α-hydroxylase deficiency associated with mood disorder. Here, the relationship between mood disorders and hypothalamo-pituitary axis is discussed in the light of the literature

  7. Mechanism-based inactivation of benzo(a)pyrene hydroxylase by aryl acetylenes and aryl olefins

    SciTech Connect

    Gan, L.S.; Lu, J.Y.L.; Alworth, W.L.

    1986-05-01

    A series of aryl acetylenes and aryl olefins have been examined as substrates and inhibitors of cytochrome P-450 dependent monooxgenases in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene, 3-ethynylperylene, 2-ethynylfluorene, methyl 1-pyrenyl acetylene, cis- and trans-1-(2-bromovinyl)pyrene, and 1-allylpyrene serve as mechanism-based irreversible inactivators (suicide inhibitors) of benzo(a)pyrene hydroxylase, while 1-vinylpyrene and phenyl 1-pyrenyl acetylene do not cause a detectable suicide inhibition of benzo(a)pyrene hydroxylase. The mechanism-based loss of benzo(a)pyrene hydroxylase caused by the aryl acetylenes is not accompanied by a corresponding loss of the P-450 content of the microsomes (suicide destruction). The suicide inhibition by these aryl acetylenes therefore does not involve covalent binding to the heme moiety of the monooxygenase. Nevertheless, in the presence of NADPH, /sup 3/H-labeled 1-ethynylpyrene becomes covalently attached to the cytochrome P-450 protein; the measured stoichiometry of binding is one 1-ethynylpyrene per P-450 heme unit. The authors conclude that the inhibition of benzo(a)pyrene hydroxylase produced by 1-ethynylpyrene may be related to the mechanism of suicide inhibition of P-450 activity by chloramphenicol rather than the mechanism of suicide destruction of P-450 previously described for acetylene and propyne.

  8. Molecular characterization of ferulate 5-hydroxylase gene from kenaf (Hibiscus cannabinus L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The purpose of this research was to clone and characterize the expression pattern of a kenaf (Hibiscus cannabinus L.) F5H gene that encodes ferulate 5-hydroxylase in the phenylpropanoid pathway. Kenaf is well known as a fast growing dicotyledonous plant, which makes it a valuable biomass plant. The ...

  9. Expression analysis of kenaf cinnamate 4-hydroxylase (C4H) ortholog during developmental and stress responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was conducted to clone and analyze the expression pattern of a C4H gene encoding cinnamate 4-hydroxylase from kenaf (Hibiscus cannabinus L.). A full-length C4H ortholog was cloned using degenerate primers and the RACE (rapid amplification of cDNA ends) method. The full-length C4H ortholog...

  10. Gallate, the component of HIF-inducing catechins, inhibits HIF prolyl hydroxylase

    SciTech Connect

    Tsukiyama, Fuyo; Nakai, Yumi; Yoshida, Masataka; Tokuhara, Takahiro; Hirota, Kiichi; Sakai, Akiko; Hayashi, Hideyuki . E-mail: hayashi@art.osaka-med.ac.jp; Katsumata, Takahiro

    2006-12-08

    Catechins have recently been reported to increase the cellular content of the hypoxia-inducible factor (HIF)-1{alpha} within mammalian cells. These catechins have a gallate moiety as a common structure. We now report that n-propyl gallate (nPG) also increases the HIF-1{alpha} protein in the rat heart-derived H9c2 cells. The increase was dose-dependent and reached a maximum at 2-4 h after the addition of nPG to the cells. nPG did not change the HIF-1{alpha} mRNA level, showing that the increase is a posttranscriptional event. Although nPG did not inhibit the HIF prolyl hydroxylase, gallate, the hydrolysis product of nPG, inhibited the enzyme completely at submillimolar concentrations. Model building studies on the human HIF prolyl hydroxylase 2 showed that the two phenolate oxygen atoms of gallate form a chelate with the active site Fe{sup 2+}, while the carboxyl group of gallate forms a strong ionic/hydrogen bonding interaction with Arg383, explaining why nPG, which has an esterified carboxyl group, is unable to inhibit the hydroxylase. Together with the observation that gallate was detected in the H9c2 cells treated with nPG, these results suggest that nPG incorporated into the cells is hydrolyzed and the released gallate inhibits the HIF prolyl hydroxylase, thereby reducing the HIF degradation rate and increasing the HIF-1{alpha} content.

  11. Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don.

    PubMed

    Vazquez-Flota, F; De Carolis, E; Alarco, A M; De Luca, V

    1997-08-01

    A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the 4-hydroxylation of desacetoxyvindoline was purified to homogeneity. Three oligopeptides isolated from a tryptic digest of the purified protein were microsequenced and one oligopeptide showed significant homology to hyoscyamine 6 beta-hydroxylase from Hyoscyamus niger. A 36-mer degenerate oligonucleotide based on this peptide sequence was used to screen a Catharanthus roseus cDNA library and three clones, cD4H-1 to -3, were isolated. Although none of the three clones were full-length, the open reading frame on each clone encoded a putative protein containing the sequence of all three peptides. Primer extension analysis suggested that cD4H-3, the longest cDNA clone, was missing 156 bp at the 5' end of the clone and sequencing of the genomic clone, gD4H-8, confirmed these results. Southern blot analysis suggested that d4h is present as a single-copy gene in C. roseus which is a diploid plant, and the significant differences in the sequence of the 3'-UTR between cD4H-1 and -3 suggest that they represent dimorphic alleles of the same hydroxylase. The identity of the clone was further confirmed when extracts of transformed Escherichia coli expressed D4H enzyme activity. The D4H clone encoded a putative protein of 401 amino acids with a calculated molecular mass of 45.5 kDa and the amino acid sequence showed a high degree of similarity with those of a growing family of 2-oxoglutarate-dependent dioxygenases of plant and fungal origin. The similarity was not restricted to the dioxygenase protein sequences but was also extended to the gene structure and organization since the 205 and 1720 bp introns of d4h were inserted around the same highly conserved amino acid consensus sequences as those for e8 protein, hyoscyamine-6 beta-hydroxylase and ethylene-forming enzyme. These results provide further support that a common ancestral gene is responsible for the appearance of this family of dioxygenases

  12. Equine cytochrome P450 aromatase exhibits an estrogen 2-hydroxylase activity in vitro.

    PubMed

    Almadhidi, J; Moslemi, S; Drosdowsky, M A; Séralini, G E

    1996-09-01

    Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water release from labelled estradiol and testosterone. Kinetic analysis of estradiol 2-hydroxylation showed an apparent K(m) of 23 microM and a V(max) of 18 nmol/min/mg, whereas the values for testosterone aromatization were a K(m) of 15.7 nM and a V(max) of 34.6 pmol/min/mg. A specific antiserum raised against purified testicular equine P450arom and known to inhibit aromatase activity [1] was also found to inhibit the estrogen hydroxylase activity of equine placental microsomes in a dose-dependent manner with an IC50 value of 15 microl serum: 0.5 ml incubate. The estrogen hydroxylase activity was inhibited in a dose-dependent manner by two classes of aromatase inhibitors, i.e. steroidal-- (4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione)--and non-steroidal--(fadrozole and miconazole). The IC50 values were approximately 300 and 890 nM for 4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione, and 92 and 285 nM, for fadrozole and miconazole, respectively. Furthermore, 4-hydroxyandrostenedione caused a time-dependent inactivation of estrogen hydroxylase activity. We conclude that equine aromatase is able to use estradiol as a substrate, and converts it to catechol estradiol in vitro, possibly using the active site of aromatization. This is the first demonstration that equine aromatase functions as an estrogen 2-hydroxylase, in addition to transforming androgens into estrogen.

  13. Induction and characterization of a cytochrome P-450-dependent camphor hydroxylase in tissue cultures of common sage (Salvia officinalis)

    SciTech Connect

    Funk, C.; Croteau, R. )

    1993-04-01

    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O[sub 2]-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl[sub 2], camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn[sup 2+]-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. 44 refs., 6 figs., 2 tabs.

  14. Induction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue Cultures of Common Sage (Salvia officinalis).

    PubMed Central

    Funk, C.; Croteau, R.

    1993-01-01

    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O2-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl2, camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn2+-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. PMID:12231778

  15. Genetic polymorphism of cholesterol 7alpha-hydroxylase (CYP7A1) and colorectal adenomas: Self Defense Forces Health Study.

    PubMed

    Tabata, Shinji; Yin, Guang; Ogawa, Shinsaku; Yamaguchi, Keizo; Mineshita, Masamichi; Kono, Suminori

    2006-05-01

    Bile acids have long been implicated in colorectal carcinogenesis, but epidemiological evidence is limited. Cholesterol 7alpha-hydroxylase (CYP7A1) is the rate-limiting enzyme producing bile acids from cholesterol. A recent case-control study showed a decreased risk of proximal colon cancer associated with the CC genotype of the CYP7A1 A-203C polymorphism. The present study examined the relationship between the CYP7A1 A-203C polymorphism and colorectal adenoma, which is a well-established precursor lesion of colorectal cancer. The study subjects comprised 446 cases of colorectal adenomas and 914 controls of normal total colonoscopy among men receiving a preretirement health examination at two hospitals of the Self Defense Forces (SDF). The CYP7A1 genotype was determined by the polymerase chain reaction-restriction fragment length polymorphism method. Statistical adjustment was made for age, hospital, rank in the SDF, smoking, alcohol use, body mass index, physical activity and parental history of colorectal cancer. The CYP7A1 polymorphism was not measurably related to the overall risk of colorectal adenomas. However, the CC genotype was associated with a decreased risk of proximal colon adenomas, but not of distal colon and rectal adenomas. Adjusted odds ratios of proximal colon adenomas (95% confidence intervals) for the AC and CC genotype versus AA genotype were 0.82 (0.54-1.24) and 0.56 (0.34-0.95), respectively. The findings add to evidence for the role of bile acids in colorectal carcinogenesis. The CC genotype of the CYP7A1 A-203C polymorphism probably renders lower activity of the enzyme synthesizing bile acids.

  16. Brain catecholamine depletion and motor impairment in a Th knock-in mouse with type B tyrosine hydroxylase deficiency.

    PubMed

    Korner, Germaine; Noain, Daniela; Ying, Ming; Hole, Magnus; Flydal, Marte I; Scherer, Tanja; Allegri, Gabriella; Rassi, Anahita; Fingerhut, Ralph; Becu-Villalobos, Damasia; Pillai, Samyuktha; Wueest, Stephan; Konrad, Daniel; Lauber-Biason, Anna; Baumann, Christian R; Bindoff, Laurence A; Martinez, Aurora; Thöny, Beat

    2015-10-01

    Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate-limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum.

  17. Evidence for a role of sterol 27-hydroxylase in glucocorticoid metabolism in vivo.

    PubMed

    Vögeli, Isabelle; Jung, Hans H; Dick, Bernhard; Erickson, Sandra K; Escher, Robert; Funder, John W; Frey, Felix J; Escher, Geneviève

    2013-11-01

    The intracellular availability of glucocorticoids is regulated by the enzymes 11β-hydroxysteroid dehydrogenase 1 (HSD11B1) and 11β-hydroxysteroid dehydrogenase 2 (HSD11B2). The activity of HSD11B1 is measured in the urine based on the (tetrahydrocortisol+5α-tetrahydrocortisol)/tetrahydrocortisone ((THF+5α-THF)/THE) ratio in humans and the (tetrahydrocorticosterone+5α-tetrahydrocorticosterone)/tetrahydrodehydrocorticosterone ((THB+5α-THB)/THA) ratio in mice. The cortisol/cortisone (F/E) ratio in humans and the corticosterone/11-dehydrocorticosterone (B/A) ratio in mice are markers of the activity of HSD11B2. In vitro agonist treatment of liver X receptor (LXR) down-regulates the activity of HSD11B1. Sterol 27-hydroxylase (CYP27A1) catalyses the first step in the alternative pathway of bile acid synthesis by hydroxylating cholesterol to 27-hydroxycholesterol (27-OHC). Since 27-OHC is a natural ligand for LXR, we hypothesised that CYP27A1 deficiency may up-regulate the activity of HSD11B1. In a patient with cerebrotendinous xanthomatosis carrying a loss-of-function mutation in CYP27A1, the plasma concentrations of 27-OHC were dramatically reduced (3.8 vs 90-140 ng/ml in healthy controls) and the urinary ratios of (THF+5α-THF)/THE and F/E were increased, demonstrating enhanced HSD11B1 and diminished HSD11B2 activities. Similarly, in Cyp27a1 knockout (KO) mice, the plasma concentrations of 27-OHC were undetectable (<1 vs 25-120 ng/ml in Cyp27a1 WT mice). The urinary ratio of (THB+5α-THB)/THA was fourfold and that of B/A was twofold higher in KO mice than in their WT littermates. The (THB+5α-THB)/THA ratio was also significantly increased in the plasma, liver and kidney of KO mice. In the liver of these mice, the increase in the concentrations of active glucocorticoids was due to increased liver weight as a consequence of Cyp27a1 deficiency. In vitro, 27-OHC acts as an inhibitor of the activity of HSD11B1. Our studies suggest that the expression of CYP27A1

  18. Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco

    NASA Technical Reports Server (NTRS)

    Blee, K.; Choi, J. W.; O'Connell, A. P.; Jupe, S. C.; Schuch, W.; Lewis, N. G.; Bolwell, G. P.

    2001-01-01

    A number of plant species contain the class II of genes encoding the cytochrome P450, CYP73, the cognate protein of which cinnamic acid 4-hydroxylase, is the second enzyme of the phenylpropanoid pathway. In order to begin to determine possible functionality, tobacco has been transformed with a truncated French bean class II cinnamate hydroxylase (CYP73A15) in the sense and antisense orientations. Signals for C4H protein could be detected in vascular tissue from wild-type plants using heterologous probes. The transformed plants showed a normal phenotype, even though detectable C4H protein was much reduced in tissue prints. Young propagated transformants displayed a range of reduced C4H activities, as well as either reduced or no phloroglucinol-stainable lignin. However, all mature tobacco plants showed the accumulation of lignin, even though its deposition was apparently delayed. This was not due to induction of tyrosine ammonia-lyase activity, which was not detected, but instead it is presumed due to sufficient C4H residual activity. Analysis of the lignin content of the plants showed reductions of up to 30% with a slightly reduced syringyl to guaiacyl ratio as compared to wild type. This reduction level was favourable in comparison with some other targets in the lignification pathway that have been manipulated including that of class I cinnamate 4-hydroxylase. It is proposed that the class II cinnamate 4-hydroxylase might also function in lignification in a number of species including French bean and tobacco, based on these data.

  19. X-ray structure of a hydroxylase-regulatory protein complex from a hydrocarbon-oxidizing multicomponent monooxygenase, Pseudomonas sp. OX1 phenol hydroxylase.

    PubMed

    Sazinsky, Matthew H; Dunten, Pete W; McCormick, Michael S; DiDonato, Alberto; Lippard, Stephen J

    2006-12-26

    Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 A resolution. PHM binds in a canyon on one side of the (alphabetagamma)2 PHH dimer, contacting alpha-subunit helices A, E, and F approximately 12 A above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more pi-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an approximately 6 A pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O2 activation, suggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH alpha-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket

  20. Splice, insertion-deletion and nonsense mutations that perturb the phenylalanine hydroxylase transcript cause phenylketonuria in India.

    PubMed

    Bashyam, Murali D; Chaudhary, Ajay K; Kiran, Manjari; Nagarajaram, Hampapathalu A; Devi, Radha Rama; Ranganath, Prajnya; Dalal, Ashwin; Bashyam, Leena; Gupta, Neerja; Kabra, Madhulika; Muranjan, Mamta; Puri, Ratna D; Verma, Ishwar C; Nampoothiri, Sheela; Kadandale, Jayarama S

    2014-03-01

    Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutational inactivation of the phenylalanine hydroxylase (PAH) gene. Missense mutations are the most common PAH mutation type detected in PKU patients worldwide. We performed PAH mutation analysis in 27 suspected Indian PKU families (including 7 from our previous study) followed by structure and function analysis of specific missense and splice/insertion-deletion/nonsense mutations, respectively. Of the 27 families, disease-causing mutations were detected in 25. A total of 20 different mutations were identified of which 7 "unique" mutations accounted for 13 of 25 mutation positive families. The unique mutations detected exclusively in Indian PKU patients included three recurrent mutations detected in three families each. The 20 mutations included only 5 missense mutations in addition to 5 splice, 4 each nonsense and insertion-deletion mutations, a silent variant in coding region and a 3'UTR mutation. One deletion and two nonsense mutations were characterized to confirm significant reduction in mutant transcript levels possibly through activation of nonsense mediated decay. All missense mutations affected conserved amino acid residues and sequence and structure analysis suggested significant perturbations in the enzyme activity of respective mutant proteins. This is probably the first report of identification of a significantly low proportion of missense PAH mutations from PKU families and together with the presence of a high proportion of splice, insertion-deletion, and nonsense mutations, points to a unique PAH mutation profile in Indian PKU patients.

  1. Cellular oxygen sensing: Importins and exportins are mediators of intracellular localisation of prolyl-4-hydroxylases PHD1 and PHD2

    SciTech Connect

    Steinhoff, Amrei; Pientka, Friederike Katharina; Moeckel, Sylvia; Kettelhake, Antje; Hartmann, Enno; Koehler, Matthias; Depping, Reinhard

    2009-10-02

    Hypoxia-inducible factors are crucial in the regulatory process of oxygen homeostasis of vertebrate cells. Inhibition of prolyl hydroxylation of HIF-{alpha} subunits by prolyl-hydroxylases (PHD1, PHD2 and PHD3) leads to transcription of a greater number of hypoxia responsive genes. We have investigated the subcellular distribution and the molecular mechanisms regulating the intracellular allocation of PHD1 and PHD2. As reported earlier we find PHD1 located exclusively in the nucleus. We demonstrate that nuclear import of PHD1 occurs importin {alpha}/{beta} dependently and relies on a nuclear localisation signal (NLS). By contrast PHD2 is cycling between nucleus and cytoplasm, and nuclear import seems to be independent of 'classical' importin {alpha}/{beta} receptors. Furthermore, we reveal that the exit of PHD2 from the nucleus requires CRM1 and the N-terminal 100 amino acids of the protein. Our findings provide new insights into the mechanisms of the regulation of the oxygen sensor cascade of PHDs in different cellular compartments.

  2. Role of cholesterol 7alpha-hydroxylase (CYP7A1) in nutrigenetics and pharmacogenetics of cholesterol lowering.

    PubMed

    Hubacek, Jaroslav A; Bobkova, Dagmar

    2006-01-01

    The relationship between dietary composition/cholesterol-lowering therapy and final plasma lipid levels is to some extent genetically determined. It is clear that these responses are under polygenic control, with multiple variants in many genes participating in the total effect (and with each gene contributing a relatively small effect). Using different experimental approaches, several candidate genes have been analyzed to date.Interesting and consistent results have been published recently regarding the A-204C promoter variant in the cholesterol 7alpha-hydroxylase (CYP7A1) gene. CYP7A1 is a rate-limiting enzyme in bile acid synthesis and therefore plays an important role in maintaining cholesterol homeostasis. CYP7A1-204CC homozygotes have the greatest decrease in total cholesterol level in response to dietary changes in different types of dietary intervention studies. In contrast, one study has reported that the effect of statins in lowering low-density lipoprotein (LDL)-cholesterol levels was slightly greater in -204AA homozygotes. The CYP7A1 A-204C variant accounts for a significant proportion of the genetic predisposition of the response of plasma cholesterol levels.

  3. Biotransformation of benzene and toluene to catechols by phenol hydroxylase from Arthrobacter sp. W1.

    PubMed

    Ma, Fang; Shi, Sheng-Nan; Sun, Tie-Heng; Li, Ang; Zhou, Ji-Ti; Qu, Yuan-Yuan

    2013-06-01

    Phenol hydroxylase gene engineered microorganism (PHIND) was used to synthesize catechols from benzene and toluene by successive hydroxylation reaction. HPLC-MS and (1)H NMR analysis proved that the products of biotransformation were the corresponding catechols via the intermediate production of phenols. It was indicated that the main products of toluene oxidation were o-cresol and p-cresol. 3-Methylcatechol was the predominant product for m-cresol biotransformation. Formation rate of catechol (25 μM/min/g cell dry weight) was 1.43-fold higher than that of methylcatechols. It was suggested that phenol hydroxylase could be successfully used to transform both benzene and toluene to catechols by successive hydroxylation.

  4. A tyrosine hydroxylase assay in microwells using coupled nonenzymatic decarboxylation of dopa

    SciTech Connect

    Bostwick, J.R.; Le, W.D. )

    1991-01-01

    A radiometric assay for tyrosine hydroxylase employing a coupled nonenzymatic decarboxylation of L-{sup 14}CDopa formed from L-{sup 14}Ctyrosine has been adapted for performance in a 96 microwell culture plate. The method uses an easily manufactured plate holder to compress blotting paper impregnated with methylbenzethonium hydroxide against the top rim of each well. This forms isolated, airtight compartments in which 14CO2 is evolved and quantitatively absorbed into the blotting paper. The method is sensitive enough to detect the production of less than 5 pmol of 14CO2. A major advantage of this system is that cells can be grown in tissue culture and subsequently assayed for tyrosine hydroxylase activity in the same well. The method is more facile than previously devised procedures, allowing for the simultaneous assay of up to 96 samples totally contained in a single, compact, portable unit.

  5. Genetic analysis of two Japanese patients with non-classical 21-hydroxylase deficiency.

    PubMed

    Imamine, Rui; Arima, Hiroshi; Kusakabe, Miho; Umeda, Hiroshi; Sato, Ikuko; Homma, Keiko; Usui, Takeshi; Oiso, Yutaka

    2009-01-01

    We report two Japanese women with androgen excess symptoms. Analyses of 21-hydroxylase gene demonstrated that a 24-year-old Japanese woman had a homozygous mutation of IVS2-13 A/C>G, while a 25-year-old Japanese woman had a compound heterozygous mutation of I172N and E245del1nt, a novel mutation which would result in completely nonfunctional enzyme due to a frame shift. As IVS2-13 A/C>G and I172N have been classified as mutations leading to severe impairment in enzyme activity, this study not only clarified a novel mutation causing 21-hydroxylase deficiency, but also demonstrated that genotype and phenotype do not correlate well in these cases.

  6. Tyrosine hydroxylase immunoreactive neurons in the forebrain of the trout: organization, cellular features and innervation.

    PubMed

    Anadón, Ramón; Rodríguez-Moldes, Isabel; González, Agustín

    We studied the segmental distribution and cellular features of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the forebrain of trout. Large differences in cell size, general morphology, and complexity of cell processes were observed between TH-ir nuclei of different regions, and a new type of complex spiny TH-ir neurons in the ventral telencephalon is described for the first time. The distribution of TH-ir fibers was also analyzed and discussed.

  7. Effect of halogenated benzenes on acetanilide esterase, acetanilide hydroxylase and procaine esterase in rats.

    PubMed

    Carlson, G P; Dziezak, J D; Johnson, K M

    1979-07-01

    1,2,4-Trichlorobenzene, 1,3,5-trichlorobenzene, hexachlorobenzene, 1,2,4-tribromobenzene, 1,3,5-tribromobenzene and hexabromobenzene were compared for their abilities to induce acetanilide esterase, acentailide hydroxylase and procaine esterase. Except for hexabromobenzene all induced acetanilide esterase whereas the hydroxylation of acetanilide was seen only with the fully halogenated benzenes and with 1,3,5-tribromobenzene. Hepatic procaine esterase activity was increased by the three chlorinated benzenes and 1,2,4-tribromobenzene.

  8. Genes encoding p-coumarate 3-hydroxylase (C3H) and methods of use

    DOEpatents

    Chapple, Clinton C. S.; Franke, Rochus; Ruegger, Max O.

    2006-07-04

    The present invention is directed to a method for altering secondary metabolism in plants, specifically phenylpropanoid metabolism. The present invention is further directed to a mutant p-coumarate 3-hydroxylase gene, referred to herein as the ref8 gene, its protein product which can be used to prepare gene constructs and transgenic plants. The gene constructs and transgenic plants are further aspects of the present invention.

  9. Sexual dimorphism of the dopamine-beta-hydroxylase-immunoreactive neurons in the rat locus ceruleus.

    PubMed

    Luque, J M; de Blas, M R; Segovia, S; Guillamón, A

    1992-06-19

    Sex differences in the noradrenaline synthesizing neurons of the locus ceruleus (LC) in rat brain were investigated immunocytochemically using an antibody to dopamine-beta-hydroxylase. Female adult rats contained a greater structural volume and average somatic area in the anterior intermediate region of the nucleus compared with males. Whether this difference is related to the endocrine status of the animals, and consequently a functionally distinct population of neurons, is yet to be determined.

  10. Induction of steroidal hydroxylase activity by plant defence compounds in the filamentous fungus Cochliobolus lunatus

    PubMed

    Vitas; Smith; Plavec; Kesselmeier; Pajic; Ferlan; Zigon; Kelly; Komel

    1999-02-01

    We investigated the hypothesis that the endogenous role of the commercially important inducible steroid hydroxylase cytochrome P450s of fungi was in defense against plant toxophores/secondary metabolites. Two plant defense compounds, the aglycones tomatidine and solanidine, the steroidal glycoalkaloid alpha-tomatine and the triterpene saponin beta-escin were tested as inducers of 11beta/14alpha-steroid hydroxylase in the filamentous fungus Cochliobolus lunatus. The extracts of saponins from the roots of Primula veris and green oat leaves were also tested as inducers of 11beta/14alpha-hydroxylation activity in progesterone biotransformation with the same fungus. Induction of steroid hydroxylase and inhibition of activity in some cases support our hypothesis that their endogenous function is in biochemical defence against secondary metabolites. 4-Pregnene-3,11,20-trione was added as a substrate for biotransformation with C. lunatus. We isolated from culture broth 14alpha-hydroxy-4-pregnene-3,11,20-trione, and the hitherto unreported compounds, 7alpha,14alpha-dihydroxy-4-pregnene-3,11,20-trione and 7alpha-hydroxy-pregna-4,8(14)-diene-3,11,20-trione.

  11. Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei B-5.

    PubMed

    Liu, Chenli; Wang, Wanpeng; Wu, Yehui; Zhou, Zhongwen; Lai, Qiliang; Shao, Zongze

    2011-05-01

    Alcanivorax dieselolei strain B-5 is a marine bacterium that can utilize a broad range of n-alkanes (C(5) -C(36) ) as sole carbon source. However, the mechanisms responsible for this trait remain to be established. Here we report on the characterization of four alkane hydroxylases from A. dieselolei, including two homologues of AlkB (AlkB1 and AlkB2), a CYP153 homologue (P450), as well as an AlmA-like (AlmA) alkane hydroxylase. Heterologous expression of alkB1, alkB2, p450 and almA in Pseudomonas putida GPo12 (pGEc47ΔB) or P. fluorescens KOB2Δ1 verified their functions in alkane oxidation. Quantitative real-time RT-PCR analysis showed that these genes could be induced by alkanes ranging from C(8) to C(36) . Notably, the expression of the p450 and almA genes was only upregulated in the presence of medium-chain (C(8) -C(16) ) or long-chain (C(22) -C(36) ) n-alkanes, respectively; while alkB1 and alkB2 responded to both medium- and long-chain n-alkanes (C(12) -C(26) ). Moreover, branched alkanes (pristane and phytane) significantly elevated alkB1 and almA expression levels. Our findings demonstrate that the multiple alkane hydroxylase systems ensure the utilization of substrates of a broad chain length range.

  12. Anti-dopamine beta-hydroxylase immunotoxin-induced sympathectomy in adult rats

    NASA Technical Reports Server (NTRS)

    Picklo, M. J.; Wiley, R. G.; Lonce, S.; Lappi, D. A.; Robertson, D.

    1995-01-01

    Anti-dopamine beta-hydroxylase immunotoxin (DHIT) is an antibody-targeted noradrenergic lesioning tool comprised of a monoclonal antibody against the noradrenergic enzyme, dopamine beta-hydroxylase, conjugated to saporin, a ribosome-inactivating protein. Noradrenergic-neuron specificity and completeness and functionality of sympathectomy were assessed. Adult, male Sprague-Dawley rats were given 28.5, 85.7, 142 or 285 micrograms/kg DHIT i.v. Three days after injection, a 6% to 73% decrease in the neurons was found in the superior cervical ganglia of the animals. No loss of sensory, nodose and dorsal root ganglia, neurons was observed at the highest dose of DHIT. In contrast, the immunotoxin, 192-saporin (142 micrograms/kg), lesioned all three ganglia. To assess the sympathectomy, 2 wk after treatment (285 micrograms/kg), rats were anesthetized with urethane (1 g/kg) and cannulated in the femoral artery and vein. DHIT-treated animals' basal systolic blood pressure and heart rate were significantly lower than controls. Basal plasma norepinephrine levels were 41% lower in DHIT-treated animals than controls. Tyramine-stimulated release of norepinephrine in DHIT-treated rats was 27% of controls. Plasma epinephrine levels of DHIT animals were not reduced. DHIT-treated animals exhibited a 2-fold hypersensitivity to the alpha-adrenergic agonist phenylephrine. We conclude that DHIT selectively delivered saporin to noradrenergic neurons resulting in destruction of these neurons. Anti-dopamine beta-hydroxylase immunotoxin administration produces a rapid, irreversible sympathectomy.

  13. Who is a carrier? Detection of unsuspected mutations in 21-hydroxylase deficiency

    SciTech Connect

    Witchel, S.S.; Lee, P.A.; Trucco, M.

    1996-01-02

    Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a common autosomal-recessive disorder. During our routine genotyping of affected individuals and their relatives using allele-specific oligonucleotide hybridization and single-strand conformational polymorphism analysis, we identified two families each segregating three mutations. In both families, a mutation known to be associated with 21-hydroxylase deficiency was identified in healthy individuals but was not detected in the propositus. The propositus in family 1 was shown to be a homozygous carrier for G at nucleotide 655, which alters the splice acceptor site at exon 3. The propositus in family 2 carried the same splicing mutation on the maternal allele and a gene deletion/conversion on the paternal allele. In both families, other clinically unaffected relatives carried the Q318X mutation in exon 8. If molecular diagnostic studies had been limited to the mutation carried by the propositi, relatives would have been misinformed regarding their status as carriers or mildly affected individuals. The findings in these two families emphasize the high frequency of alleles causing 21-hydroxylase deficiency in the population. 29 refs., 3 figs., 2 tabs.

  14. Identification of multiple steroid hydroxylases in Daphnia magna and their modulation by xenobiotics

    SciTech Connect

    Baldwin, W.S.; LeBlanc, G.A. . Dept. of Toxicology)

    1994-07-01

    Steroid hydroxylase activities were characterized in Daphnia magna and evaluated for potential use as biomarkers of xenobiotic exposure. Microsomes prepared from Daphnia magna generated as single NADPH-dependent metabolite of [[sup 14]C] testosterone. However, intact daphnids excreted at least 10 polar metabolites of [[sup 14]C] testosterone into the test medium. Six of these metabolites were identified as 2[alpha]-, 16[beta]-, 6[beta]-, 6[alpha]-, 7[alpha]-, and 15[alpha]-[[sup 14]C]hydroxytestosterone. The unidentified metabolites are also presumed to be hydroxylated products of testosterone, based on their relative migrations during TLC. The inefficient metabolism of [[sup 14]C] testosterone during the in vitro microsomal incubations may have been due to the release of P450 inhibitors during microsome preparation. Exposure of daphnids to the P450 modulators phenobarbital, [beta]-naphthoflavone, piperonyl butoxide, and malathion differentially inhibited the steroid hydroxylase activities. Results from this study indicate that Daphnia magna expresses several P450 enzymes and that these enzymes are differentially modulated by xenobiotic exposure. Steroid hydroxylase activities may serve not only as a biomarker of toxicant exposure, but also as a predictor of toxicant effects involving perturbations of steroid hormone homeostasis.

  15. Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases

    PubMed Central

    Ji, Yurui; Mao, Guannan; Wang, Yingying; Bartlam, Mark

    2013-01-01

    Environmental microbes utilize four degradation pathways for the oxidation of n-alkanes. Although the enzymes degrading n-alkanes in different microbes may vary, enzymes functioning in the first step in the aerobic degradation of alkanes all belong to the alkane hydroxylases. Alkane hydroxylases are a class of enzymes that insert oxygen atoms derived from molecular oxygen into different sites of the alkane terminus (or termini) depending on the type of enzymes. In this review, we summarize the different types of alkane hydroxylases, their degrading steps, and compare typical enzymes from various classes with regard to their three-dimensional structures, in order to provide insights into how the enzymes mediate their different roles in the degradation of n-alkanes and what determines their different substrate ranges. Through the above analyzes, the degrading mechanisms of enzymes can be elucidated and molecular biological methods can be utilized to expand their catalytic roles in the petrochemical industry or in bioremediation of oil-contaminated environments. PMID:23519435

  16. Neonatal developmental pattern of superoxide dismutase and aniline hydroxylase in rat lung

    SciTech Connect

    Kakkar, P.; Jaffery, F.N.; Viswanathan, P.N.

    1986-10-01

    The developmental biology of superoxide dismutase and aniline hydroxylase was followed in rat lungs from prenatal stage to 3 months old. Total superoxide dismutase activity as determined by spectrophotometry as well as electrophoresis was high in the prenatal rat lung, decreased in the first 24 hr postpartum, increased within 7 days, and then decreased gradually to adult levels. On polyacrylamide gel electrophoresis only two isozymic forms of superoxide dismutase were located as achromatic zones in the fetal lung. In the adult rat lung, there were three molecular forms of superoxide dismutase, two in the postmitochondrial supernatant and one in the mitochondrial fraction. Unlike superoxide dismutase, aniline hydroxylase was detectable only after 5 days of age and the activity exhibited a gradual increase afterward up to 1 month of age. The developmental pattern of superoxide dismutase and aniline hydroxylase activities in lung may be significant in understanding the mechanism of body defenses and their regulatory modulations in response to toxic air pollutants and environmental stress.

  17. Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7 alpha-hydroxylase gene transcription.

    PubMed

    Li, Tiangang; Chiang, John Y L

    2005-01-01

    Bile acids, steroids, and drugs activate steroid and xenobiotic receptor pregnane X receptor (PXR; NR1I2), which induces human cytochrome P4503A4 (CYP3A4) in drug metabolism and cholesterol 7 alpha-hydroxylase (CYP7A1) in bile acid synthesis in the liver. Rifampicin, a human PXR agonist, inhibits bile acid synthesis and has been used to treat cholestatic diseases. The objective of this study is to elucidate the mechanism by which PXR inhibits CYP7A1 gene transcription. The mRNA expression levels of CYP7A1 and several nuclear receptors known to regulate the CYP7A1 gene were assayed in human primary hepatocytes by quantitative real-time PCR (Q-PCR). Rifampicin reduced CYP7A1 and small heterodimer partner (SHP; NR02B) mRNA expression suggesting that SHP was not involved in PXR inhibition of CYP7A1. Rifampicin inhibited CYP7A1 reporter activity and a PXR binding site was localized to the bile acid response element-I. Mammalian two-hybrid assays revealed that PXR interacted with hepatic nuclear factor 4 alpha (HNF4 alpha, NR2A1) and rifampicin was required. Coimmunoprecipitation assay confirmed PXR interaction with HNF4 alpha. PXR also interacted with peroxisome proliferator-activated receptor gamma coactivator (PGC-1 alpha), which interacted with HNF4 alpha and induced CYP7A1 gene transcription. Rifampicin enhanced PXR interaction with HNF4 alpha and reduced PGC-1 alpha interaction with HNF4 alpha. Chromatin immunoprecipitation assay showed that PXR, HNF4 alpha, and PGC-1 alpha bound to CYP7A1 chromatin, and rifampicin dissociated PGC-1 alpha from chromatin. These results suggest that activation of PXR by rifampicin promotes PXR interaction with HNF4 alpha and blocks PGC-1 alpha activation with HNF4 alpha and results in inhibition of CYP7A1 gene transcription. Rifampicin inhibition of bile acid synthesis may be a protective mechanism against drug and bile acid-induced cholestasis.

  18. Cloning and characterization of tyrosine hydroxylase (TH) from the pacific white leg shrimp Litopenaeus vannamei, and its expression following pathogen challenge and hypothermal stress.

    PubMed

    Mapanao, Ratchaneegorn; Cheng, Winton

    2016-09-01

    Tyrosine hydroxylase (TH) belongs to the biopterin-dependent aromatic amino acid hydroxylase enzyme family, and it represents the first and rate-limiting step in the synthesis of catecholamines that are required for physiological and immune process in invertebrates and vertebrates. Cloned Litopenaeus vannamei TH (LvTH), containing a short alpha helix domain, a catalytic core, a regulatory domain, a phosphorylation site and two potential N-linked glycosylation sites as presented in vertebrate and insect THs without acidic region and signal peptide cleavage sites at the amino-terminal, exhibited a similarity of 60.0-61.2% and 45.0-47.0% to that of invertebrate and vertebrate THs, respectively. Further, LvTH expression was abundant in gill and haemocytes determined by quantitative real-time PCR. L. vannamei challenged with Vibrio alginolyticus at 10(5) cfu shrimp(-1) revealed significant increase of LvTH mRNA expression in haemocytes within 30-120 min and in brain within 15-30 min followed with recuperation. In addition, shrimps exposed to hypothermal stress at 18 °C significantly increased LvTH expression in haemocytes and brain within 30-60 and 15-60 min, respectively. The TH activity and haemolymph glucose level (haemocytes-free) significantly increased in pathogen challenged shrimp at 120 min and 60 min, and in hypothermal stressed shrimp at 30-60 and 30 min, respectively. These results affirm that stress response initiates in the brain while haemocytes display later response. Further, the significant elevation of TH activity in haemolymph is likely to confer by TH that released from haemocytes. In conclusion, the cloned LvTH in our current study is a neural TH enzyme appears to be involved in the physiological and immune responses of whiteleg shrimp, L. vannamei suffering stressful stimulation.

  19. Evolution of flavone synthase I from parsley flavanone 3beta-hydroxylase by site-directed mutagenesis.

    PubMed

    Gebhardt, Yvonne Helen; Witte, Simone; Steuber, Holger; Matern, Ulrich; Martens, Stefan

    2007-07-01

    Flavanone 3beta-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by site-directed mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the beta-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by syn-elimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction).

  20. Heterologous expression of the flavonoid 3',5'-hydroxylase gene of Vinca major alters flower color in transgenic Petunia hybrida.

    PubMed

    Mori, S; Kobayashi, H; Hoshi, Y; Kondo, M; Nakano, M

    2004-01-01

    Flavonoid 3',5'-hydroxylase (F3'5'H) is the key enzyme for the expression of blue or purple flower color. A full-length cDNA for the F3'5'H gene was cloned from petals of Vinca major, and its genomic clone, designated VmFH1 (accession number AB078781 in the GenBank/EMBL/DDBJ databases), was isolated from leaves by a PCR-based strategy. Nucleotide sequence analysis revealed that VmFH1 contains one intron and an open reading frame encoding a polypeptide of 506 amino acid residues. The deduced amino acid sequence shows between 51% and 83% identity with those of previously reported F3'5'H genes. Southern blot analysis showed that there are 3-4 copies of the F3'5'H gene in the genome of V. major. Transcripts of the F3'5'H gene were detected in young flower petals but not in leaves as revealed by RT-PCR analysis. When VmFH1 was expressed in transgenic Petunia hybrida under the control of the cauliflower mosaic virus 35S promoter, some transgenic plants showed drastic flower color alteration from red to deep red with deep purple sectors. These transgenic plants accumulated 3',5'-hydroxylated anthocyanins in their petals, which were never detected in non-transgenic plants by high-performance liquid chromatography analysis. These results indicate that VmFH1 isolated from V. major encodes F3'5'H and is active in a heterologous plant species.

  1. Noggin Along with a Self-Assembling Peptide Nanofiber Containing Long Motif of Laminin Induces Tyrosine Hydroxylase Gene Expression.

    PubMed

    Tavakol, Shima; Musavi, Sayed Mostafa Modaress; Tavakol, Behnaz; Hoveizi, Elham; Ai, Jafar; Rezayat, Seyed Mahdi

    2016-07-08

    Tyrosine hydroxylase (TH), a rate-limiting step in catecholamine synthesis in which its activity influences Alzheimer disease, Parkinson disease, and IQ of schizophrenia patients, has been studied for a long time. In the meantime, the present investigation assessed the effect of noggin and type of self-assembling nanofibers in TH gene over-expression by neuron-like cells derived from human endometrial-derived stromal cells (hEnSCs). Neuroblastoma cells and hEnSCs encapsulated into nanofibers including Matrigel, (RADA)4, laminin, and BMHP-1 motif bounded to (RADA)4 and their cell viability were studied for 48 h and 18 days in basal and neurogenic media, respectively, in noggin-rich media. Then, expression of neural genes and proteins has been investigated by immunocytochemistry (ICC) and real-time PCR methods, respectively. The results indicated that neuroblastoma cell and hEnSC viability is in good agreement with the level of Bcl2 and β-tubulin III gene expression; however, -BMHP-1 and -laminin nanofibers exhibited significantly higher cell viability eventually through Wnt/β-catenin signaling pathway as compared to others, respectively. The gene expression analysis of nanofibers showed that none of them induced gamma-aminobutyric acid (GABA) gene expression while glial fibrillary acidic protein (GFAP) gene just over-expressed in cells encapsulated into Matrigel with a low level of Bcl2 gene expression. However, the TH gene just had been over-expressed in cells encapsulated into -laminin nanofiber and 2D cell culture. In the absence of noggin with -laminin nanofibers, TH gene expression was suppressed. It might be concluded that although noggin through anti-BMP pathways resulted in GFAP decrement and TH gene increment, the type of scaffold that defined the final fate of cells and -laminin accompaniment might be useful for the recovery of Alzheimer and Parkinson disease patients.

  2. Molecular Cloning, Characterization, and Expression Analysis of a Prolyl 4-Hydroxylase from the Marine Sponge Chondrosia reniformis.

    PubMed

    Pozzolini, Marina; Scarfì, Sonia; Mussino, Francesca; Ferrando, Sara; Gallus, Lorenzo; Giovine, Marco

    2015-08-01

    Prolyl 4-hydroxylase (P4H) catalyzes the hydroxylation of proline residues in collagen. P4H has two functional subunits, α and β. Here, we report the cDNA cloning, characterization, and expression analysis of the α and β subunits of the P4H derived from the marine sponge Chondrosia reniformis. The amino acid sequence of the α subunit is 533 residues long with an M r of 59.14 kDa, while the β subunit counts 526 residues with an M r of 58.75 kDa. Phylogenetic analyses showed that αP4H and βP4H are more related to the mammalian sequences than to known invertebrate P4Hs. Western blot analysis of sponge lysate protein cross-linking revealed a band of 240 kDa corresponding to an α2β2 tetramer structure. This result suggests that P4H from marine sponges shares the same quaternary structure with vertebrate homologous enzymes. Gene expression analyses showed that αP4H transcript is higher in the choanosome than in the ectosome, while the study of factors affecting its expression in sponge fragmorphs revealed that soluble silicates had no effect on the αP4H levels, whereas ascorbic acid strongly upregulated the αP4H mRNA. Finally, treatment with two different tumor necrosis factor (TNF)-alpha inhibitors determined a significant downregulation of αP4H gene expression in fragmorphs demonstrating, for the first time in Porifera, a positive involvement of TNF in sponge matrix biosynthesis. The molecular characterization of P4H genes involved in collagen hydroxylation, including the mechanisms that regulate their expression, is a key step for future recombinant sponge collagen production and may be pivotal to understand pathological mechanisms related to extracellular matrix deposition in higher organisms.

  3. Assessment of the 21-hydroxylase deficiency and the adrenal functions in young females with Turner syndrome.

    PubMed

    Onder, Asan; Aycan, Zehra; Cetinkaya, Semra; Kendirci, Havva Nur Peltek; Bas, Veysel Nijat; Agladioglu, Sebahat Yilmaz

    2012-01-01

    There are few reports of an association between Turner syndrome (TS) and 21-hydroxylase deficiency. However, this association is more frequent in some populations. The aim of this study was to evaluate the incidence of 21-hydroxylase deficiency in patients with TS in our population. 21-hydroxylase deficiency was evaluated in 44 TS cases with 45X (n=20) and 24 mosaic cases. A standard dose adrenocorticotropic (ACTH) stimulation test (Synacthen, Novartis, Basel, Switzerland) was performed, and 17 hydroxyprogesterone (17OHP), dehydroepiandrosterone sulfate (DHEAS) and cortisol responses were evaluated. Patients with increased 17OHP responses in the stimulation test also underwent 21-hydroxylase gene analysis. The mean age was 14.6 +/- 4 (2.6-22.4); 37 patients were on growth hormone (GH) treatment. Nine patients were at prepubertal stage, whereas 35 were pubertal (24 on gonadal steroids and 11 spontaneously). Six patients were obese. Only one of our patients had a level of 7.5 ng/mL of 17OHP, and there was no mutation found in congenital adrenal hyperplasia (CAH) genetic analysis. In other cases, peak 17OHP levels were < or = 6 ng/mL. The mean peak 17OHP was 2.62 +/- 1.48 (1.19-7.5) ng/mL, the cortisol level was 37.6 +/- 8.43 (23.9-56.2) microg/dL and the DHEAS was 135.2+/- 87.3 (15-413) microg/dL. The increased mean basal and peak cortisol levels (20.5 +/- 10.2 and 37.6 +/- 8.4 microg/dL) were remarkable findings. Whereas basal cortisol was above 20 microg/dL in 38.7% of patients, exaggerated results up to 56.2 microg/dL were obtained in peak cortisol levels. The basal and peak 17OHP cortisol levels were not correlated with the presence of puberty, chromosome structure, gonadal steroid use, obesity or growth hormone use. This trial suggested that 21-hydroxylase deficiency was not common among patients with TS in our population. Adrenal function should be assessed, at least in the presence of clitoral enlargement in patients with TS, particularly if their karyotype

  4. Forkhead box transcription factor O1 inhibits cholesterol 7alpha-hydroxylase in human hepatocytes and in high fat diet-fed mice.

    PubMed

    Li, Tiangang; Ma, Huiyan; Park, Young Joo; Lee, Yoon-Kwang; Strom, Stephen; Moore, David D; Chiang, John Y L

    2009-10-01

    The conversion of cholesterol to bile acids is the major pathway for cholesterol catabolism. Bile acids are metabolic regulators of triglycerides and glucose metabolism in the liver. This study investigated the roles of FoxO1 in the regulation of cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in primary human hepatocytes. Adenovirus-mediated expression of a phosphorylation defective and constitutively active form of FoxO1 (FoxO1-ADA) inhibited CYP7A1 mRNA expression and bile acid synthesis, while siRNA knockdown of FoxO1 resulted in a approximately 6-fold induction of CYP7A1 mRNA in human hepatocytes. Insulin caused rapid exclusion of FoxO1 from the nucleus and resulted in the induction of CYP7A1 mRNA expression, which was blocked by FoxO1-ADA. In high fat diet-fed mice, CYP7A1 mRNA expression was repressed and inversely correlated to increase hepatic FoxO1 mRNA expression and FoxO1 nuclear retention. In conclusion, our current study provides direct evidence that FoxO1 is a strong repressor of CYP7A1 gene expression and bile acid synthesis. Impaired regulation of FoxO1 may cause down-regulation of CYP7A1 gene expression and contribute to dyslipidemia in insulin resistance.

  5. Superiority of dietary safflower oil over olive oil in lowering serum cholesterol and increasing hepatic mRnas for the LDL receptor and cholesterol 7alpha-hydroxylase in exogenously hypercholesterolemic (exHC) rats.

    PubMed

    Sato, M; Yoshida, S; Nagao, K; Imaizumi, K

    2000-06-01

    The exogenously hypercholesterolemic (ExHC) rat is a strain segregated from SD rats with a high response to dietary cholesterol. To understand the underlying mechanism(s) for this hypercholesterolemia, the interactive effects of dietary fatty acid and the susceptibility of rats to dietary cholesterol on the serum cholesterol concentration and hepatic mRNA abundance of the low-density lipoprotein (LDL) receptor, cholesterol 7alpha-hydroxylase (7alpha-hydroxylase) and 3-hydroxyl-3methylglutaryl (HMG) CoA reductase were examined. Both strains were fed on a diet supplemented with 10% each of olive, safflower or coconut oil with or without the addition of 1% cholesterol for one week. The ExHC rats fed on olive, safflower and coconut oil in combination with cholesterol respectively resulted in a 3.5-, 2.0- and 2.1-fold higher serum cholesterol concentration than that in the animals fed on the corresponding dietary fats without any supplementation of cholesterol (p < 0.01 by dietary cholesterol or type of fat). The dietary cholesterol dependent-elevation of serum cholesterol in the SD rats was less than 1.5-fold (p<0.01) and there was no dietary fat effect. The ExHC rats fed on the safflower oil-containing diet supplemented with cholesterol resulted in a higher mRNA abundance of the LDL receptor and 7alpha-hydroxylase than in the corresponding fat-fed rats without cholesterol (p<0.05). There was no dietary cholesterol-dependent change of mRNA abundance in either strain fed on olive or coconut oil, except for a decreased abundance of HMG CoA reductase mRNA in the olive oil-fed ExHC rats and coconut oil-fed Sprague-Dawley (SD) rats (p<0.05). These results indicate that the hepatic mRNA abundance of the LDL receptor and of 7alpha-hydroxylase depended on the dietary combination of cholesterol and a fatty acid and suggest that a linoleic acid-rich diet may alleviate exogenous hypercholesterolemia by activating the process involved in the hepatic uptake and biliary excretion of

  6. Transcriptome Analysis Reveals Key Flavonoid 3'-Hydroxylase and Flavonoid 3',5'-Hydroxylase Genes in Affecting the Ratio of Dihydroxylated to Trihydroxylated Catechins in Camellia sinensis.

    PubMed

    Wei, Kang; Wang, Liyuan; Zhang, Chengcai; Wu, Liyun; Li, Hailin; Zhang, Fen; Cheng, Hao

    2015-01-01

    The ratio of dihydroxylated to trihydroxylated catechins (RDTC) is an important indicator of tea quality and biochemical marker for the study of genetic diversity. It is reported to be under genetic control but the underlying mechanism is not well understood. Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are key enzymes involved in the formation of dihydroxylated and trihydroxylated catechins. The transcriptome and HPLC analysis of tea samples from Longjing43 and Zhonghuang2 under control and shading treatment were performed to assess the F3'H and F3'5'H genes that might affect RDTC. A total of 74.7 million reads of mRNA seq (2×101bp) data were generated. After de novo assembly, 109,909 unigenes were obtained, and 39,982 of them were annotated using 7 public databases. Four key F3'H and F3'5'H genes (including CsF3'5'H1, CsF3'H1, CsF3'H2 and CsF3'H3) were identified to be closely correlated with RDTC. Shading treatment had little effect on RDTC, which was attributed to the stable expression of these key F3'H and F3'5'H genes. The correlation of the coexpression of four key genes and RDTC was further confirmed among 13 tea varieties by real time PCR and HPLC analysis. The coexpression of three F3'H genes and a F3'5'H gene may play a key role in affecting RDTC in Camellia sinensis. The current results may establish valuable foundation for further research about the mechanism controlling catechin composition in tea.

  7. Intracellular ascorbate enhances hypoxia-inducible factor (HIF)-hydroxylase activity and preferentially suppresses the HIF-1 transcriptional response.

    PubMed

    Kuiper, Caroline; Dachs, Gabi U; Currie, Margaret J; Vissers, Margreet C M

    2014-04-01

    Hypoxia-inducible factor (HIF)-1 drives the transcription of hundreds of genes to support cell survival under conditions of microenvironmental and metabolic stress. HIF-1 is downregulated by iron-containing 2-oxoglutarate-dependent enzymes that require ascorbate as a cofactor. The HIF hydroxylases control both protein stability and the formation of an active transcription complex and, consequently, ascorbate could affect HIF-1α stabilization and/or gene expression, but the relative effect of ascorbate on these separate processes has not been well characterized. In this study we examined the effects of known intracellular ascorbate concentrations on both processes in response to various means of hydroxylase inhibition, including CoCl2, NiCl2, desferrioxamine, dimethyloxalylglycine, and hypoxia. Ascorbate inhibited HIF-1 activity most dramatically with all mechanisms of iron competition. In addition, HIF-1-dependent gene expression was effectively prevented by ascorbate and was inhibited even under conditions that allowed HIF-1α protein stabilization. This suggests that (1) ascorbate acts primarily to stabilize and reduce the iron atom in the hydroxylase active site and (2) the asparagine hydroxylase controlling HIF-1 transcriptional activity is particularly susceptible to fluctuations in intracellular ascorbate. These findings suggest that ascorbate plays a significant role in supporting HIF-hydroxylase function and that it could thereby modulate the cell survival response.

  8. Hypoxia-inducible Factor Prolyl 4-Hydroxylase Inhibition A TARGET FOR NEUROPROTECTION IN THE CENTRAL NERVOUS SYSTEM*

    PubMed Central

    Siddiq, Ambreena; Ayoub, Issam A.; Chavez, Juan C.; Aminova, Leila; Shah, Sapan; LaManna, Joseph C.; Patton, Stephanie M.; Connor, James R.; Cherny, Robert A.; Volitakis, Irene; Bush, Ashley I.; Langsetmo, Ingrid; Seeley, Todd; Gunzler, Volkmar; Ratan, Rajiv R.

    2008-01-01

    Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron- and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1α and HIF-2α. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1α and up-regulate HIF-dependent target genes (e.g. enolase, p21waf1/cip1, vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress. PMID:16227210

  9. In silico thermodynamics stability change analysis involved in BH4 responsive mutations in phenylalanine hydroxylase: QM/MM and MD simulations analysis.

    PubMed

    Chadha, Nidhi; Tiwari, Anjani K; Kumar, Vikas; Milton, Marilyn D; Mishra, Anil K

    2015-01-01

    The mammalian tetrahydrobiopterin (BH4)-dependent phenylalanine hydroxylases (PAH), involved in important metabolic pathways of phenylalanine, belong to non-heme iron-containing aromatic acid hydroxylases' enzyme (AAH) family. AAHs utilize BH4 as protein co-factor and thus promote hydroxylation reactions of their substrates. Any alterations in BH4 -mediated AAH's pathway or mutations in these enzymes are responsible for various disorders, and thus highlights the importance of mutational analysis to assess the effect on their biosynthetic pathways. Our present studies are aimed at single-site mutations in PAH that lead to thermodynamic stability change upon folding and further validation of designed non-reduced BH2 designed co-factors. We have presented single-site mutational analysis of PAH where single-site mutations have been identified from known literature. Further, in silico studies with the PAH, in silico mutant PAH, and crystallized known mutant A313T forms, involved QM/MM and Molecular Dynamics (MD) simulations analysis. The modified co-factor A showed high affinity with PAH and all mutant PAH with high G-score of -14.851. The best pose high affinity co-factor A subjected to QM/MM optimization which leads to square-pyramidal coordination of non-heme active site. The structural and energetic information obtained from the production phase of 20 ns MD simulation of co-factor-metalloprotein complex results helped to understand the binding mode and involvement of three molecules throughout the reaction pathways' catalysis of PAH. The free energies of binding (dG) of A were found to be -68.181 kcal/mol and -72.249 for 1DMW and 1TDW for A313T mutant. Binding of Co-factor A do not perturb the coordination environment of iron at the active site which resides in 2-Histdine and 1-Glutamate triad, and may enhance the percentage response towards co-factor-mediated therapy.

  10. Brief anoxia preconditioning and HIF prolyl-hydroxylase inhibition enhances neuronal resistance in organotypic hippocampal slices on model of ischemic damage.

    PubMed

    Lushnikova, Iryna; Orlovsky, Maxim; Dosenko, Victor; Maistrenko, Anastasiia; Skibo, Galina

    2011-04-22

    It is well known that a brief anoxia or hypoxia episodes can render brain resistant to a subsequent ischemia. Recent investigations indicate that mechanisms of such stimulated endogenous neuroprotection are related to the family of hypoxia-inducible factors (HIF), however there are still little data available on the role of HIF family members in hippocampus-a brain structure, highly sensitive to oxygen deficiency. We have used the model of cultured hippocampal slices and single-cell quantitative RT-PCR to study HIF-1α and HIF-3α mRNA expression following triple 5-min mild anoxia, 30-min oxygen-glucose deprivation and their combination. We also tested the effects of HIF prolyl-hydroxylase inhibition with 2,4-pyridinedicarboxylic acid diethyl ester pre-treatment followed by a 30-min oxygen-glucose deprivation. It was found that neuronal damage induced by oxygen-glucose deprivation was accompanied by a significant decrease in both HIF-1α and HIF-3α mRNA levels in CA1 but not CA3 neurons. Anoxia preconditioning did not affect cell viability and HIF mRNA levels but applied before oxygen-glucose deprivation prevented neuronal damage and suppression of HIF-1α and HIF-3α mRNA expression. It was also found that effects of the prolyl-hydroxylase inhibitor were similar to anoxia preconditioning. These results suggest that anoxia preconditioning increases anti-ischemic neuronal resistance which to a certain extent correlates with the changes of HIF-1α and HIF-3α expression.

  11. Crystal structure of the ectoine hydroxylase, a snapshot of the active site.

    PubMed

    Höppner, Astrid; Widderich, Nils; Lenders, Michael; Bremer, Erhard; Smits, Sander H J

    2014-10-24

    Ectoine and its derivative 5-hydroxyectoine are compatible solutes that are widely synthesized by bacteria to cope physiologically with osmotic stress. They also serve as chemical chaperones and maintain the functionality of macromolecules. 5-Hydroxyectoine is produced from ectoine through a stereo-specific hydroxylation, an enzymatic reaction catalyzed by the ectoine hydroxylase (EctD). The EctD protein is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily and is evolutionarily well conserved. We studied the ectoine hydroxylase from the cold-adapted marine ultra-microbacterium Sphingopyxis alaskensis (Sa) and found that the purified SaEctD protein is a homodimer in solution. We determined the SaEctD crystal structure in its apo-form, complexed with the iron catalyst, and in a form that contained iron, the co-substrate 2-oxoglutarate, and the reaction product of EctD, 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the EctD active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-Hydroxyectoine, however, is coordinated by EctD in manner different from that found in high affinity solute receptor proteins operating in conjunction with microbial import systems for ectoines. Our crystallographic analysis provides a detailed view into the active site of the ectoine hydroxylase and exposes an intricate network of interactions between the enzyme and its ligands that collectively ensure the hydroxylation of the ectoine substrate in a position- and stereo-specific manner.

  12. Cytotoxic and aryl hydrocarbon hydroxylase-inducing effects of laboratory rodent diets. A cell culture study

    SciTech Connect

    Toerroenen, R.; Pelkonen, K.; Kaerenlampi, S. )

    1991-01-01

    Extracts of several rodent diets were studied for their cytotoxic and aryl hydrocarbon hydroxylase-inducing properties by an in vitro method. The cell culture system based on a mouse hepatoma cell line (Hepa-1) was shown to be convenient and sensitive method for screening of diets for these parameters implying the presence of compounds potentially harmful in vivo. Considerable differences among diets and batches were detected. Smallest effects were observed with a semipurified diet and with the unrefined diet which - contrary to other four unrefined diets - contained no fish.

  13. Molecular analysis of contiguous exons of the phenylalanine hydroxylase gene: identification of a new PKU mutation.

    PubMed Central

    Dianzani, I; Camaschella, C; Saglio, G; Ferrero, G B; Ramus, S; Ponzone, A; Cotton, R G

    1993-01-01

    A modified application of the chemical cleavage of mismatch (CCM) method has been used to screen three contiguous exons (exons 9, 10, and 11) of the phenylalanine hydroxylase gene in 17 Italian PKU patients. A new nonsense heterozygous C-->G transversion within exon 11 (S359X) was identified in a single patient. Only one of the four mutations previously reported in this DNA region in Caucasians was found. This lesion, IVS X-546, was detected in five of the 34 PKU alleles examined. Our results underline the versatility of the CCM method for scanning a gene for multiple mutations. Images PMID:8097261

  14. Brain development and cognitive, psychosocial, and psychiatric functioning in classical 21-hydroxylase deficiency.

    PubMed

    Meyer-Bahlburg, Heino F L

    2011-01-01

    The disturbance of the hypothalamic-pituitary- adrenal axis characteristic of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is likely to affect brain development, yet neuroanatomic work is only beginning. Fetal hyperandrogenemia in 46, XX 21-OHD leads to masculinized brain organization and, consequently, at later stages of development, to masculinized gender-related behavior and cognitive function, including, although relatively uncommonly, gender identity. Genital masculinization as well as its surgical treatment has implications for social stigmatization and sexual functioning. CAH-associated electrolyte crises in infancy and later may result in severe cognitive impairment. Psychiatric disorders are somewhat increased, especially in patients with severe degrees of CAH.

  15. Isolation and purification of tyrosine hydroxylase from callus cultures of Portulaca grandiflora.

    PubMed

    Yamamoto Ki; Kobayashi, N; Yoshitama, K; Teramoto, S; Komamine, A

    2001-09-01

    Tyrosine hydroxylase was separated from polyphenol oxidase activity and was highly purified from betacyanin producing callus cultures of Portulaca grandiflora. The purified enzyme catalyzed the formation of DOPA (L-3,4-dihydroxyphenylalanine) from tyrosine and required the pterin compounds (6-methyl-5,6,7,8-tetrahydropterin; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterin) as coenzyme. The K(m) values for tyrosine and 6-methyl-5,6,7,8-tetrahydropterin were 0.5 mM and 0.15 mM, respectively. This enzyme was activated by Fe(2+) and Mn(2+), and inhibited by metal chelating agents.

  16. Molecular analysis of contiguous exons of the phenylalanine hydroxylase gene: identification of a new PKU mutation.

    PubMed

    Dianzani, I; Camaschella, C; Saglio, G; Ferrero, G B; Ramus, S; Ponzone, A; Cotton, R G

    1993-03-01

    A modified application of the chemical cleavage of mismatch (CCM) method has been used to screen three contiguous exons (exons 9, 10, and 11) of the phenylalanine hydroxylase gene in 17 Italian PKU patients. A new nonsense heterozygous C-->G transversion within exon 11 (S359X) was identified in a single patient. Only one of the four mutations previously reported in this DNA region in Caucasians was found. This lesion, IVS X-546, was detected in five of the 34 PKU alleles examined. Our results underline the versatility of the CCM method for scanning a gene for multiple mutations.

  17. Cholinergic and GABAergic regulation of dopamine beta-hydroxylase activity in the adrenal gland of the rat.

    PubMed

    Lima, L; Sourkes, T L

    1986-04-01

    The administration of oxotremorine together with methylatropine to rats produces a dose-dependent increase of adrenal dopamine-beta-hydroxylase (DBH) activity. This effect is abolished by denervation of the gland and by cycloheximide. The Km for tyramine is not affected by the trans-synaptic induction of DBH by oxotremorine. The induction is selective, because similar treatment does not affect adrenal dopa decarboxylase or lactate dehydrogenase in the adrenal gland. The combination of 6-hydroxydopamine i.c.v. or propranolol i.p. does not alter the effect of oxotremorine on adrenal DBH. However, propranolol reduces the tremorigenic action of the muscarinic agonist. The systemic administration of p-chlorophenylalanine or the i.c.v. injection of 5,7-dihydroxytryptamine before oxotremorine treatment does not affect the increase of adrenal DBH. Progabide, gamma-aminobutyric acid, a (GABA)A and GABAB receptor agonist that effectively crosses the blood-brain barrier, reduces the effect of oxotremorine in a dose-dependent manner. Muscimol given by either of two routes, i.c.v. at a constant rate (Alzet minipump) or i.p., produces significant decreases of adrenal DBH activity and attenuates the action of oxotremorine. Denervation of the gland abolishes the effect of muscimol i.p. in decreasing adrenal DBH activity. Baclofen, a GABAB receptor agonist, has no effect by itself or does it affect the action of oxotremorine. None of these GABA agonists has any in vitro effect on adrenal DBH activity. Bicuculline, GABAA receptor antagonist, reverses the action of progabide in oxotremorine-treated rats with respect to adrenal DBH activity, partially blocks the effect of muscimol and enhances the increase obtained with oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. LX-1031, a Tryptophan 5-hydroxylase Inhibitor, and Its Potential in Chronic Diarrhea Associated with Increased Serotonin

    PubMed Central

    Camilleri, Michael

    2010-01-01

    Background LX-1031 is an oral, small-molecule tryptophan 5-hydroxylase (TPH) inhibitor that reduces serotonin (5-HT) synthesis peripherally. It has potential for illnesses characterized by excess 5-HT, such as diarrhea-predominant irritable bowel syndrome (IBS-D) and carcinoid diarrhea. In vitro, inhibition of TPH1 occurred in 10−8 – 10−7 M range. In vivo in rodents, LX-1031 has no effect on brain 5-HT while dose-dependently reducing 5-HT, particularly in the small bowel. Pharmacokinetics After oral LX1031 in humans, systemic exposure is very low, plasma concentrations are linear in dose range 250 mg QD to 750 mg QID; the median T1/2 for elimination is ~20 hrs, and repeat administration for 14 days doubles Cmax. Pharmacodynamics In ascending-single-dose and multiple dose (14 day) trials in healthy volunteers, LX-1031 2g-4g/day significantly reduced urinary 5-hydroxyindoleacetic acid (5-HIAA) starting by day 5, and persisting over the 14 day exposure. Clinical safety and efficacy There are no dose limiting toxicities in healthy subjects or remarkable adverse effects in clinical trials to date. Over a 28-day treatment period, LX-1031 was associated with improved weekly global scores (2/4 weeks) and improved stool consistency with lower urinary 5-HIAA excretion. Conclusion LX-1031 appears promising for chronic diarrhea associated with increased 5-HT expression including IBS-D. Optimal doses, efficacy and safety in IBS clinical trials need to be fully elucidated; low systemic exposure, selectivity for TPH1 over TPH2, and lack of effect on brain 5-HT in several species suggest that LX-1031 is unlikely to cause affective disorders. PMID:21159063

  19. Human Bacterial Artificial Chromosome (BAC) Transgenesis Fully Rescues Noradrenergic Function in Dopamine β-Hydroxylase Knockout Mice

    PubMed Central

    Cubells, Joseph F.; Schroeder, Jason P.; Barrie, Elizabeth S.; Manvich, Daniel F.; Sadee, Wolfgang; Berg, Tiina; Mercer, Kristina; Stowe, Taylor A.; Liles, L. Cameron; Squires, Katherine E.; Mezher, Andrew; Curtin, Patrick; Perdomo, Dannie L.; Szot, Patricia; Weinshenker, David

    2016-01-01

    Dopamine β-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE) in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/-) mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP) in the human DBH gene promoter (-970C>T; rs1611115) is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS), which can be converted to NE by aromatic acid decarboxylase (AADC) in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a “C” or a “T” at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh

  20. Down-regulation of p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla x E. grandis leads to improved sugar release

    DOE PAGES

    Sykes, Robert W.; Gjersing, Erica L.; Foutz, Kirk; ...

    2015-08-27

    In this study, lignocellulosic materials provide an attractive replacement for food-based crops used to produce ethanol. Understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. In this study, eucalyptus down-regulated in expression of cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) or p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H, EC 1.14.13.36) were evaluated for cell wall composition and reduced recalcitrance.

  1. Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA.

    PubMed Central

    Robson, K J; Chandra, T; MacGillivray, R T; Woo, S L

    1982-01-01

    The mRNA for phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been purified from total rat liver mRNAs, of which it constitutes less than 0.25%, to greater than 10% purity in a single step by specific polysome immunoprecipitation. The purified mRNA was used for synthesis and cloning of its cDNA. Recombinant colonies containing phenylalanine hydroxylase DNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analysis. The rat cDNA clone was capable of hybridizing with human phenylalanine hydroxylase mRNA, which will permit the isolation of the corresponding human gene for analysis of phenylketonuria, a hereditary disorder in phenylalanine metabolism that causes permanent mental retardation in humans. Images PMID:6750607

  2. Cloning and characterization of a novel β-carotene hydroxylase gene from Lycium barbarum and its expression in Escherichia coli.

    PubMed

    Wu, Jiang; Ji, Jing; Wang, Gang; Li, Zhaodi; Diao, Jinjin; Wu, Guangxia

    2014-01-01

    Lycium barbarum contains high levels of zeaxanthin, which is produced by the conversion of β-carotene into zeaxanthin. β-Carotene hydroxylase catalyzes this reaction. We cloned a cDNA (chyb) encoding β-carotene hydroxylase (Chyb) from the L. barbarum leaf. A 939-bp full-length cDNA sequence was determined with 3'-rapid amplification of cDNA end assay encoding a deduced Chyb protein (34.8 kDa) with a theoretical isoelectric point of 8.36. A bioinformatics analysis showed that the L. barbarum Chyb was located in the chloroplast. Further, to investigate the catalytic activity of the L. barbarum Chyb, a complementation analysis was conducted in Escherichia coli. The results strongly demonstrated that Chyb can catalyze β-carotene to produce zeaxanthin. Thus, this study suggests that L. barbarum β-carotene hydroxylase could be a means of zeaxanthin production by genetic manipulation in E. coli.

  3. Activation of retinal tyrosine hydroxylase: tolerance induced by chronic treatment with haloperidol does not modify response to light

    SciTech Connect

    Cohen, J.; Neff, N.H.

    1982-05-01

    A single dose of haloperidol administered to rats in the dark increases the activity of retinal tyrosine hydroxylase. The ability of haloperidol to activate the enzyme is diminished 24 hr after terminating 22 to 30 days of treatment with haloperidol. The retinal enzyme is also tolerant to activation by treatment with chlorpromazine. In contrast, exposure of the animals to light activates the enzyme to the same extent in chronic haloperidol-treated and control animals. Thus, chronic haloperidol treatment does not modify the ability of the retinal enzyme system to respond to the physiological stimulus, light. Apparently, activation of retinol tyrosine hydroxylase by haloperidol and light occurs by independent mechanisms.

  4. Regulation of phenylalanine hydroxylase: conformational changes upon phosphorylation detected by H/D exchange and mass spectrometry.

    PubMed

    Li, Jun; Fitzpatrick, Paul F

    2013-07-15

    The enzyme phenylalanine hydroxylase catalyzes the hydroxylation of excess phenylalanine in the liver to tyrosine. The enzyme is regulated allosterically by phenylalanine and by phosphorylation of Ser16. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into any structural change upon phosphorylation. Peptides in both the catalytic and regulatory domains show increased deuterium incorporation into the phosphorylated protein. Deuterium is incorporated into fewer peptides than when the enzyme is activated by phenylalanine, and the incorporation is slower. This establishes that the conformational change upon phosphorylation of phenylalanine hydroxylase is different from and less extensive than that upon phenylalanine activation.

  5. In vitro screening for inhibitor of cloned Drosophila melanogaster tyramine-β-hydroxylase and docking studies.

    PubMed

    Hasan, Md Nazmul; Hosen, Mohammad Jakir; Thakur, Prasoon Kumar; Abir, Ruhshan Ahmed; Zubaer, Abdullah; Renkai, Guo; Yoshida, Mayumi; Ohta, Hiroto; Lee, Jae Man; Kusakabe, Takahiro; Hirashima, Akinori

    2016-12-01

    Biogenic amines are common biologically active substances extended within the whole animal kingdom where they play vital roles as signal transducer as well as regulator of cell functions. One of these biogenic amines called octopamine (OA) is synthesized from tyramine (TA) by the catalysis of tyramine-β-hydroxylase (TβH) originated in the insect nervous system. Both TA and OA act as neurotransmitters, neurohormones and neuromodulators in the arthropod nervous system. Herein, the inhibitory activity of 1-arylimidazole-2(3H)-thiones (AITs) was tested on cloned Drosophila tyramine-β-hydroxylase (DmTβH) expressed in Bombyx mori strain. Radiolabelled (3)H-TA was used to analyze the activity of AITs exhibited inhibitory effects on DmTβH, whose ID50 values range from 0.02 to 2511nM where DmTβH was inhibited in a dose-dependent manner at pH 7.6 and 25°C during a 30min of incubation. To understand the catalytic role of the TβH, a three dimensional structure of the TβH from Drosophila melanogaster was constructed by homology modeling using the Phyre2 web server with 100% confidence. The modeled three-dimensional structure of TβH was used to perform the docking study with AITs. This may give more insights to precise design of inhibitors for TβH to control insect's population.

  6. Combination growth hormone and gonadotropin releasing hormone analog therapy in 11beta-hydroxylase deficiency.

    PubMed

    Bajpai, Anurag; Kabra, Madhulika; Menon, P S N

    2006-06-01

    Diagnosis of 11beta-hydroxylase deficiency was made in a boy at the age of 2 1/2 years on the basis of peripheral precocious puberty, growth acceleration (height standard deviation score +4.4) with advanced skeletal maturation (bone age 8.4 years) and elevated deoxycortisol levels. Glucocorticoid supplementation led to normalization of blood pressure but was associated with progression to central precocious puberty and increase in bone age resulting in decrease in predicted adult height to 133.7 cm (target height 163 cm). The child was started on GnRH analog (triptorelin 3.75 mg every 28 days), which led to improvement in predicted adult height by 3.1 cm over 15 months. Addition of growth hormone (0.1 IU/kg/day) resulted in improvement in predicted adult height (151 cm) and height deficit (12 cm) over the next 3.6 years. Final height (151 cm) exceeded predicted height at the initiation of GnRH analog treatment by 17.3 cm. This report suggests that combination GH and GnRH analog treatment may be useful in improving height outcome in children with 11beta-hydroxylase deficiency and compromised final height.

  7. Cellular Oxygen Sensing: Crystal Structure of Hypoxia-Inducible Factor Prolyl Hydroxylase (PHD2)

    SciTech Connect

    McDonough,M.; Li, V.; Flashman, E.; Chowdhury, R.; Mohr, C.; Lienard, B.; Zondlo, J.; Oldham, N.; Clifton, I.; et al.

    2006-01-01

    Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-{alpha} subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Angstroms resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded {beta}-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

  8. Embryotoxicity, teratogenicity and aryl hydrocarbon hydroxylase activity in Forster's terns on Green Bay, Lake Michigan

    USGS Publications Warehouse

    Hoffman, D.J.; Rattner, B.A.; Sileo, L.; Docherty, D.E.; Kubiak, T.J.

    1987-01-01

    Known reproductive problems, including congenital malformations and poor hatching success, exist for the state endangered Forster's tern (Sterna forsteri) in Green Bay, Wisconsin. Twenty Forster's tern eggs were collected from separate nests at (i) a natural colony with documented reproductive problems, situated at Green Bay, Lake Michigan, and (ii) an inland colony at Lake Poygan (control) where reproduction was documented as normal. Eggs from the two locations were placed in the same laboratory incubator and candled throughout incubation. Hatching success of Green Bay eggs was 52% of that for controls. Several early embryonic deaths occurred, but most mortality occurred close to the time of hatching. Liver microsomal aryl hydrocarbon hydroxylase activity was elevated approximately threefold in Green Bay hatchlings compared to controls. Green Bay terns that hatched weighed less than controls, had an increased liver to body weight ratio, and had a shorter femur length. Two Green Bay embryos that failed to hatch had anomalies, one with a crossed beak and one with poor ossification of the foot. One Green Bay hatchling had an abnormally ossified ilium. These effects were observed in eggs where there were measureable levels of aryl hydrocarbon hydroxylase inducers including polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins.

  9. Embryotoxicity, teratogenicity, and aryl hydrocarbon hydroxylase activity in Forster's terns on Green Bay, Lake Michigan

    USGS Publications Warehouse

    Hoffman, D.J.; Rattner, B.A.; Sileo, L.; Docherty, D.; Kubiak, T.J.

    1987-01-01

    Known reproductive problems, including congenital malformations and poor hatching success, exist for the state endangered Forster's tern (Sterna forsteri) in Green Bay, Wisconsin. Twenty Forster's tern eggs were collected from separate nests at a natural colony with documented reproductive problems, situated at Green Bay, Lake Michigan, and an inland colony at Lake Poygan (control) where reproduction was documented as normal. Eggs from the two locations were placed in the same laboratory incubator and candled throughout incubation. Hatching success of Green Bay eggs was 52% of that for controls. Several early embryonic deaths occurred, but most mortality occurred close to the time of hatching. Liver microsomal aryl hydrocarbon hydroxylase activity was elevated approximately threefold in Green Bay hatchlings compared to controls. Green Bay terns that hatched weighed less than controls, had an increased liver to body weight ratio, and had a shorter femur length. Two Green Bay embryos that failed to hatch had anomalies, one with a crossed beak and one with poor ossification of the foot. One Green Bay hatchling had an abnormally ossified ilium. These effects were observed in eggs where there were measureable levels of aryl hydrocarbon hydroxylase inducers including polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins.

  10. A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis.

    PubMed

    Percy, Melanie J; Zhao, Quan; Flores, Adrian; Harrison, Claire; Lappin, Terence R J; Maxwell, Patrick H; McMullin, Mary Frances; Lee, Frank S

    2006-01-17

    The number of red blood cells is normally tightly regulated by a classic homeostatic mechanism based on oxygen sensing in the kidney. Decreased oxygen delivery resulting from anemia induces the production of erythropoietin, which increases red cell production and hence oxygen delivery. Investigations of erythropoietin regulation identified the transcription factor hypoxia-inducible factor (HIF). HIF is now recognized as being a key regulator of genes that function in a comprehensive range of processes besides erythropoiesis, including energy metabolism and angiogenesis. HIF itself is regulated through the alpha-subunit, which is hydroxylated in the presence of oxygen by a family of three prolyl hydroxylase domain proteins (PHDs)/HIF prolyl hydroxylases/egg-laying-defective nine enzymes. Hydroxylation allows capture by the von Hippel-Lindau tumor suppressor gene product, ubiquitination, and destruction by the proteasome. Here we describe an inherited mutation in a mammalian PHD enzyme. We show that this mutation in PHD2 results in a marked decrease in enzyme activity and is associated with familial erythrocytosis, identifying a previously unrecognized cause of this condition. Our findings indicate that PHD2 is critical for normal regulation of HIF in humans.

  11. Contribution of epithelial innate immunity to systemic protection afforded by prolyl hydroxylase inhibition in murine colitis

    PubMed Central

    Keely, Simon; Campbell, Eric L.; Baird, Alan W.; Hansbro, Philip M.; Shalwitz, Robert A.; Kotsakis, Anna; McNamee, Eoin N.; Eltzschig, Holger K.; Kominsky, Douglas J.; Colgan, Sean P.

    2013-01-01

    Pharmacological stabilization of hypoxia-inducible factor (HIF) through prolyl hydroxylase (PHD) inhibition limits mucosal damage associated with models of murine colitis. However, little is known about how PHD inhibitors (PHDi) influence systemic immune function during mucosal inflammation or the relative importance of immunological changes to mucosal protection. We hypothesized that PHDi enhances systemic innate immune responses to colitis-associated bacteremia. Mice with colitis induced by TNBS were treated with AKB-4924, a new HIF-1 isoform-predominant PHDi and clinical, immunological and biochemical endpoints were assessed. Administration of AKB-4924 led to significantly reduced weight loss and disease activity compared to vehicle controls. Treated groups were pyrexic, but did not become subsequently hypothermic. PHDi treatment augmented epithelial barrier function and led to an approximately 50-fold reduction in serum endotoxin during colitis. AKB-4924 also decreased cytokines involved in pyrogenesis and hypothermia, significantly reducing serum levels of IL-1β, IL-6 and TNF-α, while increasing IL-10. Treatment offered no protection against colitis in epithelial-specific HIF-1α deficient mice, strongly implicating epithelial HIF-1α as the tissue target for AKB-4924-mediated protection. Taken together, these results indicate that inhibition of prolyl hydroxylase with AKB-4924 enhances innate immunity and identifies the epithelium is a central site of inflammatory protection afforded by PHDi in murine colitis. PMID:23695513

  12. Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

    PubMed Central

    Barnes, H J; Arlotto, M P; Waterman, M R

    1991-01-01

    When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system. Images PMID:1829523

  13. Lack of Tryptophan Hydroxylase-1 in Mice Results in Gait Abnormalities

    PubMed Central

    Suidan, Georgette L.; Vanderhorst, Veronique; Hampton, Thomas G.; Wong, Siu Ling; Voorhees, Jaymie R.; Wagner, Denisa D.

    2013-01-01

    The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that Tph1 (−/−) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system. PMID:23516593

  14. Steroid 16 alpha-hydroxylase from human fetal liver; inhibition by steroids.

    PubMed

    Sano, Y; Shibusawa, H; Yoshida, N; Sekiba, K; Okinaga, S; Arai, K

    1980-01-01

    The 16 alpha-hydroxylase system in fetal liver which used dehydroepiandrosterone (DHA) or pregnenolone as substrate, was apparently inhibited by various endogenous and synthetic steriods: DHA, pregnenolone, their sulfates, androstenediol, androstenetriol, estrone, estradiol-17 beta, ethynylestradiol and chlormadinone-acetate. The inhibition constants (Ki) towards DHA were as follows; pregnenolone 22 muM, DHA-sulfate 13 muM, pregnenolone-sulfate 21 muM, androstenediol 16 muM, androstenetriol 53 muM estrone 32 muM, estradiol-17 beta 74 muM, ethynylestradiol 22 muM and chlormadinone-acetate 27 muM. The Ki values towards pregnenolone were DHA 6.3 muM, DHA-sulfate 8.3 muM, pregnenolone-sulfate 3.9 muM, androstenediol 8.7 muM, androstenetriol 14.7 muM, estradiol-17 beta 15.4 muM and ethynylestradiol 16.0 muM, respectively. The reaction products, 16 alpha OH-DHA and 16 alpha OH-pregnenolone, showed little inhibitory effect upon the 16 alpha-hydroxylase.

  15. Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene.

    PubMed

    Chang, Shu; Berman, Judit; Sheng, Yanmin; Wang, Yingdian; Capell, Teresa; Shi, Lianxuan; Ni, Xiuzhen; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

    2015-01-01

    The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity.

  16. Genistein inhibits vitamin D hydroxylases CYP24 and CYP27B1 expression in prostate cells.

    PubMed

    Farhan, Hesso; Wähälä, Kristiina; Cross, Heide S

    2003-03-01

    In human prostate cancer cells, the availability of the steroid hormone 1,25-dihydroxyvitamin D(3) for antimitotic action is determined through the activity of the two enzymes CYP24 and CYP27B1, viz. 25-hydroxyvitamin D-24-hydroxylase and 25-hydroxyvitamin D-1alpha-hydroxylase. High performance liquid chromatography (HPLC) analysis of [(3)H]25(OH)D(3) metabolism in human prostate cancer DU-145 cells revealed that genistein and other isoflavonoids, such as dihydrogenistein and daidzein, as well as the antiestrogenic compound ICI 182,780, inhibited Vitamin D-metabolizing enzyme activities. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that only in case of genistein this was due to transcriptional inhibition of CYP24 and CYP27B1 gene expressions. In case of CYP27B1, reduction of gene activity involves histone deacetylation because genistein was inactive in the presence of the histone deactylase inhibitor trichostatin A. In contrast, under the same condition, CYP24 gene activity was largely suppressed. In summary, our results suggest that a combined effect of genistein and trichostatin A could increase the responsiveness of human prostate cancer cells to the antiproliferative action of 1,25-dihydroxyvitamin D(3).

  17. The effects of Urtica dioica L. leaf extract on aniline 4-hydroxylase in mice.

    PubMed

    Ozen, Tevfik; Korkmaz, Halil

    2009-01-01

    The effects of hydroalcoholic (80% ethanol-20% water) extract of Urtica dioica L. on microsomal aniline 4-hydroxylase (A4H) were investigated in the liver of Swiss albino mice (8- 10-weeks-old) treated with two doses (50 and 100 mg/kg body weight, given orally for 14 days ). The activities of A4H showed a significant increase in the liver at both dose levels of extract treatment. The hydroalcoholic extract of Urtica dioica induced the activities of A4H that had been increased by treatment of metal ions (Mg2+ and Ca2+) and the mixture of cofactors (NADH and NADPH). At saturated concentration of cofactor, microsomal A4H exhibited significantly even higher activities in the presence of the mixture of cofactors than NADPH and NADH. Mg2+ and Ca2+ ions acted as stimulants in vitro. The present results suggest that the hydroalcoholic extract of Urtica dioica may have modalatory effect on aniline hydroxylase at least in part and enhance the activity of A4H adding metals ions and cofactors.

  18. Interferon-Inducible Cholesterol-25-Hydroxylase Inhibits Hepatitis C Virus Replication via Distinct Mechanisms

    PubMed Central

    Chen, Yongzhi; Wang, Shanshan; Yi, Zhaohong; Tian, Huabin; Aliyari, Roghiyh; Li, Yanhua; Chen, Gang; Liu, Ping; Zhong, Jin; Chen, Xinwen; Du, Peishuang; Su, Lishan; Qin, F. Xiao-Feng; Deng, Hongyu; Cheng, Genhong

    2014-01-01

    Cholesterol 25-hydroxylase (CH25H) as an interferon-stimulated gene (ISG) has recently been shown to exert broad antiviral activity through the production of 25-hydroxycholesterol (25HC), which is believed to inhibit the virus-cell membrane fusion during viral entry. However, little is known about the function of CH25H on HCV infection and replication and whether antiviral function of CH25H is exclusively mediated by 25HC. In the present study, we have found that although 25HC produced by CH25H can inhibit HCV replication, CH25H mutants lacking the hydroxylase activity still carry the antiviral activity against HCV but not other viruses such as MHV-68. Further studies have revealed that CH25H can interact with the NS5A protein of HCV and inhibit its dimer formation, which is essential for HCV replication. Thus, our work has uncovered a novel mechanism by which CH25H restricts HCV replication, suggesting that CH25H inhibits viral infection through both 25HC-dependent and independent events. PMID:25467815

  19. Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene

    PubMed Central

    Sheng, Yanmin; Wang, Yingdian; Capell, Teresa; Shi, Lianxuan; Ni, Xiuzhen; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

    2015-01-01

    The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity. PMID:26030746

  20. Dual-Action Inhibitors of HIF Prolyl Hydroxylases That Induce Binding of a Second Iron Ion

    PubMed Central

    Thalhammer, Armin; Demetriades, Marina; Chowdhury, Rasheduzzaman; Tian, Ya-Min; Stolze, Ineke; McNeill, Luke A.; Lee, Myung Kyu; Woon, Esther C. Y.; Mackeen, Mukram M.; Kawamura, Akane; Ratcliffe, Peter J.; Mecinović, Jasmin; Schofield, Christopher J.

    2015-01-01

    Inhibition of the hypoxia-inducible factor (HIF) prolyl-hydroxylases (PHD or EGLN enzymes) is of interest for the treatment of anemia and ischemia-related diseases. Most PHD inhibitors work by binding to the single ferrous ion and competing with 2-oxoglutarate (2OG) co-substrate for binding at the PHD active site. Non-specific iron chelators also inhibit the PHDs, both in vitro and in cells. We report the identification of dual action PHD inhibitors, which bind to the active site iron and also induce the binding of a second iron ion at the active site. Following analysis of small-molecule iron complexes and application of non-denaturing protein mass spectrometry to assess PHD2·iron·inhibitor stoichimetry, selected diacylhydrazines were identified as PHD2 inhibitors that induce the binding of a second iron ion. Some compounds were shown to inhibit the HIF hydroxylases in human hepatoma and renal carcinoma cell lines. PMID:23151668

  1. Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase: stereochemistry, isotope effects, and kinetic mechanism

    SciTech Connect

    Yu, Y.; Wang, L.H.; Tu, S.C.

    1987-02-24

    A neutral flavin semiquinone species was formed upon photoreduction of Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxlase whereas no flavin radical was detected by anaerobic reduction with NADH in the presence of m-hydroxybenzoate. In the latter case, the formation of flavin semiquinone is apparently thermodynamically unfavorable. A stereospecificity for the abstraction of the 4R-position hydrogen of NADH has been demonstrated for this hydroxylase. Deuterium and tritium isotope effects were observed with (4R)-(4-/sup 2/H)NADH and (4R)-(4-/sup 3/H)NADH as substrates. The /sub D/V effect indicates the existence of at least one slow step after the isotope-sensitive enzyme reduction by dihydropyridine nucleotide. A minimal kinetic mechanism has been deduced on the basis of initial velocity measurements and studies on deuterium and tritium isotope effects. Following this scheme, m-hydroxybenzoate and NADH bind to the hydroxylase in a random sequence. The flavohydroxylase is reduced by NADH, and NAD/sup +/ is released. Oxygen subsequently binds to and reacts with the reduced flavohydroxylase-m-hydroxybenzoate complex. Following the formation and release of water and gentisate, the oxidized holoenzyme is regenerated. The enzyme has a small preference for the release of NADH over m-hydroxybenzoate from the enzyme-substrates ternary complex.

  2. The Effects on Lignin Structure of Overexpression of Ferulate 5-Hydroxylase in Hybrid Poplar1[W

    PubMed Central

    Stewart, Jaclyn J.; Akiyama, Takuya; Chapple, Clint; Ralph, John; Mansfield, Shawn D.

    2009-01-01

    Poplar (Populus tremula × alba) lignins with exceedingly high syringyl monomer levels are produced by overexpression of the ferulate 5-hydroxylase (F5H) gene driven by a cinnamate 4-hydroxylase (C4H) promoter. Compositional data derived from both standard degradative methods and NMR analyses of the entire lignin component (as well as isolated lignin fraction) indicated that the C4H∷F5H transgenic's lignin was comprised of as much as 97.5% syringyl units (derived from sinapyl alcohol), the remainder being guaiacyl units (derived from coniferyl alcohol); the syringyl level in the wild-type control was 68%. The resultant transgenic lignins are more linear and display a lower degree of polymerization. Although the crucial β-ether content is similar, the distribution of other interunit linkages in the lignin polymer is markedly different, with higher resinol (β-β) and spirodienone (β-1) contents, but with virtually no phenylcoumarans (β-5, which can only be formed from guaiacyl units). p-Hydroxybenzoates, acylating the γ-positions of lignin side chains, were reduced by >50%, suggesting consequent impacts on related pathways. A model depicting the putative structure of the transgenic lignin resulting from the overexpression of F5H is presented. The altered structural features in the transgenic lignin polymer, as revealed here, support the contention that there are significant opportunities to improve biomass utilization by exploiting the malleability of plant lignification processes. PMID:19386808

  3. [Effect of butaclamol enantiomers on hypothalamic tyrosine hydroxylase in the rat brain].

    PubMed

    Mineeva, M F; Raevskiĭ, K S

    1984-10-01

    The authors demonstrate stereospecificity of the action of butaclamol enantiomers on substrate inhibition of hypothalamic tyrosine hydroxylase (TH) and regulation of the tyrosine hydroxylase response by the presynaptic membrane (presynaptic receptors) of rat hypothalamus synaptosomes under membrane activation with dopamine. The effect of (+)-butaclamol on the substrate inhibition of TH was noticeable at a concentration of 10(-8)M, reaching a maximum at 10(-5)M. (-)-Butaclamol administered at the same concentrations did not influence the substrate inhibition of the enzyme. (+)-Butaclamol added to the incubation medium containing hypothalamic synaptosomes concurrently with dopamine (10(-5)M) completely blocked the regulatory action of the latter on TH, with this action mediated via presynaptic receptors. (-)-Butaclamol (10(-5)M) antagonized the action of dopamine under the same conditions. The data obtained indicate high stereo-specificity of butaclamol enantiomers as regards their effect on presynaptic regulation of TH, suggesting that elimination of the substrate inhibition of hypothalamic TH is a stereoselective effect of neuroleptics and can be a prognostically important criterion in the appraisal of compounds with potential neuroleptic activity.

  4. Regional development of norepinephrine, dopamine-beta-hydroxylase and tyrosine hydroxylase in the rat brain subsequent to neonatal treatment with subcutaneous 6-hydroxydopamine.

    PubMed

    Schmidt, R H; Bhatnagar, R K

    1979-04-27

    Neonatal rats were injected subcutaneously with 100 mg/kg 6-hydroxydopamine (6-OHDA), or vehicle, on postnatal days 1, 2 and 3. At several times thereafter, determinations of tyrosine hydroxylase (TOH) and dopamine-beta-hydroxylase (DBH) activities, and norepinephrine (NE) concentration were made in the parietal cortex, cerebellum and pons-medulla in order to assess the extent of initial noradrenergic degeneration induced, and the rate of any ensuing regeneration. By the day following completion of the treatment (postnatal day 4), degeneration of noradrenergic terminals in the parietal cortex and cerebellum was very extensive, with NE levels and DBH activities reduced by more than 80%, and TOH activities reduced by 50%. In the parietal cortex noradrenergic degeneration remained virtually complete; and 9 and 70 days postnatal NE concentration and DBH and TOH activities were all decreased by more than 90--95%. In the cerebellum a progressive regeneration and apparent sprouting of NE fibers was observed. By postnatal day 9, NE, DBH and TOH in this tissue had all recovered to near control levels, and by day 70 these measures exceeded control levels by 95%, 115% and 50% respectively. In the pons-medulla, the initial effect of 6-OHDA on any of the measured parameters was negligible. By postnatal day 9 an increase in NE concentration was apparent, which increased further by day 70 to surpass the control level by 70%. At this same time DBH activity was increased by only 15% and TOH activity was unchanged. Separate analysis of the rostral half of the pons, which contains the locus coeruleus, revealed that on day 70 NE and DBH levels were increased much more substantially than in the whole pons-medulla, and TOH activity was also significantly elevated. This data indicates that the initial amount of degeneration induced by the 6-OHDA treatment is similar in both the parietal cortex and cerebellum, but regeneration proceeds only in the cerebellum. This suggests that

  5. Allele-specific marker development and selection efficiencies for both flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes in soybean subgenus soja.

    PubMed

    Guo, Yong; Qiu, Li-Juan

    2013-06-01

    Color is one of the phenotypic markers mostly used to study soybean (Glycine max L. Merr.) genetic, molecular and biochemical processes. Two P450-dependent mono-oxygenases, flavonoid 3'-hydroxylase (F3'H; EC1.14.3.21) and flavonoid 3',5'-hydroxylase (F3'5'H, EC1.14.13.88), both catalyzing the hydroxylation of the B-ring in flavonoids, play an important role in coloration. Previous studies showed that the T locus was a gene encoding F3'H and the W1 locus co-segregated with a gene encoding F3'5'H in soybean. These two genetic loci have identified to control seed coat, flower and pubescence colors. However, the allelic distributions of both F3'H and F3'5'H genes in soybean were unknown. In this study, three novel alleles were identified (two of four alleles for GmF3'H and one of three alleles for GmF3'5'H). A set of gene-tagged markers was developed and verified based on the sequence diversity of all seven alleles. Furthermore, the markers were used to analyze soybean accessions including 170 cultivated soybeans (G. max) from a mini core collection and 102 wild soybeans (G. soja). For both F3'H and F3'5'H, the marker selection efficiencies for pubescence color and flower color were determined. The results showed that one GmF3'H allele explained 92.2 % of the variation in tawny and two gmf3'h alleles explained 63.8 % of the variation in gray pubescence colors. In addition, two GmF3'5'H alleles and one gmF3'5'h allele explained 94.0 % of the variation in purple and 75.3 % in white flowers, respectively. By the combination of the two loci, seed coat color was determined. In total, 90.9 % of accessions possessing both the gmf3'h-b and gmf3'5'h alleles had yellow seed coats. Therefore, seed coat colors are controlled by more than two loci.

  6. Phenylalanine requirement, imbalance, and dietary excess in one-week-old chicks: growth and phenylalanine hydroxylase activity.

    PubMed

    Lartey, F M; Austic, R E

    2008-02-01

    Two experiments were performed to study Phe imbalance and toxicity in 1-wk-old Babcock B380 chicks resulting from the addition of either a mixture of indispensable amino acids lacking Phe (IAA - Phe) or excess Phe to a diet that was nutritionally adequate in Phe. Chicks received a preexperimental semipurified diet for 1 wk and experimental diets from 7 to 14 d of age. In the first experiment, the chicks were given diets with Phe levels at 0.24, 0.29, 0.34, 0.39, 0.44, and 0.49% of the diet to determine the Phe requirement. The requirement of the chicks for Phe, based on weight gain and feed efficiency, was determined to be 0.39% of the diet. In experiment 2, the IAA - Phe (10% of the diet) or excess Phe (2% of the diet) was added to a diet containing 0.44% Phe. Chicks given the IAA - Phe or excess Phe had significantly slower growth rates than chicks given the basal diet (P > or = 0.05). The activities of the major hepatic enzyme of Phe catabolism, Phe hydroxylase (PAH), were significantly higher than that of chicks fed the basal diet when the chicks were fed the diets containing IAA - Phe plus 1.1% Phe (P > or = 0.05) but not when chicks were fed the diet containing IAA - Phe alone. The activity of PAH in chicks given the excess (2%) Phe was nearly 4 times the activity of PAH in chicks given the basal diet. Adding IAA - Phe to the diet containing excess Phe also resulted in higher PAH activity than was observed in chicks fed the basal diet, although the activity was significantly lower than observed for chicks receiving the diet containing excess Phe alone (P > or = 0.05). It is concluded that hepatic PAH activity in chicks increases primarily in response to its substrate, Phe. A dietary amino acid load without Phe reduces this response to excess Phe.

  7. Brain tryptophan hydroxylase: purification of, production of antibodies to, and cellular and ultrastructural localization in serotonergic neurons of rat midbrain.

    PubMed Central

    Joh, T H; Shikimi, T; Pickel, V M; Reis, D J

    1975-01-01

    Tryptophan hydroxylase [EC 1.14.16.4; L-tryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating)], the enzyme catalyzing the rate-limiting step in the biosynthesis of serotonin, was purified 79-fold from the region of the raphe nucleus of rat midbrain by sequential column chromatography and disc-gel electrophoresis. In electrophoresis three bands were distinguished, A, B, and C, which, when separated and submitted individually to electrophoresis, reproduced the same three bands. Bands A and C were enzymatically active and inhibited by para-chlorohenylalanine. Antibodies produced to each of the three bands crossreacted by immuno double diffusion and electrophoresis with each other and homogenates of raphe nuclei; they completely inhibited enzyme activity only of tryptophan hydroxylase. Tryptophan hydroxylase was localized by light and electron immunohistochemistry to serotonin neutrons of the raphe. Ultrastructurally, in cell bodies, the enzyme was distributed in cytoplasm and in association with endoplasmic reticulum and Golgi apparatus. In dendrites and axons, it was associated with microtubules. Tryptophan hydroxylase in brain is only neuronal and cytoplasmic, exists in multiple forms, and is associated with microtubules, suggesting it may be transported from sites of synthesis in cell body into axons. Images PMID:1059145

  8. Neonatal dietary cholesterol and alleles of cholesterol 7-alpha hydroxylase affect piglet cerebrum weight, cholesterol concentration, and behavior

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This experiment was designed to test the effect of polymorphism in the cholesterol 7-alpha hydroxylase (CYP7) gene locus, and dietary cholesterol (C) on cerebrum C in neonatal pigs fed sow's milk formulas. Thirty-six pigs (18 male and 18 female) genetically selected for high (HG), or low (LG) plasma...

  9. Concomitant inhibition of prolyl hydroxylases and ROCK initiates differentiation of mesenchymal stem cells and PC12 towards the neuronal lineage.

    PubMed

    Pacary, Emilie; Petit, Edwige; Bernaudin, Myriam

    2008-12-12

    This study demonstrates that a prolyl hydroxylase inhibitor, FG-0041, is able, in combination with the ROCK inhibitor, Y-27632, to initiate differentiation of mesenchymal stem cells (MSCs) into neuron-like cells. FG-0041/Y-27632 co-treatment provokes morphological changes into neuron-like cells, increases neuronal marker expression and provokes modifications of cell cycle-related gene expression consistent with a cell cycle arrest of MSC, three events showing the engagement of MSC towards the neuronal lineage. Moreover, as we observed in our previous studies with cobalt chloride and desferroxamine, the activation of HIF-1 by this prolyl hydroxylase inhibitor is potentiated by Y-27632 which could explain at least in part the effect of this co-treatment on MSC neuronal differentiation. In addition, we show that this co-treatment enhances neurite outgrowth and tyrosine hydroxylase expression in PC12 cells. Altogether, these results evidence that concomitant inhibition of prolyl hydroxylases and ROCK represents a relevant protocol to initiate neuronal differentiation.

  10. Restricted expression of Neuroglobin in the mouse retina and co-localization with Melanopsin and Tyrosine Hydroxylase

    SciTech Connect

    Hundahl, C.A.; Fahrenkrug, J.; Luuk, H.; Hay-Schmidt, A.; Hannibal, J.

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Restricted Neuroglobin expression in the mouse retina. Black-Right-Pointing-Pointer Antibody validation using Neuroglobin-null mice. Black-Right-Pointing-Pointer Co-expression of Neuroglobin with Melanopsin and tyrosine hydroxylase. Black-Right-Pointing-Pointer No effect of Neuroglobin deficiency on neuronal survival. -- Abstract: Neuroglobin (Ngb), a neuronal specific oxygen binding heme-globin, reported to be expressed at high levels in most layers of the murine retina. Ngb's function is presently unknown, but based on its high expression level and oxygen binding capabilities Ngb was proposed to function as an oxygen reservoir facilitating oxygen metabolism in highly active neurons or to function as a neuroprotectant. In the present study, we re-examined the expression pattern of Ngb in the retina using a highly validated antibody. Furthermore, intactness of retino-hypothalamic projections and the retinal expression level of Melanopsin and Tyrosine Hydroxylase were investigated in Ngb-null mice. Ngb-immunoreactivity was found in a few neurons of the ganglion cell and inner nuclear layers co-expressing Melanopsin and Tyrosine Hydroxylase, respectively. Ngb deficiency neither affected the level of Melanopsin and Tyrosine Hydroxylase proteins nor the intactness of PACAP-positive retinohypothalamic projections in the suprachiasmatic nucleus. Based on the present results, it seems unlikely that Ngb could have a major role in retinal oxygen homeostasis and neuronal survival under normal conditions. The present study suggests that a number of previously published reports have relied on antibodies with dubious specificity.

  11. Biochemical properties of ectoine hydroxylases from extremophiles and their wider taxonomic distribution among microorganisms.

    PubMed

    Widderich, Nils; Höppner, Astrid; Pittelkow, Marco; Heider, Johann; Smits, Sander H J; Bremer, Erhard

    2014-01-01

    Ectoine and hydroxyectoine are well-recognized members of the compatible solutes and are widely employed by microorganisms as osmostress protectants. The EctABC enzymes catalyze the synthesis of ectoine from the precursor L-aspartate-β-semialdehyde. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction. This compatible solute possesses stress-protective and function-preserving properties different from those of ectoine. Hydroxylation of ectoine is carried out by the EctD protein, a member of the non-heme-containing iron (II) and 2-oxoglutarate-dependent dioxygenase superfamily. We used the signature enzymes for ectoine (EctC) and hydroxyectoine (EctD) synthesis in database searches to assess the taxonomic distribution of potential ectoine and hydroxyectoine producers. Among 6428 microbial genomes inspected, 440 species are predicted to produce ectoine and of these, 272 are predicted to synthesize hydroxyectoine as well. Ectoine and hydroxyectoine genes are found almost exclusively in Bacteria. The genome context of the ect genes was explored to identify proteins that are functionally associated with the synthesis of ectoines; the specialized aspartokinase Ask_Ect and the regulatory protein EctR. This comprehensive in silico analysis was coupled with the biochemical characterization of ectoine hydroxylases from microorganisms that can colonize habitats with extremes in salinity (Halomonas elongata), pH (Alkalilimnicola ehrlichii, Acidiphilium cryptum), or temperature (Sphingopyxis alaskensis, Paenibacillus lautus) or that produce hydroxyectoine very efficiently over ectoine (Pseudomonas stutzeri). These six ectoine hydroxylases all possess similar kinetic parameters for their substrates but exhibit different temperature stabilities and differ in their tolerance to salts. We also report the crystal structure of the Virgibacillus salexigens EctD protein in its apo

  12. Characterization of tyramine beta-hydroxylase in planarian Dugesia japonica: cloning and expression.

    PubMed

    Nishimura, Kaneyasu; Kitamura, Yoshihisa; Inoue, Takeshi; Umesono, Yoshihiko; Yoshimoto, Kanji; Taniguchi, Takashi; Agata, Kiyokazu

    2008-12-01

    The planarian Dugesia japonica has a relatively well-organized central nervous system (CNS) consisting of a brain and ventral nerve cords (VNCs), and can completely regenerate it CNS utilizing pluripotent stem cells present in the mesenchymal space. This remarkable capacity has begun to be exploited for research on neural regeneration. Recently, several kinds of molecular markers for labeling of neural subtypes have been reported in planarians. These molecular markers are useful for visualizing the distinct neural populations in planarians. In this study, we isolated a cDNA encoding tyramine beta-hydroxylase (TBH), an octopamine (OA) biosynthetic enzyme, by degenerate PCR in the planarian D. japonica, and named it DjTBH (D. japonica tyramine beta-hydroxylase). In order to examine whether DjTBH contributes to OA biosynthesis, we measured the OA content in DjTBH-knockdown planarians created by RNA interference. In addition, to examine the specificity of DjTBH for OA biosynthesis, we measured not only OA content but also noradrenaline (NA) content, because NA is synthesized by a pathway similar to that for OA. According to high-performance liquid chromatography analysis, the amount of OA, but not NA, was significantly decreased in DjTBH-knockdown planarians. In addition, we produced anti-DjTBH antibody to visualize the octopaminergic neural network. As shown by immunofluorescence analysis using anti-DjTBH antibody, DjTBH-immunopositive neurons were mainly distributed in the head region, and elongated their dendrites and/or axons along the VNCs. In order to visualize octopaminergic and dopaminergic nervous systems (phenolamine/catecholamine nervous system) in the planarian CNS, double-immunofluorescence analysis was carried out using both anti-DjTBH antibody and anti-DjTH (a planarian tyrosine hydroxylase) antibody. DjTBH-immunopositive neurons and DjTH-immunopositive neurons mainly formed distinct neural networks in the head region. Here, we demonstrated that Dj

  13. Classroom Demonstration of a Spot Test for Pbenylpyruvic Acid and Its Relationship to Phenylketonuria

    ERIC Educational Resources Information Center

    Halkides, Christopher J.

    2004-01-01

    Classical phenylketonuria (PKU) is caused by a lack activity in the enzyme phenylalanine hydroxylase, leading to elevated concentrations of phenylalanine in the blood. A simple demonstration and three advanced demonstrations of a spot test for phenylpyruvic acid and its relationship to phenylketonuria are given.

  14. Tyrosine hydroxylase (TH), its cofactor tetrahydrobiopterin (BH4), other catecholamine-related enzymes, and their human genes in relation to the drug and gene therapies of Parkinson's disease (PD): historical overview and future prospects.

    PubMed

    Nagatsu, Toshiharu; Nagatsu, Ikuko

    2016-11-01

    Tyrosine hydroxylase (TH), which was discovered at the National Institutes of Health (NIH) in 1964, is a tetrahydrobiopterin (BH4)-requiring monooxygenase that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines (CAs), such as dopamine, noradrenaline, and adrenaline. Since deficiencies of dopamine and noradrenaline in the brain stem, caused by neurodegeneration of dopamine and noradrenaline neurons, are mainly related to non-motor and motor symptoms of Parkinson's disease (PD), we have studied human CA-synthesizing enzymes [TH; BH4-related enzymes, especially GTP-cyclohydrolase I (GCH1); aromatic L-amino acid decarboxylase (AADC); dopamine β-hydroxylase (DBH); and phenylethanolamine N-methyltransferase (PNMT)] and their genes in relation to PD in postmortem brains from PD patients, patients with CA-related genetic diseases, mice with genetically engineered CA neurons, and animal models of PD. We purified all human CA-synthesizing enzymes, produced their antibodies for immunohistochemistry and immunoassay, and cloned all human genes, especially the human TH gene and the human gene for GCH1, which synthesizes BH4 as a cofactor of TH. This review discusses the historical overview of TH, BH4-, and other CA-related enzymes and their genes in relation to the pathophysiology of PD, the development of drugs, such as L-DOPA, and future prospects for drug and gene therapy for PD, especially the potential of induced pluripotent stem (iPS) cells.

  15. Biodegradation of methyl parathion and p-nitrophenol: evidence for the presence of a p-nitrophenol 2-hydroxylase in a Gram-negative Serratia sp. strain DS001.

    PubMed

    Pakala, Suresh B; Gorla, Purushotham; Pinjari, Aleem Basha; Krovidi, Ravi Kumar; Baru, Rajasekhar; Yanamandra, Mahesh; Merrick, Mike; Siddavattam, Dayananda

    2007-01-01

    A soil bacterium capable of utilizing methyl parathion as sole carbon and energy source was isolated by selective enrichment on minimal medium containing methyl parathion. The strain was identified as belonging to the genus Serratia based on a phylogram constructed using the complete sequence of the 16S rRNA. Serratia sp. strain DS001 utilized methyl parathion, p-nitrophenol, 4-nitrocatechol, and 1,2,4-benzenetriol as sole carbon and energy sources but could not grow using hydroquinone as a source of carbon. p-Nitrophenol and dimethylthiophosphoric acid were found to be the major degradation products of methyl parathion. Growth on p-nitrophenol led to release of stoichiometric amounts of nitrite and to the formation of 4-nitrocatechol and benzenetriol. When these catabolic intermediates of p-nitrophenol were added to resting cells of Serratia sp. strain DS001 oxygen consumption was detected whereas no oxygen consumption was apparent when hydroquinone was added to the resting cells suggesting that it is not part of the p-nitrophenol degradation pathway. Key enzymes involved in degradation of methyl parathion and in conversion of p-nitrophenol to 4-nitrocatechol, namely parathion hydrolase and p-nitrophenol hydroxylase component "A" were detected in the proteomes of the methyl parathion and p-nitrophenol grown cultures, respectively. These studies report for the first time the existence of a p-nitrophenol hydroxylase component "A", typically found in Gram-positive bacteria, in a Gram-negative strain of the genus Serratia.

  16. Sequence variation at the phenylalanine hydroxylase gene in the British Isles.

    PubMed Central

    Tyfield, L A; Stephenson, A; Cockburn, F; Harvie, A; Bidwell, J L; Wood, N A; Pilz, D T; Harper, P; Smith, I

    1997-01-01

    Using mutation and haplotype analysis, we have examined the phenylalanine hydroxylase gene in the phenylketonuria populations of four geographical areas of the British Isles: the west of Scotland, southern Wales, and southwestern and southeastern England. The enormous genetic diversity of this locus within the British Isles is demonstrated in the large number of different mutations characterized and in the variety of genetic backgrounds on which individual mutations are found. Allele frequencies of the more common mutations exhibited significant nonrandom distribution in a north/south differentiation. Differences between the west of Scotland and southwestern England may be related to different events in the recent and past histories of their respective populations. Similarities between southern Wales and southeastern England are likely to reflect the heterogeneity that is seen in and around two large capital cities. Finally, comparison with more recently colonized areas of the world corroborates the genealogical origin by range expansion of several mutations. PMID:9012412

  17. Overexpression, crystallization and preliminary X-ray crystallographic analysis of the ectoine hydroxylase from Sphingopyxis alaskensis.

    PubMed

    Hoeppner, Astrid; Widderich, Nils; Bremer, Erhard; Smits, Sander H J

    2014-04-01

    The ectoine hydroxylase (EctD) is a member of the non-haem-containing iron(II)- and 2-oxoglutarate-dependent dioxygenase superfamily. Its mononuclear iron centre is a prerequisite for the activity of this enzyme and promotes the O2-dependent oxidative decarboxylation of 2-oxoglutarate, which is coupled to a two-electron oxidation of the substrate ectoine to yield 5-hydroxyectoine. An expression and purification protocol for the EctD enzyme from Sphingopyxis alaskensis was developed and the protein was crystallized using the sitting-drop vapour-diffusion method. This resulted in two different crystal forms, representing the apo and iron-bound forms of the enzyme.

  18. Variation in hepatic aryl hydrocarbon hydroxylase activity in flounder, Platichthys flesus: A baseline study

    NASA Astrophysics Data System (ADS)

    Tarlebø, J.; Solbakken, J. E.; Palmork, K. H.

    1985-06-01

    This investigation is concerned with the natural variations in aryl hydrocarbon hydroxylase (AHH) activity of flounder ( Platichthys flesus L.) throughout the year. A general trend towards higher activity in males was observed. It became significant during gonadal maturation, a period during which the activity in females was inhibited. Addition of α-naphthoflavone inhibited AHH activity except in females with maturing gonads. Enzyme activity measured at the optimal temperature for incubation showed highest activity during spring followed by a significant decrease during summer. Activity increased again during autumn, followed by a second decrease in the winter season. When activity was calculated based on ambient water temperature at time of sampling, smaller fluctuations between different seasons were observed; the only significant variation was high activity in June. The results obtained indicate that AHH activity is affected by both exogenous and endogenous factors, which should be taken into consideration if AHH activity is used as a biological indicator of marine pollution effects.

  19. Overexpression of cinnamate 4-hydroxylase gene enhances biosynthesis of decursinol angelate in Angelica gigas hairy roots.

    PubMed

    Park, Nam Il; Park, Jee Hee; Park, Sang Un

    2012-02-01

    Angelica gigas is a medicinal plant that produces pyranocoumarins, including decursin (D) and decursinol angelate (DA), which have neuroprotective, anticancer, and antiandrogenic effects. In this study, the coumarin biosynthetic pathway was engineered to increase the production of DA. Specifically, a vector was constructed which contained the A. gigas phenylalanine ammonia-lyase (AgPAL) and cinnamate 4-hydroxylase (AgC4H) genes that were driven by the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic hairy roots that overexpressed AgPAL or AgC4H genes were obtained by using an Agrobacterium rhizogenes-mediated transformation system. Among them, only AgC4H-transgenic hairy root lines produced more DA than control transgenic hairy root lines. The enhanced gene expression corresponded to elevated C4H activities. This study showed the importance of C4H in the production of DA in A. gigas hairy root culture.

  20. Towards the identification of the allosteric Phe-binding site in phenylalanine hydroxylase.

    PubMed

    Carluccio, Carla; Fraternali, Franca; Salvatore, Francesco; Fornili, Arianna; Zagari, Adriana

    2016-01-01

    The enzyme phenylalanine hydroxylase (PAH) is defective in the inherited disorder phenylketonuria. PAH, a tetrameric enzyme, is highly regulated and displays positive cooperativity for its substrate, Phe. Whether Phe binds to an allosteric site is a matter of debate, despite several studies worldwide. To address this issue, we generated a dimeric model for Phe-PAH interactions, by performing molecular docking combined with molecular dynamics simulations on human and rat wild-type sequences and also on a human G46S mutant. Our results suggest that the allosteric Phe-binding site lies at the dimeric interface between the regulatory and the catalytic domains of two adjacent subunits. The structural and dynamical features of the site were characterized in depth and described. Interestingly, our findings provide evidence for lower allosteric Phe-binding ability of the G46S mutant than the human wild-type enzyme. This also explains the disease-causing nature of this mutant.

  1. Xenon and halogenated alkanes track putative substrate binding cavities in the soluble methane monooxygenase hydroxylase.

    PubMed

    Whittington, D A; Rosenzweig, A C; Frederick, C A; Lippard, S J

    2001-03-27

    To investigate the role of protein cavities in facilitating movement of the substrates, methane and dioxygen, in the soluble methane monooxygenase hydroxylase (MMOH), we determined the X-ray structures of MMOH from Methylococcus capsulatus (Bath) cocrystallized with dibromomethane or iodoethane, or by using crystals pressurized with xenon gas. The halogenated alkanes bind in two cavities within the alpha-subunit that extend from one surface of the protein to the buried dinuclear iron active site. Two additional binding sites were located in the beta-subunit. Pressurization of two crystal forms of MMOH with xenon resulted in the identification of six binding sites located exclusively in the alpha-subunit. These results indicate that hydrophobic species bind preferentially in preexisting cavities in MMOH and support the hypothesis that such cavities may play a functional role in sequestering and enhancing the availability of the physiological substrates for reaction at the active site.

  2. Noradrenergic lesioning with an anti-dopamine beta-hydroxylase immunotoxin

    NASA Technical Reports Server (NTRS)

    Picklo, M. J.; Wiley, R. G.; Lappi, D. A.; Robertson, D.

    1994-01-01

    Sympathectomy has been achieved by a variety of methods but each has its limitations. These include lack of tissue specificity, incomplete lesioning, and the age range of susceptibility to the lesioning. To circumvent these drawbacks, an immunotoxin was constructed using a monoclonal antibody against the noradrenergic specific enzyme dopamine beta-hydroxylase (D beta H) coupled via a disulfide bond to saporin, a ribosomal inactivating protein. Three days after intravenous injection of the anti-D beta H immunotoxin (50 micrograms) into adult Sprague-Dawley rats, 66% of neurons in the superior cervical ganglia were chromatolytic. Superior cervical ganglia neurons were poisoned in 1 day old and 1 week old (86% of neurons) neonatal rats following subcutaneous injection of 3.75 and 15 micrograms, respectively. The anti-D beta H immunotoxin will be a useful tool in the study of the peripheral noradrenergic system in adult and neonatal animals.

  3. Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases.

    PubMed

    Quinlan, Rena F; Jaradat, Tahhan T; Wurtzel, Eleanore T

    2007-02-15

    Carotenoids and their derivatives are essential for growth, development, and signaling in plants and have an added benefit as nutraceuticals in food crops. Despite the importance of the biosynthetic pathway, there remain open questions regarding some of the later enzymes in the pathway. The CYP97 family of P450 enzymes was predicted to function in carotene ring hydroxylation, to convert provitamin A carotenes to non-provitamin A xanthophylls. However, substrate specificity was difficult to investigate directly in plants, which mask enzyme activities by a complex and dynamic metabolic network. To characterize the enzymes more directly, we amplified cDNAs from a model crop, Oryza sativa, and used functional complementation in Escherichia coli to test activity and specificity of members of Clans A and C. This heterologous system will be valuable for further study of enzyme interactions and substrate utilization needed to understand better the role of CYP97 hydroxylases in plant carotenoid biosynthesis.

  4. Immobilization of tryptophan hydroxylase by immune adsorption: a method to study regulation of catalytic activity.

    PubMed

    Johansen, P A; Jennings, I; Cotton, R G; Kuhn, D M

    1992-12-01

    Tryptophan hydroxylase (TPH) can be immobilized by adsorption to Pansorbin after binding to the monoclonal antibody PH8. This method yields recoveries of 35%-40% of total TPH activity in crude extracts and can be completed in 1.5 h. The immobilized form of TPH retains the essential kinetic properties of the native enzyme and responds to activators (phosphatidylserine) and inhibitors (catechol compounds) as does the native enzyme. Unlike TPH in brain extracts, immobilized TPH is not activated by calcium-stimulated phosphorylating conditions. When extracts from which TPH has been precipitated, and which contain calcium-calmodulin dependent protein kinase are added to immobilized TPH, the activation of TPH is restored. This method of immobilization of TPH via immune-adsorption allows for the highly specific and rapid preparation of affinity purified TPH that can be used to study the regulation of this enzyme by a variety of effectors, especially protein kinases.

  5. Regulation of tyrosine hydroxylase transcription by hnRNP K and DNA secondary structure

    PubMed Central

    Banerjee, Kasturi; Wang, Meng; Cai, Elizabeth; Fujiwara, Nana; Baker, Harriet; Cave, John W.

    2014-01-01

    Regulation of tyrosine hydroxylase gene (Th) transcription is critical for specifying and maintaining the dopaminergic neuronal phenotype. Here we define a molecular regulatory mechanism for Th transcription conserved in tetrapod vertebrates. We show that heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transactivator of Th transcription. It binds to previously unreported and evolutionarily conserved G:C-rich regions in the Th proximal promoter. hnRNP K directly binds C-rich single DNA strands within these conserved regions and also associates with double-stranded sequences when proteins, such as CREB, are bound to an adjacent cis-regulatory element. The single DNA strands within the conserved G:C-rich regions adopt either G-quadruplex or i-motif secondary structures. We also show that small molecule-mediated stabilization of these secondary structures represses Th promoter activity. These data suggest that these secondary structures are targets for pharmacological modulation of the dopaminergic phenotype. PMID:25493445

  6. The effects of anti-inflammatory agents on skin tumor initiation and aryl hydrocarbon hydroxylase.

    PubMed

    Slaga, T J; Viaje, A; Bracken, W

    1977-02-01

    The effects of various clinically used anti-inflammatory agents on mouse skin tumorigenesis and aryl hydrocarbon hydroxylase (AHH) were investigated. Oxyphenbutazone, a nonsteroidal anti-inflammatory agent, inhibited 3-methylcholanthrene (MC) tumor initiation but was less effective than the steroidal anti-inflammatory agent, dexamethasone. Oxyphenbutazone was not found to induce AHH activity in mouse epidermis, whereas indomethacin and Seclazone had a slight inducing effect. When these agents were added directly to the in vitro AHH assay, they did not inhibit AHH activity. However, additional experiments have shown a decreased epidermally mediated covalent binding of MC to DNA in vitro when the epidermal homogenates were isolated from mice pretreated with either dexamethasone or oxyphenbutazone and MC at 3 or 12 hr before killing.

  7. Reproductive outcomes of female patients with congenital adrenal hyperplasia due to 21-hydroxylase defi ciency

    PubMed Central

    Mnif, Mouna Feki; Kamoun, Mahdi; Kacem, Faten Hadj; Mnif, Fatma; Charfi, Nadia; Naceur, Basma Ben; Rekik, Nabila; Abid, Mohamed

    2013-01-01

    Fertility in women with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) appears to be reduced, especially in women with the classic salt-wasting type. Several factors have been suggested to contribute to this subfertility such as androgen excess, adrenal progesterone hypersecretion, consequences of genital reconstructive surgery, secondary polycystic ovaries syndrome, and psychosexual factors. In contrast to this subfertility, pregnancies are commonly normal and uneventful. Adequate glucocorticoid therapy and improvement of surgical and psychological management could contribute to optimize fertility in CAH female patients, even among women with the classic variant. This review provides current information regarding the reproductive outcomes of women with CAH due to 21-OHD and the fertility and pregnancy issues in this population. PMID:24083158

  8. Purification of p-hydroxyphenylpyruvate hydroxylase from rat liver--requirement for cofactors.

    PubMed

    Lin, K T; Crawhall, J C

    1976-05-01

    The enzyme p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27) from rat liver was studied with the assay method which measures the release of 14CO2 from p-hydroxyphenyl [carboxy-14C]pyruvate. Extensive dialysis of the crude enzyme extract against Tris buffer or purification involving ammonium sulfate, gel filtration, and ion exchange results in loss of enzyme activity that can be reactivated by Fe2+, dichlorophenolindophenol, and various other agents. The effect of these activators depends critically on their final concentration in the assay media. A 70-fold purification of the enzyme fraction yielded a preparation which behaved as a single protein band in Sephadex G-150. It had an isoelectric point at 5.85 and molecular weight of 63 000. The enzyme obtained appears to be different in some respects from those described by other workers from the liver of dog, human, chicken, and frog.

  9. Pyrithione Zn selectively inhibits hypoxia-inducible factor prolyl hydroxylase PHD3.

    PubMed

    Na, Yu-Ran; Woo, Dustin J; Kim, So Yeon; Yang, Eun Gyeong

    2016-04-01

    Increasing evidence emphasizes the role of the hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) isoforms in regulating non-HIF substrates, but isoform selective PHD inhibitors under physiological conditions have not yet been reported. Here we have identified pyrithione Zn (PZ) as a potent, isoform-selective PHD3 inhibitor. The IC50 value of PZ was determined as 0.98 μM for PHD3, while it did not show any inhibitory activity toward full length and truncated PHD2 up to 1 mM. The selective efficacy of PZ was further demonstrated at the cellular level by observing inhibition of the PHD3-dependent DNA damage response pathway without stabilization of HIF-1α.

  10. Distribution of neurons expressing tyrosine hydroxylase in the human cerebral cortex

    PubMed Central

    Benavides-Piccione, Ruth; DeFelipe, Javier

    2007-01-01

    Since the very first detailed description of the different types of cortical interneurons by Cajal, the tremendous variation in the morphology, physiology and neurochemical properties of these cells has become apparent. However, it still remains unclear whether all types of interneurons are present in all cortical areas and species. Here we have focused on tyrosine hydroxylase (TH)-immunoreactive cortical interneurons, which although only present in certain species, are particularly abundant in the human neocortex. We argue that this type of interneuron is more widespread in the human neocortex than in any other species examined so far and that, therefore, it is probably involved in a larger variety of cortical circuits. In addition, notable regional variation can be seen in relation to these interneurons. These differences further emphasize the variability in the design of microcircuits between cortical areas and species, and they probably reflect an evolutionary adaptation of cortical circuits to particular functions. PMID:17593221

  11. Serum dopamine-beta-hydroxylase activity in rat during post-natal development.

    PubMed

    Lamprecht, F; Wooten, G F

    1976-01-01

    Rat serum dopamine-beta-hydroxylase (DBH) activity is quite high in the immediate post-natal period reaching peak activity (75 units) at 16 days of age. Activity then decreases rapidly over the following weeks to approach adult levels (10 units) by seven weeks of age. Also, specific activity of DBH in heart increased rapidly during the first 2 1/2 weeks of life attaining adult levels by 18 days of age. In contrast, heart weight and total DBH activity in whole heart increased in a coordinate fashion at a relatively constant rate throughout the first seven weeks of life. Serum levels of non-copper sensitive endogenous inhibitor (s) of DBH increased throughout the first seven weeks of life while no change in copper sensitive inhibition was observed. Also, the rapid phase of decrease in serum DBH activity corresponded to the period of the most rapid increase in body weight.

  12. Current advances in the novel functions of hypoxia-inducible factor and prolyl hydroxylase in invertebrates.

    PubMed

    Wang, L; Cui, S; Ma, L; Kong, L; Geng, X

    2015-12-01

    Oxygen is essential for aerobic life, and hypoxia has very severe consequences. Organisms need to overcome low oxygen levels to maintain biological functions during normal development and in disease states. The mechanism underlying the hypoxic response has been widely investigated in model animals such as Drosophila melanogaster and Caenorhabditis elegans. Hypoxia-inducible factor (HIF), a key gene product in the response to oxygen deprivation, is primarily regulated by prolyl hydroxylase domain enzymes (PHDs). However, recent findings have uncovered novel HIF-independent functions of PHDs. This review provides an overview of how invertebrates are able to sustain hypoxic damages, and highlights some recent discoveries in the regulation of cellular signalling by PHDs. Given that some core genes and major pathways are evolutionarily conserved, these research findings could provide insight into oxygen-sensitive signalling in mammals, and have biomedical implications for human diseases.

  13. Chronic stress effects on working memory: association with prefrontal cortical tyrosine hydroxylase.

    PubMed

    Lee, Young-A; Goto, Yukiori

    2015-06-01

    Chronic stress causes deficits in cognitive function including working memory, for which transmission of such catecholamines as dopamine and noradrenaline transmission in the prefrontal cortex (PFC) are crucial. Since catecholamine synthesis depends on the rate-limiting enzyme, tyrosine hydroxylase (TH), TH is thought to play an important role in PFC function. In this study, we found that two distinct population existed in Sprague-Dawley rats in terms of working memory capacity, one with higher working memory capacity, and the other with low capacity. This distinction of working memory capacity became apparent after rats were exposed to chronic stress. In addition, such working memory capacity and alterations of working memory function by chronic stress were associated with TH expression in the PFC.

  14. Procollagen Lysyl Hydroxylase 2 is Essential for Hypoxia-Induced Breast Cancer Metastasis

    PubMed Central

    Gilkes, Daniele; Bajpai, Saumendra; Wong, Carmen Chak-Lui; Chaturvedi, Pallavi; Hubbi, Maimon E.; Wirtz, Denis; Semenza, Gregg L.

    2013-01-01

    Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix in the metastatic process may lead to the development of improved therapies to treat cancer patients. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. We found that in hypoxic breast cancer cells, HIF-1 activates transcription of the PLOD1 and PLOD2 genes encoding procollagen lysyl hydroxylases that are required for the biogenesis of collagen, which is a major constituent of the extracellular matrix. High PLOD2 expression in breast cancer biopsies is associated with increased risk of mortality. We demonstrate that PLOD2 is critical for fibrillar collagen formation by breast cancer cells, increases tumor stiffness, and is required for metastasis to lymph nodes and lungs. PMID:23378577

  15. Elevation of molybdenum hydroxylase levels in rabbit liver after ingestion of phthalazine or its hydroxylated metabolite.

    PubMed

    Johnson, C; Stubley-Beedham, C; Stell, J G

    1984-11-15

    Oral administration of phthalazine (50 mg/kg/day) or 1-hydroxyphthalazine (10 mg/kg/day) to female rabbits caused an increase in the specific activity of the hepatic molybdenum hydroxylases aldehyde oxidase and xanthine oxidase, whereas no effect on microsomal cytochrome P-450 activity was observed. The rise in the specific activity of purified aldehyde oxidase fractions was accompanied by a similar increase in molybdenum content. A significant lowering of the Km value for phthalazine was demonstrated with enzyme from treated rabbits whereas Km values for structurally similar substrates such as isoquinoline were unchanged from control values. Iso-electric focusing of DEAE-cellulose fractions showed the presence of an additional band of activity indicating that genuine induction of aldehyde oxidase had occurred in rabbits treated with phthalazine or 1-hydroxyphthalazine.

  16. Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases

    PubMed Central

    Quinlan, Rena F.; Jaradat, Tahhan T.; Wurtzel, Eleanore T.

    2007-01-01

    Carotenoids and their derivatives are essential for growth, development, and signaling in plants and have an added benefit as nutraceuticals in food crops. Despite the importance of the biosynthetic pathway, there remain open questions regarding some of the later enzymes in the pathway. The CYP97 family of P450 enzymes was predicted to function in carotene ring hydroxylation, to convert provitamin A carotenes to nonprovitamin A xanthophylls. However, substrate specificity was difficult to investigate directly in plants, which mask enzyme activities by a complex and dynamic metabolic network. To characterize the enzymes more directly, we amplified cDNAs from a model crop, Oryza sativa, and used functional complementation in Escherichia coli to test activity and specificity of members of Clans A and C. This heterologous system will be valuable for further study of enzyme interactions and substrate utilization needed to understand better the role of CYP97 hydroxylases in plant carotenoid biosynthesis. PMID:17196929

  17. An exploratory evaluation of tyrosine hydroxylase inhibition in planaria as a model for parkinsonism.

    PubMed

    Prokai, David; Nguyen, Thinh; Kamrowski, Kurt; Chandra, Ashwin; Talamantes, Tatjana; Baxter, Lewis R; Prokai, Laszlo

    2013-11-26

    Planaria are the simplest organisms with bilateral symmetry and a central nervous system (CNS) with cephalization; therefore, they could be useful as model organisms to investigate mechanistic aspects of parkinsonism and to screen potential therapeutic agents. Taking advantage of the organism's anti-tropism towards light, we measured a significantly reduced locomotor velocity in planaria after exposure to 3-iodo-L-tyrosine, an inhibitor of tyrosine hydroxylase that is an enzyme catalyzing the first and rate-limiting step in the biosynthesis of catecholamines. A simple semi-automatic assay using videotaped experiments and subsequent evaluation by tracking software was also implemented to increase throughput. The dopaminergic regulation of locomotor velocity was confirmed by bromocriptine, a drug whose mechanisms of action to treat Parkinson's disease is believed to be through the stimulation of nerves that control movement.

  18. To Cheat or Not To Cheat: Tryptophan Hydroxylase 2 SNP Variants Contribute to Dishonest Behavior.

    PubMed

    Shen, Qiang; Teo, Meijun; Winter, Eyal; Hart, Einav; Chew, Soo H; Ebstein, Richard P

    2016-01-01

    Although, lying (bear false witness) is explicitly prohibited in the Decalogue and a focus of interest in philosophy and theology, more recently the behavioral and neural mechanisms of deception are gaining increasing attention from diverse fields especially economics, psychology, and neuroscience. Despite the considerable role of heredity in explaining individual differences in deceptive behavior, few studies have investigated which specific genes contribute to the heterogeneity of lying behavior across individuals. Also, little is known concerning which specific neurotransmitter pathways underlie deception. Toward addressing these two key questions, we implemented a neurogenetic strategy and modeled deception by an incentivized die-under-cup task in a laboratory setting. The results of this exploratory study provide provisional evidence that SNP variants across the tryptophan hydroxylase 2 (TPH2) gene, that encodes the rate-limiting enzyme in the biosynthesis of brain serotonin, contribute to individual differences in deceptive behavior.

  19. To Cheat or Not To Cheat: Tryptophan Hydroxylase 2 SNP Variants Contribute to Dishonest Behavior

    PubMed Central

    Shen, Qiang; Teo, Meijun; Winter, Eyal; Hart, Einav; Chew, Soo H.; Ebstein, Richard P.

    2016-01-01

    Although, lying (bear false witness) is explicitly prohibited in the Decalogue and a focus of interest in philosophy and theology, more recently the behavioral and neural mechanisms of deception are gaining increasing attention from diverse fields especially economics, psychology, and neuroscience. Despite the considerable role of heredity in explaining individual differences in deceptive behavior, few studies have investigated which specific genes contribute to the heterogeneity of lying behavior across individuals. Also, little is known concerning which specific neurotransmitter pathways underlie deception. Toward addressing these two key questions, we implemented a neurogenetic strategy and modeled deception by an incentivized die-under-cup task in a laboratory setting. The results of this exploratory study provide provisional evidence that SNP variants across the tryptophan hydroxylase 2 (TPH2) gene, that encodes the rate-limiting enzyme in the biosynthesis of brain serotonin, contribute to individual differences in deceptive behavior. PMID:27199691

  20. Hypotensive but not normotensive haemorrhage increases tryptophan hydroxylase-2 mRNA in caudal midline medulla.

    PubMed

    Brown, Heidi J; Henderson, Luke A; Keay, Kevin A

    2006-05-08

    Severe blood loss triggers shock, a precipitous hypotension and bradycardia. The integrity of (i) neurons in the vasodepressor region of the caudal midline medulla and (ii) central 5-HT neurotransmission are critical for the expression of haemorrhagic shock. This study investigated whether progressive blood loss triggers altered synthesis of 5-HT in the vasodepressor region of the caudal midline medulla by measuring changes in relative expression levels of tryptophan hydroxylase 2 (TpH 2) mRNA, the rate-limiting enzyme in the synthesis of neuronal 5-HT. Hypotensive but not normotensive haemorrhage triggered a significant increase in TpH 2 mRNA in the vasodepressor region of the caudal midline medulla, identifying an important role for 5-HT-containing caudal midline medullary neurons in haemorrhagic shock.

  1. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases

    NASA Astrophysics Data System (ADS)

    Chowdhury, Rasheduzzaman; Leung, Ivanhoe K. H.; Tian, Ya-Min; Abboud, Martine I.; Ge, Wei; Domene, Carmen; Cantrelle, François-Xavier; Landrieu, Isabelle; Hardy, Adam P.; Pugh, Christopher W.; Ratcliffe, Peter J.; Claridge, Timothy D. W.; Schofield, Christopher J.

    2016-08-01

    The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1-3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel-Lindau protein (VHL)-elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.

  2. Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines.

    PubMed

    Tomiya, Noboru; Narang, Someet; Lee, Yuan C; Betenbaugh, Michael J

    2004-01-01

    enzymes responsible for generating the essential donor sugar nucleotide, CMP- N -acetylneuraminic acid, required for sialylation. Inhibition of N -acetylglucosaminidase has also been applied to alter N-glycan processing in insect cells. This review summarizes current knowledge on N-glycan processing in lepidopteran insect cell lines, and recent progress in glycoengineering lepidopteran insect cells to produce glycoproteins containing complex N-glycans.

  3. [Congenital adrenal hyperplasia due to 21-hydroxylase deficiency--management in adults].

    PubMed

    Ambroziak, Urszula; Bednarczuk, Tomasz; Ginalska-Malinowska, Maria; Małunowicz, Ewa Maria; Grzechocińska, Barbara; Kamiński, Paweł; Bablok, Leszek; Przedlacki, Jerzy; Bar-Andziak, Ewa

    2010-01-01

    Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common autosomal recessive hereditary diseases. The impairment of cortisol synthesis leads to excessive stimulation of the adrenal glands by adrenocorticotropic hormone (ACTH), adrenal hyperplasia, and excessive androgen synthesis. The syndrome is characterised by a considerable correlation between the genotype and the phenotype with the type of CYP21A2 gene mutation affecting the severity of 21-hydroxylase deficiency. The clinical manifestations of CAH in adults result from adrenocortical and adrenomedullary insufficiency, hyperandrogenism, and the adverse effects of glucocorticosteroids used for the treatment of the condition. Non-classic CAH may sometimes be asymptomatic. In patients with classic CAH obesity, hyperinsulinaemia, insulin resistance, and hyperleptinaemia are more often seen than in the general population. These abnormalities promote the development of metabolic syndrome and its sequelae, including endothelial dysfunction, and cardiovascular disease. Long-term glucocorticosteroid treatment is also a known risk factor for osteoporosis. Patients with CAH require constant monitoring of biochemical parameters (17a-hydroxyprogesterone and androstenedione), clinical parameters (body mass, waist circumference, blood pressure, glucose, and lipids), and bone mineral density by densitometry. The principal goal of treatment in adults with CAH is to improve quality of life, ensure that they remain fertile, reduce the manifestations of hyperandrogenisation in females, and minimise the adverse effects of glucocorticosteroid treatment. Patients with classic CAH require treatment with glucocorticosteroids and, in cases of salt wasting, also with a mineralocorticosteroid. Radical measures, such as bilateral adrenalectomy, are very rarely needed. Asymptomatic patients with non-classic CAH require monitoring: treatment is not always necessary. Medical care for patients with CAH

  4. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency - management in adults.

    PubMed

    Ambroziak, Urszula; Bednarczuk, Tomasz; Ginalska-Malinowska, Maria; Małunowicz, Ewa Maria; Grzechocińska, Barbara; Kamiński, Paweł; Bablok, Leszek; Przedlacki, Jerzy; Bar-Andziak, Ewa

    2010-01-01

    Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common autosomal recessive hereditary diseases. The impairment of cortisol synthesis leads to excessive stimulation of the adrenal glands by adrenocorticotropic hormone (ACTH), adrenal hyperplasia, and excessive androgen synthesis. The syndrome is characterised by a considerable correlation between the genotype and the phenotype with the type of CYP21A2 gene mutation affecting the severity of 21-hydroxylase deficiency. The clinical manifestations of CAH in adults result from adrenocortical and adrenomedullary insufficiency, hyperandrogenism, and the adverse effects of glucocorticosteroids used for the treatment of the condition. Non-classic CAH may sometimes be asymptomatic. In patients with classic CAH obesity, hyperinsulinaemia, insulin resistance, and hyperleptinaemia are more often seen than in the general population. These abnormalities promote the development of metabolic syndrome and its sequelae, including endothelial dysfunction, and cardiovascular disease. Long-term glucocorticosteroid treatment is also a known risk factor for osteoporosis. Patients with CAH require constant monitoring of biochemical parameters (17a-hydroxyprogesterone [17-OHP] and androstenedione), clinical parameters (body mass, waist circumference, blood pressure, glucose, and lipids), and bone mineral density by densitometry. The principal goal of treatment in adults with CAH is to improve quality of life, ensure that they remain fertile, reduce the manifestations of hyperandrogenisation in females, and minimise the adverse effects of glucocorticosteroid treatment. Patients with classic CAH require treatment with glucocorticosteroids and, in cases of salt wasting, also with a mineralocorticosteroid. Radical measures, such as bilateral adrenalectomy, are very rarely needed. Asymptomatic patients with non-classic CAH require monitoring: treatment is not always necessary. Medical care for patients

  5. Osmotically induced synthesis of the compatible solute hydroxyectoine is mediated by an evolutionarily conserved ectoine hydroxylase.

    PubMed

    Bursy, Jan; Pierik, Antonio J; Pica, Nathalie; Bremer, Erhard

    2007-10-26

    By using natural abundance (13)C NMR spectroscopy, we investigated the types of compatible solutes synthesized in a variety of Bacilli under high salinity growth conditions. Glutamate, proline, and ectoine were the dominant compatible solutes synthesized by the various Bacillus species. The majority of the inspected Bacilli produced the tetrahydropyrimidine ectoine in response to high salinity stress, and a subset of these also synthesized a hydroxylation derivative of ectoine, 5-hydroxyectoine. In Salibacillus salexigens, a representative of the ectoine- and 5-hydroxyectoine-producing species, ectoine production was linearly correlated with the salinity of the growth medium and dependent on an ectABC biosynthetic operon. The formation of 5-hydroxyectoine was primarily a stationary growth phase phenomenon. The enzyme responsible for ectoine hydroxylation (EctD) was purified from S. salexigens to apparent homogeneity. The EctD protein was shown in vitro to directly hydroxylate ectoine in a reaction dependent on iron(II), molecular oxygen, and 2-oxoglutarate. We identified the structural gene (ectD) for the ectoine hydroxylase in S. salexigens. Northern blot analysis showed that the transcript levels of the ectABC and ectD genes increased as a function of salinity. Many EctD-related proteins can be found in data base searches in various Bacteria. Each of these bacterial species also contains an ectABC ectoine biosynthetic gene cluster, suggesting that 5-hydroxyectoine biosynthesis strictly depends on the prior synthesis of ectoine. Our data base searches and the biochemical characterization of the EctD protein from S. salexigens suggest that the EctD-related ectoine hydroxylases are members of a new subfamily within the non-heme-containing, iron(II)- and 2-oxoglutarate-dependent dioxygenase superfamily (EC 1.14.11).

  6. Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase

    PubMed Central

    Pallan, Pradeep S.; Wang, Chunxue; Lei, Li; Yoshimoto, Francis K.; Auchus, Richard J.; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

    2015-01-01

    Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613–10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C–H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 107 m−1 s−1) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 107 m−1 s−1) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants. PMID:25855791

  7. Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase.

    PubMed

    Coulombel, Lydie; Nolan, Louise C; Nikodinovic, Jasmina; Doyle, Evelyn M; O'Connor, Kevin E

    2011-03-01

    Escherichia coli cells, expressing 4-hydroxyphenylacetate 3-hydroxylase, fully transformed 4-halogenated phenols to their equivalent catechols as single products in shaken flasks. 4-Fluorophenol was transformed at a rate 1.6, 1.8, and 3.4-fold higher than the biotransformation of 4-chloro-, 4-bromo-, and 4-iodo-phenol, respectively. A scale-up from shaken flask to a 5 L stirred tank bioreactor was undertaken to develop a bioprocess for the production of 4-substituted halocatechols at higher concentrations and scale. In a stirred tank reactor, the optimized conditions for induction of 4-HPA hydroxylase expression were at 37 °C for 3 h. The rate of biotransformation of 4-fluorophenol to 4-fluorocatechol by stirred tank bioreactor grown cells was the same at 1 and 4.8 mM (5.13 μmol/min/g CDW) once the ratio of biocatalyst (E. coli CDW) to substrate concentration (mM) was maintained at 2:1. At 10.8 mM 4-fluorophenol, the rate of 4-fluorocatechol formation decreased by 4.7-fold. However, the complete transformation of 1.3 g of 4-fluorophenol (10.8 mM) to 4-fluorocatechol was achieved within 7 h in a 1 L reaction volume. Similar to 4-fluorophenol, other 4-substituted halophenols were completely transformed to 4-halocatechols at 2 mM within a 1-2 h period. An increase in 4-halophenol concentration to 4.8 mM resulted in a 2.5-20-fold decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol products followed by column chromatography resulted in the production of purified products with a final yield of between 33% and 38%.

  8. Alkane hydroxylase gene (alkB) phylotype composition and diversity in northern Gulf of Mexico bacterioplankton

    PubMed Central

    Smith, Conor B.; Tolar, Bradley B.; Hollibaugh, James T.; King, Gary M.

    2013-01-01

    Natural and anthropogenic activities introduce alkanes into marine systems where they are degraded by alkane hydroxylases expressed by phylogenetically diverse bacteria. Partial sequences for alkB, one of the structural genes of alkane hydroxylase, have been used to assess the composition of alkane-degrading communities, and to determine their responses to hydrocarbon inputs. We present here the first spatially extensive analysis of alkB in bacterioplankton of the northern Gulf of Mexico (nGoM), a region that experiences numerous hydrocarbon inputs. We have analyzed 401 partial alkB gene sequences amplified from genomic extracts collected during March 2010 from 17 water column samples that included surface waters and bathypelagic depths. Previous analyses of 16S rRNA gene sequences for these and related samples have shown that nGoM bacterial community composition and structure stratify strongly with depth, with distinctly different communities above and below 100 m. Although we hypothesized that alkB gene sequences would exhibit a similar pattern, PCA analyses of operational protein units (OPU) indicated that community composition did not vary consistently with depth or other major physical-chemical variables. We observed 22 distinct OPUs, one of which was ubiquitous and accounted for 57% of all sequences. This OPU clustered with AlkB sequences from known hydrocarbon oxidizers (e.g., Alcanivorax and Marinobacter). Some OPUs could not be associated with known alkane degraders, however, and perhaps represent novel hydrocarbon-oxidizing populations or genes. These results indicate that the capacity for alkane hydrolysis occurs widely in the nGoM, but that alkane degrader diversity varies substantially among sites and responds differently than bulk communities to physical-chemical variables. PMID:24376439

  9. The upregulation of immune responses in tyrosine hydroxylase (TH) silenced Litopenaeus vannamei.

    PubMed

    Mapanao, Ratchaneegorn; Chang, Chin-Chyuan; Cheng, Winton

    2017-02-01

    Catecholamines (CAs) play a crucial role in maintaining physiological and immune homeostasis in invertebrates and vertebrates under stressful conditions. Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in CA synthesis. To develop an effective CA-related immunological defense system against stress and pathogen infection, various criteria, were evaluated in TH double-stranded (ds) RNA-injected white shrimp, Litopenaeus vannamei. Specifically, the relative transcript quantification of TH, dopamine β-hydroxylase (DBH), crustacean hyperglycemic hormone (CHH), and other immune-related genes; TH activity in the haemolymph; and the estimation of l-dihydroxyphenylalanine (l-DOPA), glucose, and lactate levels in the haemolymph were examined. TH depletion revealed a significant increase in the total haemocyte count; granular cells; semigranular cells; respiratory bursts (RBs, release of superoxide anion); superoxide dismutase (SOD) activity; phagocytic activity and clearance efficiency; and the expression of lipopolysaccharide and β-1,3-glucan-binding protein and peroxinectin, SOD, crustin, and lysozyme genes. In addition, the reduction of TH gene expression and activity was accompanied by a decline of phenoloxidase (PO) activity per granulocyte, lower glucose and lactate levels, and significantly low expression of DBH and CHH genes. However, the number of hyaline cells, activity of PO, RBs per haemocyte, and expression of POI and POII genes were not significantly different in the LvTH-silenced shrimp. Notably, the survival ratio of LvTH-silenced shrimp was significantly higher than that of shrimp injected with diethyl pyrocarbonate-water and nontargeting dsRNA when challenged with Vibrio alginolyticus. Therefore, the depletion of TH can enhance disease resistance in shrimp by upregulating specific immune parameters but downregulating the levels of carbohydrate metabolites.

  10. Deregulation of the lysyl hydroxylase matrix cross-linking system in experimental and clinical bronchopulmonary dysplasia

    PubMed Central

    Witsch, Thilo J.; Turowski, Paweł; Sakkas, Elpidoforos; Niess, Gero; Becker, Simone; Herold, Susanne; Mayer, Konstantin; Vadász, István; Roberts, Jesse D.; Seeger, Werner

    2013-01-01

    Bronchopulmonary dysplasia (BPD) is a common and serious complication of premature birth, characterized by a pronounced arrest of alveolar development. The underlying pathophysiological mechanisms are poorly understood although perturbations to the maturation and remodeling of the extracellular matrix (ECM) are emerging as candidate disease pathomechanisms. In this study, the expression and regulation of three members of the lysyl hydroxylase family of ECM remodeling enzymes (Plod1, Plod2, and Plod3) in clinical BPD, as well as in an experimental animal model of BPD, were addressed. All three enzymes were localized to the septal walls in developing mouse lungs, with Plod1 also expressed in the vessel walls of the developing lung and Plod3 expressed uniquely at the base of developing septa. The expression of plod1, plod2, and plod3 was upregulated in the lungs of mouse pups exposed to 85% O2, an experimental animal model of BPD. Transforming growth factor (TGF)-β increased plod2 mRNA levels and activated the plod2 promoter in vitro in lung epithelial cells and in lung fibroblasts. Using in vivo neutralization of TGF-β signaling in the experimental animal model of BPD, TGF-β was identified as the regulator of aberrant plod2 expression. PLOD2 mRNA expression was also elevated in human neonates who died with BPD or at risk for BPD, compared with neonates matched for gestational age at birth or chronological age at death. These data point to potential roles for lysyl hydroxylases in normal lung development, as well as in perturbed late lung development associated with BPD. PMID:24285264

  11. Bile acids: regulation of synthesis.

    PubMed

    Chiang, John Y L

    2009-10-01

    Bile acids are physiological detergents that generate bile flow and facilitate intestinal absorption and transport of lipids, nutrients, and vitamins. Bile acids also are signaling molecules and inflammatory agents that rapidly activate nuclear receptors and cell signaling pathways that regulate lipid, glucose, and energy metabolism. The enterohepatic circulation of bile acids exerts important physiological functions not only in feedback inhibition of bile acid synthesis but also in control of whole-body lipid homeostasis. In the liver, bile acids activate a nuclear receptor, farnesoid X receptor (FXR), that induces an atypical nuclear receptor small heterodimer partner, which subsequently inhibits nuclear receptors, liver-related homolog-1, and hepatocyte nuclear factor 4alpha and results in inhibiting transcription of the critical regulatory gene in bile acid synthesis, cholesterol 7alpha-hydroxylase (CYP7A1). In the intestine, FXR induces an intestinal hormone, fibroblast growth factor 15 (FGF15; or FGF19 in human), which activates hepatic FGF receptor 4 (FGFR4) signaling to inhibit bile acid synthesis. However, the mechanism by which FXR/FGF19/FGFR4 signaling inhibits CYP7A1 remains unknown. Bile acids are able to induce FGF19 in human hepatocytes, and the FGF19 autocrine pathway may exist in the human livers. Bile acids and bile acid receptors are therapeutic targets for development of drugs for treatment of cholestatic liver diseases, fatty liver diseases, diabetes, obesity, and metabolic syndrome.

  12. Mechanisms for increased expression of cholesterol 7α-hydroxylase (Cyp7a1) in lactating rats

    PubMed Central

    Wooton-Kee, Clavia Ruth; Coy, Donna J; Athippozhy, Antony T; Zhao, Tianyong; Jones, Brett R; Vore, Mary

    2009-01-01

    Cholesterol 7α-hydroxylase (Cyp7a1) and the bile acid pool size are increased 2–3 fold in lactating postpartum rats. We investigated the interaction of nuclear receptors with the Cyp7a1 proximal promoter and the expression of regulatory signaling pathways in postpartum rats at day 10 (PPd10) vs female controls to identify the mechanisms of increased expression of Cyp7a1, which is maximal at 16 h. Liver X receptor (LXRα) and RNA Polymerase II (RNA Pol II) recruitment to Cyp7a1 chromatin were increased 1.5- and 2.5-fold, respectively, at 16 h on PPd10. Expression of nuclear receptors farnesoid X receptor (FXR), LXRα, liver receptor homologue (LRH-1), hepatocyte nuclear factor 4α (HNF4α), and short heterodimer partner (SHP) mRNA and co-activator peroxisome proliferators-activated receptor γ coactivator-1α (PGC-1α) mRNA was unchanged in PPd10 vs controls at 16 h, while chicken ovalbumin upstream transcription factor II (COUP-TFII) was decreased 40% at 16 h. Investigation of a repressive signaling pathway, the cJun-N-terminal kinase (JNK) signaling pathway in PPd10 vs controls, showed decreased mRNA expression of hepatocyte growth factor (HGF; decreased 60% at 16 h) and tyrosine kinase receptor cMet (decreased 44–50% at 16 h), but these were not accompanied by decreased expression of phosphorylated c-Jun. Importantly, expression of Fibroblast Growth Factor 15 (FGF15) mRNA in the ileum was decreased 70% in PPd10 vs controls, while phosphorylated mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (Erk1/2) protein expression in liver was decreased 88% at 16 h. Conclusion The increased recruitment of LXRα, a Cyp7a1 stimulatory pathway, and decreased expression of FGF15 and phosphorylated Erk1/2, a Cyp7a1 repressive pathway, combined to increase Cyp7a1 expression during lactation. PMID:19957370

  13. The FAAH inhibitor URB597 efficiently reduces tyrosine hydroxylase expression through CB1- and FAAH-independent mechanisms

    PubMed Central

    Bosier, Barbara; Muccioli, Giulio G; Lambert, Didier M

    2013-01-01

    Background Anandamide and 2-arachidonoylglycerol are neuromodulatory lipids interacting with cannabinoid receptors, whose availability is regulated by the balance between ‘on demand’ generation and enzymatic degradation [by fatty acid amide hydrolase (FAAH)/monoacylglycerol lipase]. Given the reported effects of anandamide on dopamine transmission, we investigated the influence of endocannabinoids and URB597, a well-known FAAH inhibitor, on the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. Experimental Approach We investigated TH expression in N1E115 neuroblastoma using a reporter gene assay, as well as mRNA and protein quantifications. FAAH inhibition was confirmed by measuring radiolabelled substrate hydrolysis and endogenous endocannabinoids. Key Results Anandamide decreased TH promoter activity in N1E115 cells through CB1 receptor activation. Unexpectedly, URB597 reduced TH expression (pEC50 = 8.7 ± 0.2) through FAAH-independent mechanisms. Indeed, four structurally unrelated inhibitors of FAAH had no influence on TH expression, although all the inhibitors increased endocannabinoid levels. At variance with the endocannabinoid responses, the use of selective antagonists indicated that the URB597-mediated decrease in TH expression was not directed by the CB1 receptor, but rather by abnormal-cannabidiol-sensitive receptors and PPARs. Further supporting the physiological relevance of these in vitro data, URB597 administration resulted in reduced TH mRNA levels in mice brain. Conclusions While confirming the implication of endocannabinoids on the modulation of TH, we provide strong evidence for additional physiologically relevant off-target effects of URB597. In light of the numerous preclinical studies involving URB597, particularly in anxiety and depression, the existence of non-CB1 and non-FAAH mediated influences of URB597 on key enzymes of the catecholaminergic transmission system should be taken into account when

  14. Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9α-Hydroxylase Enzymes

    PubMed Central

    Petrusma, Mirjan; van der Geize, Robert

    2012-01-01

    Rieske nonheme monooxygenase 3-ketosteroid 9α-hydroxylase (KSH) enzymes play a central role in bacterial steroid catabolism. KSH is a two-component iron-sulfur-containing enzyme, with KshA representing the terminal oxygenase component and KshB the reductase component. We previously reported that the KshA1 and KshA5 homologues of Rhodococcus rhodochrous DSM43269 have clearly different substrate preferences. KshA protein sequence alignments and three-dimensional crystal structure information for KshAH37Rv of Mycobacterium tuberculosis H37Rv served to identify a variable region of 58 amino acids organized in a β sheet that is part of the so-called helix-grip fold of the predicted KshA substrate binding pocket. Exchange of the β sheets between KshA1 and KshA5 resulted in active chimeric enzymes with substrate preferences clearly resembling those of the donor enzymes. Exchange of smaller parts of the KshA1 and KshA5 β-sheet regions revealed that a highly variable loop region located at the entrance of the active site strongly contributes to KSH substrate preference. This loop region may be subject to conformational changes, thereby affecting binding of different substrates in the active site. This study provides novel insights into KshA structure-function relationships and shows that KSH monooxygenase enzymes are amenable to protein engineering for the development of biocatalysts with improved substrate specificities. PMID:22020644

  15. Differential expression of flavonoid 3'-hydroxylase during fruit development establishes the different B-ring hydroxylation patterns of flavonoids in Fragaria × ananassa and Fragaria vesca.

    PubMed

    Thill, Jana; Miosic, Silvija; Gotame, Tek Prasad; Mikulic-Petkovsek, Maja; Gosch, Christian; Veberic, Robert; Preuss, Anja; Schwab, Wilfried; Stampar, Franci; Stich, Karl; Halbwirth, Heidi

    2013-11-01

    Flavonoid 3'-hydroxylase (F3'H) was studied for the first time in different Fragaria species. The cDNA clones isolated from unripe and ripe fruits of Fragaria x ananassa (garden strawberry) and Fragaria vesca (wild strawberry) showed high similarity (99% at the amino acid level) to the publically available F. vesca genome sequence and no significant differences could be identified between species and developmental stages of the fruits. In contrast, the genomic F3'H clones showed differences in the non-coding regions and 5'-flanking elements. The recombinant F3'Hs were functionally active and showed high specificity for naringenin, dihydrokaempferol, and kaempferol, whereas apigenin was only a minor substrate. During fruit development, a clear difference in the F3'H expression was observed between F. × ananassa and F. vesca. While a drastic decline of F3'H expression occurred during fruit ripening in F. × ananassa, F3'H in F. vesca was highly expressed in all stages. This was reflected by the anthocyanin composition, which showed a prevalence of pelargonidin in ripe fruits of F. × ananassa, whereas F. vesca had a high content of cyanidin. Screening of 17 berry species for their anthocyanin and flavonol composition showed that the prevalence of monohydroxylated anthocyanins makes garden strawberry unique among all other fruit species indicating that selection of bright red color during strawberry breeding, which consumers typically associate with freshness and ripeness, has selected phenotypes with a special biochemical background.

  16. Biosynthesis and metabolism of salicylic acid.

    PubMed Central

    Lee, H I; León, J; Raskin, I

    1995-01-01

    Pathways of salicylic acid (SA) biosynthesis and metabolism in tobacco have been recently identified. SA, an endogenous regulator of disease resistance, is a product of phenylpropanoid metabolism formed via decarboxylation of trans-cinnamic acid to benzoic acid and its subsequent 2-hydroxylation to SA. In tobacco mosaic virus-inoculated tobacco leaves, newly synthesized SA is rapidly metabolized to SA O-beta-D-glucoside and methyl salicylate. Two key enzymes involved in SA biosynthesis and metabolism: benzoic acid 2-hydroxylase, which converts benzoic acid to SA, and UDPglucose:SA glucosyltransferase (EC 2.4.1.35), which catalyzes conversion of SA to SA glucoside have been partially purified and characterized. Progress in enzymology and molecular biology of SA biosynthesis and metabolism will provide a better understanding of signal transduction pathway involved in plant disease resistance. PMID:11607533

  17. Biosynthesis and metabolism of salicylic acid

    SciTech Connect

    Lee, H.; Leon, J.; Raskin, I.

    1995-05-09

    Pathways of salicylic acid (SA) biosynthesis and metabolism in tobacco have been recently identified. SA, an endogenous regulator of disease resistance, is a product of phenylpropanoid metabolism formed via decarboxylation of trans-cinnamic acid to benzoic acid and its subsequent 2-hydroxylation to SA. In tobacco mosaic virus-inoculated tobacco leaves, newly synthesized SA is rapidly metabolized to SA O-{beta}-D-glucoside and methyl salicylate. Two key enzymes involved in SA biosynthesis and metabolism: benzoic acid 2-hydroxylase, which converts benzoic acid to SA, and UDPglucose:SA glucosyltransferase (EC 2.4.1.35), which catalyzes conversion of SA to SA glucoside have been partially purified and characterized. Progress in enzymology and molecular biology of SA biosynthesis and metabolism will provide a better understanding of signal transduction pathway involved in plant disease resistance. 62 refs., 1 fig.

  18. Evolution of rosmarinic acid biosynthesis.

    PubMed

    Petersen, Maike; Abdullah, Yana; Benner, Johannes; Eberle, David; Gehlen, Katja; Hücherig, Stephanie; Janiak, Verena; Kim, Kyung Hee; Sander, Marion; Weitzel, Corinna; Wolters, Stefan

    2009-01-01

    Rosmarinic acid and chlorogenic acid are caffeic acid esters widely found in the plant kingdom and presumably accumulated as defense compounds. In a survey, more than 240 plant species have been screened for the presence of rosmarinic and chlorogenic acids. Several rosmarinic acid-containing species have been detected. The rosmarinic acid accumulation in species of the Marantaceae has not been known before. Rosmarinic acid is found in hornworts, in the fern family Blechnaceae and in species of several orders of mono- and dicotyledonous angiosperms. The biosyntheses of caffeoylshikimate, chlorogenic acid and rosmarinic acid use 4-coumaroyl-CoA from the general phenylpropanoid pathway as hydroxycinnamoyl donor. The hydroxycinnamoyl acceptor substrate comes from the shikimate pathway: shikimic acid, quinic acid and hydroxyphenyllactic acid derived from l-tyrosine. Similar steps are involved in the biosyntheses of rosmarinic, chlorogenic and caffeoylshikimic acids: the transfer of the 4-coumaroyl moiety to an acceptor molecule by a hydroxycinnamoyltransferase from the BAHD acyltransferase family and the meta-hydroxylation of the 4-coumaroyl moiety in the ester by a cytochrome P450 monooxygenase from the CYP98A family. The hydroxycinnamoyltransferases as well as the meta-hydroxylases show high sequence similarities and thus seem to be closely related. The hydroxycinnamoyltransferase and CYP98A14 from Coleus blumei (Lamiaceae) are nevertheless specific for substrates involved in RA biosynthesis showing an evolutionary diversification in phenolic ester metabolism. Our current view is that only a few enzymes had to be "invented" for rosmarinic acid biosynthesis probably on the basis of genes needed for the formation of chlorogenic and caffeoylshikimic acid while further biosynthetic steps might have been recruited from phenylpropanoid metabolism, tocopherol/plastoquinone biosynthesis and photorespiration.

  19. Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase.

    PubMed

    Turner, Glenn W; Croteau, Rodney

    2004-12-01

    We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.

  20. The effect of butaclamol and of other neuroleptic agents on the apomorphine-elicited inhibition of synaptosomal tyrosine hydroxylase activity.

    PubMed

    Bronaugh, R L; Tabak, J; Ohashi, T; Goldstein, M

    1975-01-01

    The effects of the two enantiomers of butaclamol and of several neuroleptics on the apomorphine-elicited inhibition of synaptosomal tyrosine hydroxylase activity was investigated. The (+) but not the (-) enantiomer of butaclamol reverses the apomorphine-elicited enzyme inhibition. (+) Butaclamol is more potent than the other tested neuroleptics. All the tested neuroleptics reverse the apomorphine-elicited enzyme inhibition but their relative potency differs. Using two criteria, namely the concentrations of neuroleptics required to reverse enzyme inhibition maximally or by 25%, the order of decreasing potency is as follows: (+) butaclamol, fluphenazine, haloperidol, pimozide, chlorpromazine. The results suggest that the reversal of apomorphine-elicited inhibition of synaptosomal tyrosine hydroxylase activity is a valid test model for screening antipsychotic drugs.

  1. Mutation R96W in cytochrome P450c17 gene causes combined 17{alpha}-hydroxylase/17-20-lyase deficiency in two french canadian patients

    SciTech Connect

    LaFlamme, N.; Leblanc, J.F.; Mailloux, J.

    1996-01-01

    Congenital adrenal hyperplasia (CAH) is the most frequent cause of adrenal insufficiency and ambiguous genitalia in newborn children. In contrast to CAH caused by 21{alpha}-hydroxylase and 11{beta}-hydroxylase deficiencies, which impairs steroid formation in the adrenal exclusively, 17{alpha}-hydroxylase/17,20-lyase deficiency impairs steroid biosynthesis in the adrenals and gonads. The sequence of CYP17 gene was determined by direct sequencing of asymmetric PCR products in two French-Canadian 46,XY pseudohermaphrodite siblings suffering from combined 17{alpha}-hydroxylase/17,20-lyase deficiency. The two patients are homozygous for the novel missense mutation R96W caused by a C to T transition converting codon Arg{sup 96} (CGG) into a Trp (TGG) in exon 1. Both parents are heterozygous for this missense mutation. We assessed the effect of the R96W mutation on 17{alpha}-hydroxylase/17,20-lyase activity by analysis of mutant enzyme, generated by site-directed mutagenesis, expressed in COS-1 cells. The presence of R96W substitution almost completely abolished the activity of the mutant protein. The present findings provide a molecular explanation for the signs and symptoms of combined 17 {alpha}-hydroxylase/17,20-lyase deficiency in these two patients and provide useful information on the structure-activity relationships of the P450c17 enzyme. 31 refs., 4 figs., 1 tab.

  2. Comparison of Aryl Hydrocarbon Hydroxylase Induction in Cultured Blood Lymphocytes and Pulmonary Macrophages

    PubMed Central

    McLemore, Theodore L.; Martin, R. Russell; Toppell, Kenneth L.; Busbee, David L.; Cantrell, Elroy T.

    1977-01-01

    Aryl hydrocarbon hydroxylase induction was studied in cultured peripheral blood lymphocytes and pulmonary alveolar macrophages from 15 smokers and 8 nonsmokers with a variety of pulmonary diseases. Enzyme levels in lymphocytes from cigarette smokers cultured in medium without an inducing agent were 57±6 mU/106 cells (mean±SEM), while enzyme levels in lymphocytes from nonsmokers were 20±2 mU/106 cells (P < 0.001). When lymphocytes were cultured in the presence of the inducing agent, benzo-(a)anthracene, enzyme activity was increased to 168±23 mU/106 cells in smokers' cells and 99±22 mU/106 cells in lymphocytes from nonsmokers (P < 0.04). When noninduced enzyme values in cultured macrophages were compared, smokers' cells had enzyme levels of 45±5 mU/106 cells, whereas nonsmokers had enzyme activity of 24±2 mU/106 cells (P < 0.002). However, pulmonary macrophages from smokers or nonsmokers, cultured in the presence of benzo(a)-anthracene, had similar levels of induced enzyme activity (P > 0.1). A positive correlation was observed for nonsmokers (r = 0.596, P > 0.1 <0.2) or smokers (r = 0.640, P < 0.04), when enzyme values for noninduced cultures of macrophages and lymphocytes from individual patients were simultaneously compared. Enzyme values for macrophages and lymphocytes cultured in the presence of an inducer also revealed a positive correlation for individual smokers (r = 0.801, P < 0.001) or nonsmokers (r = 0.785, P < 0.01). Inducibility (expressed as fold-induction) for macrophages and lymphocytes from individual patients was also positively correlated (r = 0.889, P < 0.001 for nonsmokers and r = 0.942, P < 0.001 for smokers). These results indicate that the capacity for aryl hydrocarbon hydroxylase induction is similar whether tested in lymphocytes or pulmonary macrophages from this group of pulmonary disease patients. PMID:908748

  3. New, tritium-release assay for 25-hydroxyvitamin D-1. cap alpha. -hydroxylase

    SciTech Connect

    Brown, A.J.; Perlman, K.; DeLuca, H.F.

    1986-05-01

    A new, rapid assay for 25-hydroxyvitamin D (25-OH-D)-1..cap alpha..-hydroxylase has been developed using 25-OH-(1..cap alpha..-/sup 3/H)D/sub 3/ as substrate. This compound was prepared by reduction of 1-oxo-25-hydroxycyclovitamin D/sub 3/ with (/sup 3/H)NaBH/sub 4/, separation of the 1..cap alpha..- and 1..beta..-hydroxy products by HPLC, subsequent treatments with methylsulfonylchloride and lithium aluminum hydride, cycloreversion, and saponification. The 1..cap alpha..- and 1..beta..-tritiated substrates were tested in the solubilized and reconstituted chick 1..cap alpha..-hydroxylase system. After incubation, the reaction mixture was passed through a reversed phase silica cartridge to separate (/sup 3/H)H/sub 2/O from the labeled substrate. The cartridges were then washed with methanol to elute all vitamin D metabolites, and the amount of 1,25-(OH)/sub 2/(/sup 3/H)D/sub 3/ was measured by HPLC. In addition, identical reaction mixtures using 25-OH-(26,27-/sup 3/H)D/sub 3/ as substrate were extracted and analyzed by HPLC for 1,25-(OH)/sub 2/(/sup 3/H)D/sub 3/. Reactions with 25-OH-(1..cap alpha..-/sup 3/H)D/sub 3/ produced (/sup 3/H)H/sub 2/O comparable to the amount of 1,25-(OH)/sub 2/(26,27-/sup 3/H)D/sub 3/ and negligible (/sup 3/H) in 1,25-(OH)/sub 2/D/sub 3/. Conversely, reactions with 25-OH-(1..beta..-/sup 3/H)D/sub 3/ produced negligible (/sup 3/H)H/sub 2/O but produced 1,25-(OH)/sub 2/(/sup 3/H)D/sub 3/ comparable to that from reactions with 25-OH-(26,27-/sup 3/H)D/sub 3/. The results indicate that 1..cap alpha..-hydroxylation specifically displaces the 1..cap alpha..-hydrogen of 25-OH-D/sub 3/ and that the release of the 1..cap alpha..-/sup 3/H provides an accurate measure of vitamin D 1..cap alpha..-hydroxylation.

  4. Inhibition of prolyl 4-hydroxylase decreases muscle fibrosis following chronic rotator cuff tear

    PubMed Central

    Gumucio, J. P.; Flood, M. D.; Bedi, A.; Kramer, H. F.; Russell, A. J.

    2017-01-01

    Objectives Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1α is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1α following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics. Methods Chronic supraspinatus tears were induced in adult rats, and repaired 28 days later. Rats received 0 mg/kg, 3 mg/kg, or 10 mg/kg GSK1120360A daily. Collagen content, contractility, fibre type distribution and size, the expression of genes involved in fibrosis, lipid accumulation, atrophy and inflammation, and the mechanical properties of the enthesis were then assessed two weeks following surgical repair. Results At two weeks following repair, treatment groups showed increased muscle mass but there was a 15% decrease in force production in the 10 mg/kg group from controls, and no difference between the 0 mg/kg and the 3 mg/kg groups. There was a decrease in the expression of several gene transcripts related to matrix accumulation and fibrosis, and a 50% decrease in collagen content in both treated groups compared with controls. Additionally, the expression of inflammatory genes was reduced in the treated groups compared with controls. Finally, PHD inhibition improved the maximum stress and displacement to failure in repaired tendons. Conclusions GSK1120360A resulted in improved enthesis mechanics with variable effects on muscle function. PHD inhibition may be beneficial for connective tissue injuries in which muscle atrophy has not occurred. Cite this article: J. P. Gumucio

  5. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

    PubMed

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2016-03-29

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo.

  6. Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations.

    PubMed

    Menabò, Soara; Polat, Seher; Baldazzi, Lilia; Kulle, Alexandra E; Holterhus, Paul-Martin; Grötzinger, Joachim; Fanelli, Flaminia; Balsamo, Antonio; Riepe, Felix G

    2014-05-01

    Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11β-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling.

  7. Regulation of rat liver phenylalanine hydroxylase. II. Substrate binding and the role of activation in the control of enzymatic activity.

    PubMed

    Shiman, R; Xia, T; Hill, M A; Gray, D W

    1994-10-07

    Activation by phenylalanine and reduction by the co-factor (6R)-tetrahydrobiopterin (BH4) are required for formation of active liver phenylalanine hydroxylase. This work describes effects of the activation and redox state on substrate and effector recognition of this enzyme, it establishes relationships among the pterin and phenylalanine binding sites on the different forms of the enzyme, and it provides a quantitative description of the enzyme's presumptive regulatory and catalytic sites. BH4, 7,8-dihydrobiopterin (BH2), 6-methyltetrahydropterin, and 5-deaza-6-methyltetrahydropterin were found to bind to unactivated phenylalanine hydroxylase with a stoichiometry of 1/enzyme subunit and with hyperbolic kinetics; all appear to compete for the same binding site on the enzyme, and all appear to bind in the proximity of, but not to, the enzyme's non-heme iron. In the transition from unactivated to activated enzyme, phenylalanine and pterin binding is modified, a new site for phenylalanine is formed, and the pterin site is replaced by a site of greatly decreased affinity for BH4 and BH2, one which does not appear to recognize the dihydroxypropyl side chain of BH4 and BH2. The pterin- and phenylalanine-binding sites on activated phenylalanine hydroxylase appear to be part of the enzyme's active site. Despite large effects on substrate binding, neither chelator binding ability nor solvent accessibility of the iron are affected by activation; activation appears to affect the nearby environment of the enzyme's iron but not the iron itself. Studies of oxidized and reduced phenylalanine hydroxylase indicate that the redox state is not a major determinant of pterin and phenylalanine association with enzyme.

  8. Tuning the Transcriptional Response to Hypoxia by Inhibiting Hypoxia-inducible Factor (HIF) Prolyl and Asparaginyl Hydroxylases*

    PubMed Central

    Chan, Mun Chiang; Ilott, Nicholas E.; Schödel, Johannes; Sims, David; Tumber, Anthony; Lippl, Kerstin; Mole, David R.; Pugh, Christopher W.; Ratcliffe, Peter J.; Ponting, Chris P.; Schofield, Christopher J.

    2016-01-01

    The hypoxia-inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a tissue context-dependent manner. The major hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1–3) and an asparaginyl hydroxylase (factor-inhibiting HIF (FIH)). PHD catalysis regulates HIFα levels, and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of specific HIF target gene transcription is unknown. We report studies using small molecule HIF hydroxylase inhibitors that investigate the extent to which HIF target gene expression is induced by PHD or FIH inhibition. The results reveal substantial differences in the role of prolyl and asparaginyl hydroxylation in regulating hypoxia-responsive genes in cells. PHD inhibitors with different structural scaffolds behave similarly. Under the tested conditions, a broad-spectrum 2-oxoglutarate dioxygenase inhibitor is a better mimic of the overall transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in the transcriptional induction of a subset of genes not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable. PMID:27502280

  9. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

    PubMed Central

    Molitor, Christian; Mauracher, Stephan Gerhard

    2016-01-01

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze the o-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme’s interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate–enzyme complexes were performed, and a key residue was identified that influences the plant PPO’s acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their—so far unknown—natural substrates in vivo. PMID:26976571

  10. A novel profibrotic mechanism mediated by TGF-β-stimulated collagen prolyl hydroxylase expression in fibrotic lung mesenchymal cells

    PubMed Central

    Luo, Yongfeng; Xu, Wei; Chen, Hui; Warburton, David; Dong, Rachel; Qian, Bangping; Selman, Moisés; Gauldie, Jack; Kolb, Martin; Shi, Wei

    2015-01-01

    Idiopathic pulmonary fibrosis is a severe chronic lung disease with a high mortality rate. Excessive TGF-β signaling is recognized as a central player in lung fibrosis. However, the related mechanisms remain unclear. Herein we used a novel Tbx4 lung enhancer-driven Tet-On transgenic system to inhibit TGF-β signaling in mouse lung resident mesenchymal cells at different stages of bleomycin-induced fibrosis by conditionally knocking out TGF-β receptor II or expressing a dominant-negative TGF-β receptor II. Abrogation of mesenchymal TGF-β signaling markedly attenuated bleomycin-induced fibrotic pathology, which was independent of altered early inflammation. Furthermore, a novel TGF-β downstream target gene P4HA3 (an α-subunit of collagen prolyl hydroxylase) was identified, and its expression was significantly increased in fibroblastic foci of both bleomycin-induced fibrotic mouse lungs and idiopathic pulmonary fibrosis patients’ lungs. The relationship between activated TGF-β signaling, upregulation of P4HA3, as well as increased hydroxyproline/collagen production was further verified in cultured lung fibroblasts. Moreover, inhibition of collagen prolyl hydroxylase by pyridine-2,5-dicarboxylate attenuated both TGF-β-stimulated collagen production in cultured fibroblasts and bleomycin-induced mouse lung fibrosis. These data indicate that increased expression and activity of collagen prolyl hydroxylase is one of the important mechanisms underlying TGF-β-mediated profibrotic effects. Inhibition of collagen prolyl hydroxylase may be a new promising approach for preventing and treating pulmonary fibrosis. PMID:25779936

  11. Pituitary-dependent masculinization of hepatic hexobarbital hydroxylase in Crl:CD-1(ICR)BR mice.

    PubMed

    Shapiro, B H

    1985-03-25

    The sexual dimorphism in hepatic drug metabolism found in Crl:CD-1 mice is due to the normally repressive effects of testicular androgens on the activities of hepatic monooxygenases. The ability of testosterone to elevate the Michaelis constant (Km) and reduce the maximum velocity (Vmax) of hepatic hexobarbital hydroxylase is dependent upon the pituitary, so that in the hypophysectomized mouse androgens have no repressive effects on the activities of hepatic monooxygenases.

  12. Effect of Water Miscible Organic Solvents on p-Nitrophenol Hydroxylase (CYP2E1) Activity in Rat Liver Microsomes

    PubMed Central

    Patil, Pranali G.; Kamble, S. H.; Shah, T. S.; Iyer, K. R.

    2015-01-01

    Organic solvents used for solubilization of the substrates/NCEs are known to affect the activity of cytochrome P450 enzymes. Further, this effect varies with the solvents used, the substrates and CYP450 isoforms in question. In the present study, we have investigated the effect of ten commonly used water miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, acetone, dimethyl sulphoxide, N,N-dimethyl formamide, dioxane and polyethylene glycol 400) on p-nitrophenol hydroxylase activity at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. All the solvents studied showed concentration dependent inhibition of the p-nitrophenol hydroxylase activity except acetonitrile which showed activation of the activity at concentration range studied. Out of ten solvents studied, dioxane was found to be the most inhibitory solvent (inhibition >90% at 0.25% v/v concentration). Overall, solvents like dimethyl sulphoxide, dimethyl formamide and dioxane appeared to be unsuitable for characterizing p-nitrophenol hydroxylase (CYP2E1-mediated) reactions due to a high degree of inhibition. On the other hand, methanol and acetonitrile at concentrations <0.5% v/v appeared to be appropriate solvents for substrate solubilization while evaluating CYP2E1-mediated catalysis. The results of this study imply that caution should be exercised while choosing solvents for dissolution of substrate during enzyme studies in liver microsomes. PMID:26180273

  13. A late 17α-hydroxylase deficiency diagnosis that leads to the discovery of a new CYP17 gene mutation.

    PubMed

    Guenego, Agathe; Morel, Yves; Ionesco, Oana; Mallet, Delphine; Priou-Guesdon, Melanie

    2015-02-01

    17α-Hydroxylase deficiency is a rare form of congenital adrenal hyperplasia. It leads to a reduced production of cortisol and sex steroids and thus an increase in adrenocorticotrophic hormone and gonadotrophins levels. High adrenocorticotrophic hormone levels result in an accumulation of 17-deoxysteroids, such as deoxycorticosterone and corticosterone. Deoxycorticosterone and corticosterone have an important mineralocorticoid activity. We report the case of a 66-year-old woman who presented with hypertension and symptomatic hypokalaemia. Primary hyperaldosteronism was suspected and a right adrenal mass was removed. After surgery, the patient was referred to the endocrinology department for persistant hypokalaemia. Actually, she presented some signs of hypogonadism (impuberism, primary amenorrhea, infertility). Cortisol and 17OH-progesterone serum levels were low. Deoxycorticosterone and corticosterone were markedly elevated. The hypothesis of 17α-hydroxylase deficiency was considered and confirmed by genetic exploration. A non-sense mutation c.938G>A (p.Trp313X) in exon 5 of the CYP17 gene was found that had never been reported so far to our knowledge. Moreover, the patient's karyotype found a mosaic Turner syndrome. This case is particularly interesting because of the delay of diagnosis. The 17α-hydroxylase deficiency diagnosis is to be considered when hypertension is associated with hypokalaemia and hypogonadism, even in adult patients.

  14. Conversion of β-carotene into astaxanthin: Two separate enzymes or a bifunctional hydroxylase-ketolase protein?

    PubMed Central

    Martín, Juan F; Gudiña, Eduardo; Barredo, José L

    2008-01-01

    Astaxanthin is a xanthophyll of great interest in animal nutrition and human health. The market prospect in the nutraceutics industries for this health-protective molecule is very promising. Astaxanthin is synthesized by several bacteria, algae and plants from β-carotene by the sequential action of two enzymes: a β-carotene, 3,3'-hydroxylase that introduces an hydroxyl group at the 3 (and 3') positions of each of the two β-ionone rings of β-carotene, and a β-carotene ketolase that introduces keto groups at carbons 4 and 4' of the β-ionone rings. Astaxanthin is also produced by the yeast-like basidiomycete Xanthophyllomyces dendrorhous. A gene crtS involved in the conversion of β-carotene to astaxanthin has been cloned simultaneously by two research groups. Complementation studies of X. dendrorhous mutants and expression analysis in Mucor circinelloides reveals that the CrtS enzyme is a β-carotene hydroxylase of the P-450 monooxygenase family that converts β-carotene to the hydroxylated derivatives β-cryptoxanthin and zeaxanthin, but it does not form astaxanthin or the ketolated intermediates in this fungus. A bifunctional β-carotene hydroxylase-ketolase activity has been proposed for the CrtS protein. The evidence for and against this hypothesis is analyzed in detail in this review. PMID:18289382

  15. miR-190 Enhances HIF-Dependent Responses to Hypoxia in Drosophila by Inhibiting the Prolyl-4-hydroxylase Fatiga

    PubMed Central

    De Lella Ezcurra, Ana Laura; Bertolin, Agustina Paola; Kim, Kevin; Gándara, Lautaro; Luschnig, Stefan; Perrimon, Norbert; Melani, Mariana; Wappner, Pablo

    2016-01-01

    Cellular and systemic responses to low oxygen levels are principally mediated by Hypoxia Inducible Factors (HIFs), a family of evolutionary conserved heterodimeric transcription factors, whose alpha- and beta-subunits belong to the bHLH-PAS family. In normoxia, HIFα is hydroxylated by specific prolyl-4-hydroxylases, targeting it for proteasomal degradation, while in hypoxia the activity of these hydroxylases decreases due to low oxygen availability, leading to HIFα accumulation and expression of HIF target genes. To identify microRNAs required for maximal HIF activity, we conducted an overexpression screen in Drosophila melanogaster, evaluating the induction of a HIF transcriptional reporter. miR-190 overexpression enhanced HIF-dependent biological responses, including terminal sprouting of the tracheal system, while in miR-190 loss of function embryos the hypoxic response was impaired. In hypoxic conditions, miR-190 expression was upregulated and required for induction of HIF target genes by directly inhibiting the HIF prolyl-4-hydroxylase Fatiga. Thus, miR-190 is a novel regulator of the hypoxia response that represses the oxygen sensor Fatiga, leading to HIFα stabilization and enhancement of hypoxic responses. PMID:27223464

  16. miR-190 Enhances HIF-Dependent Responses to Hypoxia in Drosophila by Inhibiting the Prolyl-4-hydroxylase Fatiga.

    PubMed

    De Lella Ezcurra, Ana Laura; Bertolin, Agustina Paola; Kim, Kevin; Katz, Maximiliano Javier; Gándara, Lautaro; Misra, Tvisha; Luschnig, Stefan; Perrimon, Norbert; Melani, Mariana; Wappner, Pablo

    2016-05-01

    Cellular and systemic responses to low oxygen levels are principally mediated by Hypoxia Inducible Factors (HIFs), a family of evolutionary conserved heterodimeric transcription factors, whose alpha- and beta-subunits belong to the bHLH-PAS family. In normoxia, HIFα is hydroxylated by specific prolyl-4-hydroxylases, targeting it for proteasomal degradation, while in hypoxia the activity of these hydroxylases decreases due to low oxygen availability, leading to HIFα accumulation and expression of HIF target genes. To identify microRNAs required for maximal HIF activity, we conducted an overexpression screen in Drosophila melanogaster, evaluating the induction of a HIF transcriptional reporter. miR-190 overexpression enhanced HIF-dependent biological responses, including terminal sprouting of the tracheal system, while in miR-190 loss of function embryos the hypoxic response was impaired. In hypoxic conditions, miR-190 expression was upregulated and required for induction of HIF target genes by directly inhibiting the HIF prolyl-4-hydroxylase Fatiga. Thus, miR-190 is a novel regulator of the hypoxia response that represses the oxygen sensor Fatiga, leading to HIFα stabilization and enhancement of hypoxic responses.

  17. Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction

    SciTech Connect

    Day, D.J.; Barany, F.; Speiser, P.W.

    1995-09-01

    Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia, an inherited inability to synthesize cortisol that occurs in 1 in 10,000-15,000 births. Affected females are born with ambiguous genitalia, a condition that can be ameliorated by administering dexamethasone to the mother for most of gestation. Prenatal diagnosis is required for accurate treatment of affected females as well as for genetic counseling purposes. Approximately 95% of mutations causing this disorder result from recombinations between the gene encoding the 21-hydroxylase enzyme (CYP21) and a linked, highly homologous pseudogene (CYP21P). Approximately 20% of these mutations are gene deletions, and the remainder are gene conversions that transfer any of nine deleterious mutations from the CYP21P pseudogene to CYP21. We describe a methodology for genetic diagnosis of 21-hydroxylase deficiency that utilizes gene-specific PCR amplification in conjunction with thermostable DNA ligase to discriminate single nucleotide variations in a multiplexed ligation detection assay. The assay has been designed to be used with either fluorescent or radioactive detection of ligation products by electrophoresis on denaturing acrylamide gels and is readily adaptable for use in other disease systems. 30 refs., 5 figs.

  18. Point mutation of Arg440 to his in cytochrome P450c17 causes severe 17{alpha}-hydroxylase deficiency

    SciTech Connect

    Fardella, C.E.; Hum, D.W.; Miller, W.L.; Homoki, J.

    1994-07-01

    Genetic disorders in the gene encoding P450c17 cause 17{alpha}-hydroxylase deficiency. The consequent defects in the synthesis of cortisol and sex steroids cause sexual infantilism and a female phenotype in both genetic sexes as well as mineralorcorticoid excess and hypertension. A 15-yr-old patient from Germany was seen for absent pubertal development and mild hypertension with hypokalemia, high concentrations of 17-deoxysteroids, and hypergonadotropic hypogonadism. Analysis of her P450c17 gene by polymerase chain reaction amplification and direct sequencing showed mutation of codon 440 from CGC (Arg) to CAC (His). Expression of a vector encoding this mutated form of P450c17 in transfected nonsteroidogenic COS-1 cells showed that the mutant P450c17 protein was produced, but it lacked both 17{alpha}-hydroxylase and 17,20-lyase activities. To date, 15 different P450c17 mutations have been described in 23 patients with 17{alpha}-hydroxylase deficiency, indicating that mutations in this gene are due to random events. 36 refs., 3 figs., 2 tabs.

  19. Effect of Water Miscible Organic Solvents on p-Nitrophenol Hydroxylase (CYP2E1) Activity in Rat Liver Microsomes.

    PubMed

    Patil, Pranali G; Kamble, S H; Shah, T S; Iyer, K R

    2015-01-01

    Organic solvents used for solubilization of the substrates/NCEs are known to affect the activity of cytochrome P450 enzymes. Further, this effect varies with the solvents used, the substrates and CYP450 isoforms in question. In the present study, we have investigated the effect of ten commonly used water miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, acetone, dimethyl sulphoxide, N,N-dimethyl formamide, dioxane and polyethylene glycol 400) on p-nitrophenol hydroxylase activity at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. All the solvents studied showed concentration dependent inhibition of the p-nitrophenol hydroxylase activity except acetonitrile which showed activation of the activity at concentration range studied. Out of ten solvents studied, dioxane was found to be the most inhibitory solvent (inhibition >90% at 0.25% v/v concentration). Overall, solvents like dimethyl sulphoxide, dimethyl formamide and dioxane appeared to be unsuitable for characterizing p-nitrophenol hydroxylase (CYP2E1-mediated) reactions due to a high degree of inhibition. On the other hand, methanol and acetonitrile at concentrations <0.5% v/v appeared to be appropriate solvents for substrate solubilization while evaluating CYP2E1-mediated catalysis. The results of this study imply that caution should be exercised while choosing solvents for dissolution of substrate during enzyme studies in liver microsomes.

  20. Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR I from Croton stellatopilosus Ohba.

    PubMed

    Sintupachee, Siriluk; Promden, Worrawat; Ngamrojanavanich, Nattaya; Sitthithaworn, Worapan; De-Eknamkul, Wanchai

    2015-10-01

    While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase(designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0 kDa,respectively. Amino acid sequence comparison showed that both CYP76F45 (63–73%) and CsCPR I (79–83%) share relatively high sequence identities with homologous proteins in other plant species.Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins,showed that only simultaneous incubation of the membrane bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levelsof the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19-Evallesamine,that accumulated in these plant parts. These results suggested that CYP76F45 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus [corrected].

  1. Classical forms of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in adults.

    PubMed

    Bachelot, Anne; Chakthoura, Zeina; Rouxel, Agnès; Dulon, Jérome; Touraine, Philippe

    2008-01-01

    During childhood, the main aims of the medical treatment of congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase are to prevent salt loss and virilization and to attain normal stature and normal puberty. As such, there is a narrow therapeutic window through which the intended results can be achieved. In adulthood, the clinical management has received little attention, but recent studies have shown the relevance of long-term follow-up of these patients. The aims here are to review the multiple clinical, hormonal and metabolic abnormalities that could be found in adult CAH patients as such a decrease in bone mineral density, overweight and disturbed reproductive functions. In women with classic CAH, a low fertility rate is reported, and is probably the consequence of multiple factors including neuroendocrine and hormonal factors, feminizing surgery, and psychological factors. Men with CAH may present hypogonadism either through the effect of adrenal rests or from suppression of gonadotropins resulting in infertility. Therefore a multidisciplinary team with knowledge of CAH should carefully follow up these patients, from childhood through to adulthood, to avoid these complications and to ensure treatment compliance and tight control of the adrenal androgens.

  2. Approach to the adult with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

    PubMed

    Merke, Deborah P

    2008-03-01

    Congenital adrenal hyperplasia (CAH) describes a group of autosomal recessive disorders where there is impairment of cortisol biosynthesis. CAH due to 21-hydroxylase deficiency accounts for 95% of cases and shows a wide range of clinical severity. Treatment of the classic or severe form of CAH is targeted at replacing cortisol and aldosterone and effectively controlling excess androgen symptoms by using the lowest possible glucocorticoid dose. Treatment of the mild or nonclassic form is targeted at controlling excess androgen symptoms and may or may not involve glucocorticoid therapy. Hydrocortisone is the treatment of choice for children, but there is no consensus on how patients should be treated as adults. Current glucocorticoid therapy is suboptimal because it is often difficult to reduce excess androgen without giving excess glucocorticoid, and patients may experience hypercortisolism, androgen excess, or a combination of these states. Treatment of CAH, especially in the adult patient, remains controversial given the lack of prospective randomized controlled trials comparing treatment regimens. Nevertheless, patients benefit from careful individualized therapy with avoidance of Cushingoid side effects and optimization of reproductive, sexual, and bone health.

  3. Hormonal treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

    PubMed

    Bachelot, A; Chakhtoura, Z; Rouxel, A; Dulon, J; Touraine, P

    2007-09-01

    During childhood, the main aims of the medical treatment of congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase deficiency, are to prevent salt loss and virilization and to achieve normal stature and normal puberty. As such, there is a narrow therapeutic window through which the intended results can be achieved. In adulthood, the clinical management has received little attention, but recent studies have shown the relevance of long-term follow-up of these patients. Indeed, long-term evaluation of adult CAH patients enables the identification of multiple clinical, hormonal and metabolic abnormalities as bone mineral density alteration, overweight and disturbed reproductive functions. In women with classic CAH, low fertility rate is reported, and is probably the consequence of multiple factors, including neuroendocrine and hormonal factors, feminizing surgery, and psychological factors. Men with CAH may present hypogonadism either through the effect of adrenal rests or from suppression of gonadotropins resulting in infertility. These patients should therefore be carefully followed-up, from childhood through to adulthood, to avoid these complications and to ensure treatment compliance and tight control of the adrenal androgens, by multidisciplinary teams who have knowledge of CAH.

  4. Mutant PINK1 upregulates tyrosine hydroxylase and dopamine levels, leading to vulnerability of dopaminergic neurons.

    PubMed

    Zhou, Zhi Dong; Refai, Fathima Shaffra; Xie, Shao Ping; Ng, Shin Hui; Chan, Christine Hui Shan; Ho, Patrick Ghim Hoe; Zhang, Xiao Dong; Lim, Tit Meng; Tan, Eng King

    2014-03-01

    PINK1 mutations cause autosomal recessive forms of Parkinson disease (PD). Previous studies suggest that the neuroprotective function of wild-type (WT) PINK1 is related to mitochondrial homeostasis. PINK1 can also localize to the cytosol; however, the cytosolic function of PINK1 has not been fully elucidated. In this study we demonstrate that the extramitochondrial PINK1 can regulate tyrosine hydroxylase (TH) expression and dopamine (DA) content in dopaminergic neurons in a PINK1 kinase activity-dependent manner. We demonstrate that overexpression of full-length (FL) WT PINK1 can downregulate TH expression and DA content in dopaminergic neurons. In contrast, overexpression of PD-linked G309D, A339T, and E231G PINK1 mutations upregulates TH and DA levels in dopaminergic neurons and increases their vulnerability to oxidative stress. Furthermore transfection of FL WT PINK1 or PINK1 fragments with the PINK1 kinase domain can inhibit TH expression, whereas kinase-dead (KD) FL PINK1 or KD PINK1 fragments upregulate TH level. Our findings highlight a potential novel function of extramitochondrial PINK1 in dopaminergic neurons. Deregulation of these functions of PINK1 may contribute to PINK1 mutation-induced dopaminergic neuron degeneration. However, deleterious effects caused by PINK1 mutations may be alleviated by iron-chelating agents and antioxidant agents with DA quinone-conjugating capacity.

  5. Expression mediated by three partial sequences of the human tyrosine hydroxylase promoter in vivo

    PubMed Central

    Rolland, Anne-Sophie; Kareva, Tatyana; Yarygina, Olga; Kholodilov, Nikolai; Burke, Robert E

    2016-01-01

    The use of viral vectors to transfect postmitotic neurons has provided an important research tool, and it offers promise for treatment of neurologic disease. The utility of vectors is enhanced by the use of selective promoters that permit control of the cellular site of expression. One potential clinical application is in the neurorestorative treatment of Parkinson’s disease by the induction of new axon growth. However, many of the genes with an ability to restore axons have oncogenic potential. Therefore, clinical safety would be enhanced by restriction of expression to neurons affected by the disease, particularly dopamine neurons. To achieve this goal we have evaluated in vivo three partial sequences of the promoter for human tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis. All sequences induced expression in dopamine neurons. None of them induced expression in glia or in nondopaminergic neurons in striatum or cortex. We conclude that these sequences have potential use for targeting dopamine neurons in research and clinical applications. PMID:27689101

  6. Putaminal Mosaic Visualized by Tyrosine Hydroxylase Immunohistochemistry in the Human Neostriatum

    PubMed Central

    Morigaki, Ryoma; Goto, Satoshi

    2016-01-01

    Among the basal ganglia-thalamocortical circuits, the putamen plays a critical role in the “motor” circuits that control voluntary movements and motor learning. The human neostriatum comprises two functional subdivisions known as the striosome (patch) and matrix compartments. Accumulating evidence suggests that compartment-specific dysregulations of dopamine activity might be involved in the disease-specific pathology and symptoms of human striatal diseases including movement disorders. This study was undertaken to examine whether or how striatal dopaminergic innervations are organized into the compartmentalized architecture found in the putamen of adult human brains. For this purpose, we used a highly sensitive immunohistochemistry (IHC) technique to identify tyrosine hydroxylase (TH; EC 1.14.16.2), a marker for striatal dopaminergic axons and terminals, in formalin-fixed paraffin-embedded (FFPE) tissues obtained from autopsied human brains. Herein, we report that discrete compartmentalization of TH-labeled innervations occurs in the putamen, as in the caudate nucleus (CN), with a higher density of TH labeling in the matrix compared to the striosomes. Our results provide anatomical evidence to support the hypothesis that compartment-specific dysfunction of the striosome-matrix dopaminergic systems might contribute to the genesis of movement disorders. PMID:27092059

  7. In-crystal reaction cycle of a toluene-bound diiron hydroxylase.

    PubMed

    Acheson, Justin F; Bailey, Lucas J; Brunold, Thomas C; Fox, Brian G

    2017-03-27

    Electrophilic aromatic substitution is one of the most important and recognizable classes of organic chemical transformation. Enzymes create the strong electrophiles that are needed for these highly energetic reactions by using O2, electrons, and metals or other cofactors. Although the nature of the oxidants that carry out electrophilic aromatic substitution has been deduced from many approaches, it has been difficult to determine their structures. Here we show the structure of a diiron hydroxylase intermediate formed during a reaction with toluene. Density functional theory geometry optimizations of an active site model reveal that the intermediate is an arylperoxo Fe(2+)/Fe(3+) species with delocalized aryl radical character. The structure suggests that a carboxylate ligand of the diiron centre may trigger homolytic cleavage of the O-O bond by transferring a proton from a metal-bound water. Our work provides the spatial and electronic constraints needed to propose a comprehensive mechanism for diiron enzyme arene hydroxylation that accounts for many prior experimental results.

  8. Catechol and aldehyde moieties of 3,4-dihydroxyphenylacetaldehyde contribute to tyrosine hydroxylase inhibition and neurotoxicity.

    PubMed

    Vermeer, Lydia M M; Florang, Virginia R; Doorn, Jonathan A

    2012-09-20

    Parkinson's disease (PD) is a progressive neurodegenerative disorder which leads to the selective loss of dopaminergic neurons. This causes a decrease in the important neurotransmitter dopamine (DA), which is essential for coordinated movement. Previous studies have implicated the monoamine oxidase metabolite of DA, 3,4-dihydroxphenylacetaldehyde (DOPAL), in the pathogenesis of PD and have shown it to be a reactive intermediate capable of protein modification. DOPAL also has demonstrated the ability to cause mitochondrial dysfunction and lead to significant inhibition of the rate-limiting enzyme in DA synthesis, tyrosine hydroxylase (TH). The current study was undertaken to investigate four analogs of DOPAL, including a novel nitrile analog, to determine how the structure of DOPAL is related to its toxicity and inhibition of TH. Both mitochondrial function and inhibition of TH in cell lysate were investigated. Furthermore, a novel whole cell assay was designed to determine the consequence to enzyme action when DOPAL levels were elevated. The results presented here demonstrate that changes to DOPAL structure lead to a decrease in toxicity and inhibition of enzyme activity as compared to the parent compound. Furthermore, the production of superoxide anion but not hydrogen peroxide increased in the presence of elevated DOPAL. These results reveal the toxicity of DOPAL and demonstrate that both the catechol and aldehyde are required to potently inhibit TH activity.

  9. Species-dependent expression of the hyoscyamine 6 beta-hydroxylase gene in the pericycle.

    PubMed

    Kanegae, T; Kajiya, H; Amano, Y; Hashimoto, T; Yamada, Y

    1994-06-01

    The tropane alkaloid scopolamine is synthesized in the pericycle of branch roots in certain species of the Solanaceae. The enzyme responsible for the synthesis of scopolamine from hyoscyamine is hyoscyamine 6 beta-hydroxylase (H6H). The gene for H6H was isolated from Hyoscyamus niger. It has an exon/intron organization very similar to those for ethylene-forming enzymes, suggesting a common evolutionary origin. The 827-bp 5' flanking region of the H6H gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to three solanaceous species by Agrobacterium-mediated transformation systems: H. niger and belladonna (Atropa belladonna), which have high and low levels, respectively, of H6H mRNA in the root, and tobacco (Nicotiana tabacum), which has no endogenous H6H gene. Histochemical analysis showed that GUS expression occurred in the pericycle and at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of hairy roots and plants of transgenic tobacco. In transgenic hairy roots and regenerated plants of belladonna, the root meristem was stained with GUS activity, except for a few transformants in which the vascular cylinder was also stained. These studies indicate that the cell-specific expression of the H6H gene is controlled by some genetic regulation specific to scopolamine-producing plants.

  10. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons

    PubMed Central

    Liu, Zhenyi; Brown, Andrew; Fisher, Dan; Wu, Yumei; Warren, Joe; Cui, Xiaoxia

    2016-01-01

    The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson’s disease. PMID:26886559

  11. An improved procedure for the purification of catalytically active alkane hydroxylase from Pseudomonas putida GPo1.

    PubMed

    Xie, Meng; Alonso, Hernan; Roujeinikova, Anna

    2011-10-01

    Bacterial alkane hydroxylases are of high interest for bioremediation applications as they allow some bacteria to grow in oil-contaminated environments. Furthermore, they have tremendous biotechnological potential as they catalyse the stereo- and regio-specific hydroxylation of chemically inert alkanes, which can then be used in the synthesis of pharmaceuticals and other high-cost chemicals. Despite their potential, progress on the detailed characterization of these systems has so far been slow mainly due to the lack of a robust procedure to purify its membrane protein component, monooxygenase AlkB, in a stable and active form. This study reports a new method for isolating milligramme amounts of recombinant Pseudomonas putida GPo1 AlkB in a folded, catalytically active form to purity levels above 90%. AlkB solubilised and purified in the detergent lauryldimethylamine oxide was demonstrated to be active in catalysing the epoxidation reaction of 1-octene with an estimated K (m) value of 0.2 mM.

  12. Tyrosine Hydroxylase is crucial for maintaining pupal tanning and immunity in Anopheles sinensis

    PubMed Central

    Qiao, Liang; Du, Minghui; Liang, Xin; Hao, Youjin; He, Xiu; Si, Fengling; Mei, Ting; Chen, Bin

    2016-01-01

    Tyrosine hydroxylase (TH), the initial enzyme in the melanin pathway, catalyzes tyrosine conversion into Dopa. Although expression and regulation of TH have been shown to affect cuticle pigmentation in insects, no direct functional studies to date have focused on the specific physiological processes involving the enzyme during mosquito development. In the current study, silencing of AsTH during the time period of continuous high expression in Anopheles sinensis pupae led to significant impairment of cuticle tanning and thickness, imposing a severe obstacle to eclosion in adults. Meanwhile, deficiency of melanin in interference individuals led to suppression of melanization, compared to control individuals. Consequently, the ability to defend exogenous microorganisms declined sharply. Accompanying down-regulation of the basal expression of five antimicrobial peptide genes resulted in further significant weakening of immunity. TH homologs as well as the composition of upstream transcription factor binding sites at the pupal stage are highly conserved in the Anopheles genus, implying that the TH-mediated functions are crucial in Anopheles. The collective evidence strongly suggests that TH is essential for Anopheles pupae tanning and immunity and provides a reference for further studies to validate the utility of the key genes involved in the melanization pathway in controlling mosquito development. PMID:27416870

  13. Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan.

    PubMed

    Lin, Yuheng; Sun, Xinxiao; Yuan, Qipeng; Yan, Yajun

    2014-07-18

    5-Hydroxytryptophan (5-HTP) is a drug that is clinically effective against depression, insomnia, obesity, chronic headaches, etc. It is only commercially produced by the extraction from the seeds of Griffonia simplicifolia because of a lack of synthetic methods. Here, we report the efficient microbial production of 5-HTP via combinatorial protein and metabolic engineering approaches. First, we reconstituted and screened prokaryotic phenylalanine 4-hydroxylase activity in Escherichia coli. Then, sequence- and structure-based protein engineering dramatically shifted its substrate preference, allowing for efficient conversion of tryptophan to 5-HTP. Importantly, E. coli endogenous tetrahydromonapterin (MH4) could be utilized as the coenzyme, when a foreign MH4 recycling mechanism was introduced. Whole-cell bioconversion allowed the high-level production of 5-HTP (1.1-1.2 g/L) from tryptophan in shake flasks. On this basis, metabolic engineering efforts were further made to achieve the de novo 5-HTP biosynthesis from glucose. This work not only holds great scale-up potential but also demonstrates a strategy for expanding the native metabolism of microorganisms.

  14. Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain.

    PubMed

    Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J; Yue, Wyatt W

    2016-04-06

    The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme.

  15. Genotype-phenotype correlations analysis of mutations in the phenylalanine hydroxylase (PAH) gene.

    PubMed

    Bercovich, Dani; Elimelech, Arava; Zlotogora, Joel; Korem, Sigal; Yardeni, Tal; Gal, Nurit; Goldstein, Nurit; Vilensky, Bela; Segev, Roni; Avraham, Smadar; Loewenthal, Ron; Schwartz, Gerard; Anikster, Yair

    2008-01-01

    The aims of our research were to define the genotype-phenotype correlations of mutations in the phenylalanine hydroxylase (PAH) gene that cause phenylketonuria (PKU) among the Israeli population. The mutation spectrum of the PAH gene in PKU patients in Israel is described, along with a discussion on genotype-phenotype correlations. By using polymerase chain reaction/denaturing high-performance liquid chromatography (PCR/dHPLC) and DNA sequencing, we screened all exons of the PAH gene in 180 unrelated patients with four different PKU phenotypes [classic PKU, moderate PKU, mild PKU, and mild hyperphenylalaninemia (MHP)]. In 63.2% of patient genotypes, the metabolic phenotype could be predicted, though evidence is also found for both phenotypic inconsistencies among subjects with more than one type of mutation in the PAH gene. Data analysis revealed that about 25% of patients could participate in the future in (6R)-L: -erythro-5, 6, 7, 8-tetrahydrobiopterin (BH4) treatment trials according to their mutation genotypes. This study enables us to construct a national database in Israel that will serve as a valuable tool for genetic counseling and a prognostic evaluation of future cases of PKU.

  16. Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase.

    PubMed

    Zhang, Shengnan; Fitzpatrick, Paul F

    2016-04-01

    Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25-117) is the regulatory domain of PheH lacking residues 1-24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25-117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25-117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH.

  17. The interplay between genotype, metabolic state and cofactor treatment governs phenylalanine hydroxylase function and drug response.

    PubMed

    Staudigl, Michael; Gersting, Søren W; Danecka, Marta K; Messing, Dunja D; Woidy, Mathias; Pinkas, Daniel; Kemter, Kristina F; Blau, Nenad; Muntau, Ania C

    2011-07-01

    The discovery of a pharmacological treatment for phenylketonuria (PKU) raised new questions about function and dysfunction of phenylalanine hydroxylase (PAH), the enzyme deficient in this disease. To investigate the interdependence of the genotype, the metabolic state (phenylalanine substrate) and treatment (BH(4) cofactor) in the context of enzyme function in vitro and in vivo, we (i) used a fluorescence-based method for fast enzyme kinetic analyses at an expanded range of phenylalanine and BH(4) concentrations, (ii) depicted PAH function as activity landscapes, (iii) retraced the analyses in eukaryotic cells, and (iv) translated this into the human system by analyzing the outcome of oral BH(4) loading tests. PAH activity landscapes uncovered the optimal working range of recombinant wild-type PAH and provided new insights into PAH kinetics. They demonstrated how mutations might alter enzyme function in the space of varying substrate and cofactor concentrations. Experiments in eukaryotic cells revealed that the availability of the active PAH enzyme depends on the phenylalanine-to-BH(4) ratio. Finally, evaluation of data from BH(4) loading tests indicated that the patient's genotype influences the impact of the metabolic state on drug response. The results allowed for visualization and a better understanding of PAH function in the physiological and pathological state as well as in the therapeutic context of cofactor treatment. Moreover, our data underscore the need for more personalized procedures to safely identify and treat patients with BH(4)-responsive PAH deficiency.

  18. Molecular characterization of phenylketonuria and tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency in Japan.

    PubMed

    Okano, Yoshiyuki; Kudo, Satoshi; Nishi, Yasuaki; Sakaguchi, Tomoko; Aso, Kazuyoshi

    2011-04-01

    Phenylketonuria (PKU) is a heterogeneous metabolic disorder caused by a deficiency in hepatic phenylalanine hydroxylase (PAH). On the basis of phenotype/genotype correlations, determination of phenylketonuric genotype is important for classification of the clinical phenotype and treatment of PKU, including tetrahydrobiopterin therapy. We characterized the genotypes of 203 Japanese patients with PKU and hyperphenylalaninemia using the following systems: (1) denaturing high-performance liquid chromatography with a GC-clamped primer; (2) direct sequencing; and, (3) multiplex ligation-dependent probe amplification. Of 406 mutant alleles, 390 (96%) were genotyped; 65 mutations were identified, including 22 new mutations. R413P, R241C, IVS4-1g>a, R111X and R243Q were prevalent mutations. Mutations prevalent in the Japanese cohort are also common in Korean and Northern Chinese populations, suggesting same origin. The spectrum of prevalent mutations was not significantly different among six Japanese districts, indicating that Japan comprises a relatively homogeneous ethnic group. We classified the mutations by clinical phenotypes and in vivo PAH activity and estimated the mutations with potential tetrahydrobiopterin (BH(4)) responsiveness. The frequency of BH(4) responsiveness based on the genotype was 29.1% in Japanese PKU patients. A catalog of PKU genotypes would be useful for predicting clinical phenotype, deciding on the subsequent treatment of PKU including BH(4) therapy, and genetic counseling in East Asia.

  19. Role of NonO-histone interaction in TNFalpha-suppressed prolyl-4-hydroxylase alpha1.

    PubMed

    Zhang, Cheng; Zhang, Ming-Xiang; Shen, Ying H; Burks, Jared K; Li, Xiao-Nan; LeMaire, Scott A; Yoshimura, Koichi; Aoki, Hiroki; Matsuzaki, Masunori; An, Feng-Shuang; Engler, David A; Matsunami, Risë K; Coselli, Joseph S; Zhang, Yun; Wang, Xing Li

    2008-08-01

    Inflammation is a key process in cardiovascular diseases. The extracellular matrix (ECM) of the vasculature is a major target of inflammatory cytokines, and TNFalpha regulates ECM metabolism by affecting collagen production. In this study, we have examined the pathways mediating TNFalpha-induced suppression of prolyl-4 hydroxylase alpha1 (P4Halpha1), the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Using human aortic smooth muscle cells, we found that TNFalpha activated the MKK4-JNK1 pathway, which induced histone (H) 4 lysine 12 acetylation within the TNFalpha response element in the P4Halpha1 promoter. The acetylated-H4 then recruited a transcription factor, NonO, which, in turn, recruited HDACs and induced H3 lysine 9 deacetylation, thereby inhibiting transcription of the P4Halpha1 promoter. Furthermore, we found that TNFalpha oxidized DJ-1, which may be essential for the NonO-P4Halpha1 interaction because treatment with gene specific siRNA to knockout DJ-1 eliminated the TNFalpha-induced NonO-P4Halpha1 interaction and its suppression. Our findings may be relevant to aortic aneurysm and dissection and the stability of the fibrous cap of atherosclerotic plaque in which collagen metabolism is important in arterial remodeling. Defining this cytokine-mediated regulatory pathway may provide novel molecular targets for therapeutic intervention in preventing plaque rupture and acute coronary occlusion.

  20. FoxO1 in dopaminergic neurons regulates energy homeostasis and targets tyrosine hydroxylase

    PubMed Central

    Doan, Khanh V.; Kinyua, Ann W.; Yang, Dong Joo; Ko, Chang Mann; Moh, Sang Hyun; Shong, Ko Eun; Kim, Hail; Park, Sang-Kyu; Kim, Dong-Hoon; Kim, Inki; Paik, Ji-Hye; DePinho, Ronald A.; Yoon, Seul Gi; Kim, Il Yong; Seong, Je Kyung; Choi, Yun-Hee; Kim, Ki Woo

    2016-01-01

    Dopaminergic (DA) neurons are involved in the integration of neuronal and hormonal signals to regulate food consumption and energy balance. Forkhead transcriptional factor O1 (FoxO1) in the hypothalamus plays a crucial role in mediation of leptin and insulin function. However, the homoeostatic role of FoxO1 in DA system has not been investigated. Here we report that FoxO1 is highly expressed in DA neurons and mice lacking FoxO1 specifically in the DA neurons (FoxO1 KODAT) show markedly increased energy expenditure and interscapular brown adipose tissue (iBAT) thermogenesis accompanied by reduced fat mass and improved glucose/insulin homoeostasis. Moreover, FoxO1 KODAT mice exhibit an increased sucrose preference in concomitance with higher dopamine and norepinephrine levels. Finally, we found that FoxO1 directly targets and negatively regulates tyrosine hydroxylase (TH) expression, the rate-limiting enzyme of the catecholamine synthesis, delineating a mechanism for the KO phenotypes. Collectively, these results suggest that FoxO1 in DA neurons is an important transcriptional factor that directs the coordinated control of energy balance, thermogenesis and glucose homoeostasis. PMID:27681312

  1. A Retrospective Analysis of the Growth Pattern in Patients with Salt-wasting 21-Hydroxylase Deficiency

    PubMed Central

    Kawano, Atsuko; Kohno, Hitoshi; Miyako, Kenichi

    2014-01-01

    Abstract The objective of this study was to investigate the growth pattern of children with the salt-wasting form of congenital adrenal hyperplasia caused by 21-hydroxylase deficiency (21-OHD). We reviewed the medical records of 13 patients in whom salt-wasting 21-OHD was diagnosed during the first 2 mo of life at our hospital from 1980 through 2008. Six reached adult height. Growth patterns, bone age, biochemical data, and the hydrocortisone dose at each growth stage were analyzed retrospectively. The mean adult height was 155.1 ± 6.5 cm (mean ± SD) in females and 158.1 ± 7.1 cm in males. Although length at birth was normal or longer than the national mean in almost all patients, the mean height SD score of both boys and girls decreased to below 0 SD during infancy. Subsequently, both boys and girls transiently showed growth acceleration and reached their peak growth velocity at 3–10 yr of age. In conclusion, in addition to suppression of growth during infancy, there was inappropriate growth acceleration during childhood. Especially from 3 mo to 3 yr of age, decreasing the hydrocortisone dose in patients who exhibit slower growth may lead to satisfactory height outcomes. Also, strict adjustment of the hydrocortisone dose to avoid accelerated growth from childhood to adolescence might improve adult height outcomes of patients with 21-OHD. PMID:24790384

  2. Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

    PubMed Central

    Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J.; Yue, Wyatt W.

    2016-01-01

    The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme. PMID:27049649

  3. The crystal structure of human dopamine β-hydroxylase at 2.9 Å resolution.

    PubMed

    Vendelboe, Trine V; Harris, Pernille; Zhao, Yuguang; Walter, Thomas S; Harlos, Karl; El Omari, Kamel; Christensen, Hans E M

    2016-04-01

    The norepinephrine pathway is believed to modulate behavioral and physiological processes, such as mood, overall arousal, and attention. Furthermore, abnormalities in the pathway have been linked to numerous diseases, for example hypertension, depression, anxiety, Parkinson's disease, schizophrenia, Alzheimer's disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine β-monooxygenase N-terminal) domain, also found in >1600 other proteins, reveals a possible metal-binding site and a ligand-binding pocket. The catalytic core structure shows two different conformations: an open active site, as also seen in another member of this enzyme family [the peptidylglycine α-hydroxylating (and α-amidating) monooxygenase], and a closed active site structure, in which the two copper-binding sites are only 4 to 5 Å apart, in what might be a coupled binuclear copper site. The dimerization domain adopts a conformation that bears no resemblance to any other known protein structure. The structure provides new molecular insights into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system.

  4. Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

    NASA Technical Reports Server (NTRS)

    Kim, Chun-Hyung; Zabetian, Cyrus P.; Cubells, Joseph F.; Cho, Sonhae; Biaggioni, Italo; Cohen, Bruce M.; Robertson, David; Kim, Kwang-Soo

    2002-01-01

    Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

  5. Tyrosine Hydroxylase Binding to Phospholipid Membranes Prompts Its Amyloid Aggregation and Compromises Bilayer Integrity

    PubMed Central

    Baumann, Anne; Jorge-Finnigan, Ana; Jung-KC, Kunwar; Sauter, Alexander; Horvath, Istvan; Morozova-Roche, Ludmilla A.; Martinez, Aurora

    2016-01-01

    Tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of catecholamine neurotransmitters and hormones, binds to negatively charged phospholipid membranes. Binding to both large and giant unilamellar vesicles causes membrane permeabilization, as observed by efflux and influx of fluorescence dyes. Whereas the initial protein-membrane interaction involves the N-terminal tail that constitutes an extension of the regulatory ACT-domain, prolonged membrane binding induces misfolding and self-oligomerization of TH over time as shown by circular dichroism and Thioflavin T fluorescence. The gradual amyloid-like aggregation likely occurs through cross-β interactions involving aggregation-prone motives in the catalytic domains, consistent with the formation of chain and ring-like protofilaments observed by atomic force microscopy in monolayer-bound TH. PC12 cells treated with the neurotoxin 6-hydroxydopamine displayed increased TH levels in the mitochondrial fraction, while incubation of isolated mitochondria with TH led to a decrease in the mitochondrial membrane potential. Furthermore, cell-substrate impedance and viability assays showed that supplementing the culture media with TH compromises cell viability over time. Our results revealed that the disruptive effect of TH on cell membranes may be a cytotoxic and pathogenic factor if the regulation and intracellular stability of TH is compromised. PMID:28004763

  6. Comparative effects of indole and aminoacetonitrile derivatives on dimethylnitrosamine-demethylase and aryl hydrocarbon hydroxylase activities.

    PubMed

    Arcos, J C; Myers, S C; Neuburger, B J; Argus, M F

    1980-04-01

    The effect of in vivo administration of indole and five 3-indolyl derivatives including L-tryptophan, as well as of aminoacetonitrile and 3 of its derivatives, were studied on the carcinogen-metabolizing hepatic mixed-function oxidases dimethylnitrosamine (DMN)-demethylase I and II and aryl hydrocarbon hydroxylase (AHH). Indole, 3-indolylmethanol, 3-indolyl-acetonitrile, 3-indolylacetone and L-tryptophan induce AHH activity from 3- to 6-fold of the control level, whereas beta-3-indolylethanol has no effect; the latter compound produces a 21% decrease of the endoplasmic reticulum content in the tissue. Only L-tryptophan induces DMN-demethylase I and only L-tryptophan and 3-indolylmethanol induce DMN-demethylase II, representing a doubling of enzyme activity in all 3 instances. Aminoacetonitrile is a potent repressor of DMN-demethylase I. Substitutions on the amino group bring about strong decrease or abolishment of mixed-function oxidase repressor activity; thus, iminodiacetonitrile has only about 1/5th the repressor activity of the parent compound, whereas nitrilotriacetonitrile and dimethylaminoacetonitrile appear to be inactive. Aminoacetonitrile and its derivatives studied have no effect on DMN-demethylase II and AHH activities. The mixed-function oxidase-modifying effects of the indole compounds and of aminoacetonitrile and its derivatives illustrate the potential complexity of effects of dietary constituents on the carcinogenic responses.

  7. Reactivating HIF prolyl hydroxylases under hypoxia results in metabolic catastrophe and cell death.

    PubMed

    Tennant, D A; Frezza, C; MacKenzie, E D; Nguyen, Q D; Zheng, L; Selak, M A; Roberts, D L; Dive, C; Watson, D G; Aboagye, E O; Gottlieb, E

    2009-11-12

    Cells exposed to low-oxygen conditions (hypoxia) alter their metabolism to survive. This response, although vital during development and high-altitude survival, is now known to be a major factor in the selection of cells with a transformed metabolic phenotype during tumorigenesis. It is thought that hypoxia-selected cells have increased invasive capacity and resistance to both chemo- and radiotherapies, and therefore represent an attractive target for antitumor therapy. Hypoxia inducible factors (HIFs) are responsible for the majority of gene expression changes under hypoxia, and are themselves controlled by the oxygen-sensing HIF prolyl hydroxylases (PHDs). It was previously shown that mutations in succinate dehydrogenase lead to the inactivation PHDs under normoxic conditions, which can be overcome by treatment with alpha-ketoglutarate derivatives. Given that solid tumors contain large regions of hypoxia, the reactivation of PHDs in these conditions could induce metabolic catastrophe and therefore prove an effective antitumor therapy. In this report we demonstrate that derivatized alpha-ketoglutarate can be used as a strategy for maintaining PHD activity under hypoxia. By increasing intracellular alpha-ketoglutarate and activating PHDs we trigger PHD-dependent reversal of HIF1 activation, and PHD-dependent hypoxic cell death. We also show that derivatized alpha-ketoglutarate can permeate multiple layers of cells, reducing HIF1alpha levels and its target genes in vivo.

  8. Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma

    PubMed Central

    Chen, Yulong; Terajima, Masahiko; Yang, Yanan; Sun, Li; Ahn, Young-Ho; Pankova, Daniela; Puperi, Daniel S.; Watanabe, Takeshi; Kim, Min P.; Blackmon, Shanda H.; Rodriguez, Jaime; Liu, Hui; Behrens, Carmen; Wistuba, Ignacio I.; Minelli, Rosalba; Scott, Kenneth L.; Sanchez-Adams, Johannah; Guilak, Farshid; Pati, Debananda; Thilaganathan, Nishan; Burns, Alan R.; Creighton, Chad J.; Martinez, Elisabeth D.; Zal, Tomasz; Grande-Allen, K. Jane; Yamauchi, Mitsuo; Kurie, Jonathan M.

    2015-01-01

    Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde–derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde–derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma. PMID:25664850

  9. Cell wall modifications triggered by the down-regulation of Coumarate 3-hydroxylase-1 in maize.

    PubMed

    Fornalé, Silvia; Rencoret, Jorge; Garcia-Calvo, Laura; Capellades, Montserrat; Encina, Antonio; Santiago, Rogelio; Rigau, Joan; Gutiérrez, Ana; Del Río, José-Carlos; Caparros-Ruiz, David

    2015-07-01

    Coumarate 3-hydroxylase (C3H) catalyzes a key step of the synthesis of the two main lignin subunits, guaiacyl (G) and syringyl (S) in dicotyledonous species. As no functional data are available in regards to this enzyme in monocotyledonous species, we generated C3H1 knock-down maize plants. The results obtained indicate that C3H1 participates in lignin biosynthesis as its down-regulation redirects the phenylpropanoid flux: as a result, increased amounts of p-hydroxyphenyl (H) units, lignin-associated ferulates and the flavone tricin were detected in transgenic stems cell walls. Altogether, these changes make stem cell walls more degradable in the most C3H1-repressed plants, despite their unaltered polysaccharide content. The increase in H monomers is moderate compared to C3H deficient Arabidopsis and alfalfa plants. This could be due to the existence of a second maize C3H protein (C3H2) that can compensate the reduced levels of C3H1 in these C3H1-RNAi maize plants. The reduced expression of C3H1 alters the macroscopic phenotype of the plants, whose growth is inhibited proportionally to the extent of C3H1 repression. Finally, the down-regulation of C3H1 also increases the synthesis of flavonoids, leading to the accumulation of anthocyanins in transgenic leaves.

  10. Prolyl hydroxylase domain proteins regulate bone mass through their expression in osteoblasts.

    PubMed

    Zhu, Ke; Song, Pingping; Lai, Yumei; Liu, Chuanju; Xiao, Guozhi

    2016-12-05

    The roles of prolyl hydroxylase domain proteins (PHDs) in bone are incompletely understood. Here we deleted the expression of genes encoding PHD1, PHD2, and PHD3 in osteoblasts in mice by breeding the floxed Phd1-3 mice with Col1a1-Cre transgenic mice. Results showed that mice lacking PHD1-3 in osteoblasts (Phd1-3ob-/-) had increased bone mass. Bone parameters such as bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were increased, while trabecular spacing (Tb.Sp) was decreased in Phd1-3ob-/- relative to wild-type (WT) femurs. In contrast, loss of PHD1-3 in osteoblasts did not alter cortical thickness (Cort.Th). The mineralization apposition rate (MAR) was increased in Phd1-3ob-/- bone compared to that of wild-type (WT) bone, demonstrating an enhancement of osteoblast function. Loss of PHD1-3 increased the number of osteoblast progenitors (CFU-OBs) in bone marrow cultures. Interestingly, deleting Phd1-3 genes in osteoblasts increased osteoclast formation in vitro and in bone.

  11. Recent advances in diagnosis, treatment, and outcome of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

    PubMed

    Riepe, Felix G; Sippell, Wolfgang G

    2007-12-01

    Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is an autosomal-recessive disease causing cortisol deficiency, aldosterone deficiency and hyperandrogenism. Diagnosis of 21-OHD is confirmed by steroid analysis in newborn screening or later on. Standard medical treatment consists of oral glucocorticoid and mineralocorticoid administration in order to suppress adrenal androgens and to compensate for adrenal steroid deficiencies. However, available treatment is far from ideal, and not much is known about the long-term outcome in CAH as trials in patients in adulthood or old age are rare. Here we briefly describe the pathophysiology, clinical picture, genetics and epidemiology of 21-OHD. This is followed by a comprehensive review of the recent advances in diagnosis, treatment and outcome. Novel insights have been gained in the fields of newborn screening, specific steroid measurement utilizing mass spectrometry, genetics, glucocorticoid stress dosing, additive medical therapy, prenatal treatment, side-effects of medical treatment, adrenomedullary involvement, metabolic morbidity, fertility and gender identity. However, many issues are still unresolved, and novel questions, which will have to be answered in the future, arise with every new finding.

  12. Tryptophan hydroxylase Is Required for Eye Melanogenesis in the Planarian Schmidtea mediterranea.

    PubMed

    Lambrus, Bramwell G; Cochet-Escartin, Olivier; Gao, Jiarong; Newmark, Phillip A; Collins, Eva-Maria S; Collins, James J

    2015-01-01

    Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian Schmidtea mediterranea. We characterize the tryptophan hydroxylase (tph) gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that tph is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in S. mediterranea, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in tph knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP), or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems.

  13. Loss of prolyl hydroxylase-2 in myeloid cells and T-lymphocytes impairs tumor development.

    PubMed

    Mamlouk, Soulafa; Kalucka, Joanna; Singh, Rashim Pal; Franke, Kristin; Muschter, Antje; Langer, Anika; Jakob, Christiane; Gassmann, Max; Baretton, Gustavo B; Wielockx, Ben

    2014-02-15

    The tumor microenvironment plays a pivotal role during cancer development and progression. The balance between suppressive and cytotoxic responses of the tumor immune microenvironment has been shown to have a direct effect on the final outcome in various human and experimental tumors. Recently, we demonstrated that the oxygen sensor HIF-prolyl hydroxylase-2 (PHD2) plays a detrimental role in tumor cells, stimulating systemic growth and metastasis in mice. In our current study, we show that the conditional ablation of PHD2 in the hematopoietic system also leads to reduced tumor volume, intriguingly generated by an imbalance between enhanced cell death and improved proliferation of tumor cells. This effect seems to rely on the overall downregulation of protumoral as well as antitumoral cytokines. Using different genetic approaches, we were able to confine this complex phenotype to the crosstalk of PHD2-deficient myeloid cells and T-lymphocytes. Taken together, our findings reveal a multifaceted role for PHD2 in several hematopoietic lineages during tumor development and might have important implications for the development of tumor therapies in the future.

  14. Loss of epithelial hypoxia-inducible factor prolyl hydroxylase 2 accelerates skin wound healing in mice.

    PubMed

    Kalucka, Joanna; Ettinger, Andreas; Franke, Kristin; Mamlouk, Soulafa; Singh, Rashim Pal; Farhat, Katja; Muschter, Antje; Olbrich, Susanne; Breier, Georg; Katschinski, Dörthe M; Huttner, Wieland; Weidemann, Alexander; Wielockx, Ben

    2013-09-01

    Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β3-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.

  15. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases

    PubMed Central

    Chowdhury, Rasheduzzaman; Leung, Ivanhoe K. H.; Tian, Ya-Min; Abboud, Martine I.; Ge, Wei; Domene, Carmen; Cantrelle, François-Xavier; Landrieu, Isabelle; Hardy, Adam P.; Pugh, Christopher W.; Ratcliffe, Peter J.; Claridge, Timothy D. W.; Schofield, Christopher J.

    2016-01-01

    The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1–3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel–Lindau protein (VHL)–elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors. PMID:27561929

  16. Overexpression of a tomato flavanone 3-hydroxylase-like protein gene improves chilling tolerance in tobacco.

    PubMed

    Meng, Chen; Zhang, Song; Deng, Yong-Sheng; Wang, Guo-Dong; Kong, Fan-Ying

    2015-11-01

    Flavonoids are secondary metabolites found in plants with a wide range of biological functions, such as stress protection. This study investigated the functions of a tomato (Solanum lycopersicum) flavanone 3-hydroxylase-like protein gene SlF3HL by using transgenic tobacco. The expression of the gene was up-regulated under chilling (4 °C), heat (42 °C), salt (NaCl) and oxidative (H2O2) stresses. The transgenic plants that displayed high SlF3HL mRNA and protein levels showed higher flavonoid content than the WT plants. Moreover, the expression of three flavonoid biosynthesis-related structural genes, namely, chalcone synthase (CHS), chalcone isomerase (CHI) and flavonol synthase (FLS) was also higher in the transgenic plants than in the WT plants. Under chilling stress, the transgenic plants showed not only faster seed germination, better survival and growth, but also lower malondialdehyde (MDA) accumulation, relative electrical conductivity (REC) and H2O2 and O2(·-) levels compared with WT plants. These results suggested that SlF3HL stimulated flavonoid biosynthesis in response to chilling stress.

  17. Copper reduces striatal protein nitration and tyrosine hydroxylase inactivation induced by MPP+ in rats.

    PubMed

    Rubio-Osornio, M; Montes, S; Pérez-Severiano, F; Aguilera, P; Floriano-Sánchez, E; Monroy-Noyola, A; Rubio, C; Ríos, C

    2009-06-01

    Striatal administration of 1-methyl-4-phenylpyridinium (MPP(+)), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson's disease. Copper acts as a prosthetic group of several antioxidant enzymes and recent data show that copper attenuated MPP(+)-evoked neurotoxicity. We evaluated the effect of copper (as a supplement) upon proteins nitration (60 kDa) and tyrosine hydroxylase (TH) inactivation induced by MPP(+) (10 microg/8 microL) injection into the rat striatum. Copper pretreatment (10 micromol/kg i.p.) prevented both MPP(+)-induced proteins nitration and TH inactivation. Copper treatment also prevented the dopamine-depleting effect of MPP(+) injection. Those results were accompanied by a significant reduction of enzymatic activity of the constitutive nitric oxide synthase (cNOS), whereas, the protein levels of the three isoforms of NOS remained unchanged. Results indicate that the effect of copper against MPP(+)-induced proteins nitration and TH inactivation in the striatum of rat may be mediated by a reduction of cNOS activity.

  18. Intake of melatonin increases tryptophan hydroxylase type 1 activity in aged rats: Preliminary study.

    PubMed

    Moranta, D; Barceló, P; Aparicio, S; Garau, C; Sarubbo, F; Ramis, M; Nicolau, C; Esteban, S

    2014-01-01

    Pineal melatonin is important not only for synchronization of biological rhythms, but also in the ageing process as a potential drug to relieve oxidative damage. During ageing, the nocturnal melatonin production decreases resulting in an increased incidence of disorders. Present in vivo experiments were performed to study the effects of exogenous melatonin chronically administered to old rats on the pineal biosynthesis of melatonin and the precursor serotonin (5-HT) mediated by tryptophan hydroxylase type 1 (TPH-1). Accumulation of 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition was used as a measure of the TPH-1 activity. 5-HT and its metabolite 5-HIAA were also quantified by HPLC-ED. As expected, ageing resulted in worsening of different neurochemical parameters. However, chronic intake of melatonin (1mg/kg/day, diluted in drinking water, 4 weeks) increased TPH-1 activity and significantly improved the age-induced deficits in nocturnal melatonin content in the pineal gland. Results suggest that melatonin intake (or melatonin rich foods) may contribute to recover the pineal function preventing the nocturnal descent of 5-HT and melatonin biosynthesis that normally occur in pineal gland as a consequence of ageing.

  19. Variability of the tryptophan hydroxylase gene: study in victims of violent suicide.

    PubMed

    Stefulj, Jasminka; Kubat, Milovan; Balija, Melita; Skavic, Josip; Jernej, Branimir

    2005-03-30

    Tryptophan hydroxylase (TPH), the enzyme controlling serotonin synthesis, is considered to be a potential contributor to the biological substrate of suicide. The association of the promoter (-7065CT) and intron 7 (218AC) polymorphisms, and the related haplotype, of the Tph1 gene with suicidal behavior was investigated in a sample of 160 victims of violent suicide and 284 healthy controls. All individuals were males of Croatian (Slavic) origin. Allele frequencies of both polymorphisms in Croatian controls were similar to control values reported for other European populations. Alleles at the two loci demonstrated highly significant linkage disequilibrium. No differences between controls and victims for the Tph1 genetic variation, either at single loci, or at a haplotypic level, were demonstrated, albeit there was a tendency, not reaching statistical significance, towards an increase of the intron 7CC genotype in the suicide group. Negative association results on the individual Tph1 loci, in accordance with the majority of previous reports, confirmed the lack of their major effect also in the Slavic ethnicity. Haplotypic results, on the other hand, opposing the previous positive finding, point to the possible influence of ethnicity (or gender) on the association between the Tph1 gene polymorphism and suicide.

  20. Effect of LPS treatment on tyrosine hydroxylase expression and Parkinson-like behaviors.

    PubMed

    Girard-Joyal, Olivier; Ismail, Nafissa

    2016-12-24

    Puberty is a critical period of development during which the brain undergoes reorganizing and remodeling. Exposure to stress during this period is thought to interfere with normal brain development and increase susceptibility to mental illnesses. In female mice, pubertal exposure to lipopolysaccharide (LPS), a bacterial endotoxin, has been shown to alter sexual, anxiety-like, and depression-like behaviors and cognition in an enduring manner. However, the mechanisms underlying these effects remain unknown. The present study examined age and sex difference in tyrosine hydroxylase (TH) expression and dopamine-dependent and Parkinson-like behaviors following LPS treatment. The results show that LPS treatment during adulthood causes an enduring increase in TH expression in many of the brain regions examined. In contrast, there is no change in TH expression following LPS treatment during puberty. However, pubertal LPS treatment induces enduring behavioral deficits in tests of Parkinson-like behaviors, more so in male than in female mice. These results suggest that the low levels of TH following exposure to pubertal immune challenge may predispose mice to Parkinson-like behavior. These findings add to our understanding of stress and immune responses during puberty and their impact on mental health later in life.

  1. The crystal structure of human dopamine β-hydroxylase at 2.9 Å resolution

    PubMed Central

    Vendelboe, Trine V.; Harris, Pernille; Zhao, Yuguang; Walter, Thomas S.; Harlos, Karl; El Omari, Kamel; Christensen, Hans E. M.

    2016-01-01

    The norepinephrine pathway is believed to modulate behavioral and physiological processes, such as mood, overall arousal, and attention. Furthermore, abnormalities in the pathway have been linked to numerous diseases, for example hypertension, depression, anxiety, Parkinson’s disease, schizophrenia, Alzheimer’s disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine β-monooxygenase N-terminal) domain, also found in >1600 other proteins, reveals a possible metal-binding site and a ligand-binding pocket. The catalytic core structure shows two different conformations: an open active site, as also seen in another member of this enzyme family [the peptidylglycine α-hydroxylating (and α-amidating) monooxygenase], and a closed active site structure, in which the two copper-binding sites are only 4 to 5 Å apart, in what might be a coupled binuclear copper site. The dimerization domain adopts a conformation that bears no resemblance to any other known protein structure. The structure provides new molecular insights into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system. PMID:27152332

  2. Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

    PubMed Central

    Bezem, Maria T.; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I.

    2016-01-01

    Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

  3. Aryl hydrocarbon hydroxylase (AHH) induction by polybrominated biphenyls (PBBs): enchancement by photolysis.

    PubMed

    Robertson, L W; Parkinson, A; Chittim, B; Bandiera, S; Sawyer, T W; Safe, S

    1981-01-01

    Irradiation of the commercial polybrominated biphenyl (PBB( mixture, fireMaster BP-6, in cyclohexane solution at 300 nm for 930 min resulted in a marked diminution of the major components of the mixture. Administration of the photolyzed PBB mixture of fireMaster BP-6 to immature male Wistar rats caused both dose-related decreases in thymus weight and increase in hepatic microsomal benzo[a]pyrene hydroxylase (AHH), 4-dimethylaminoantipyrine N-demethylase and NADPH-cytochrome c reductase activities and cytochrome P-450 content. The dose effecting half-maximal AHH induction for the photolyzed PBBs (9 mg . KG-1) was approximately 6 times lower than that of fireMaster BP-6 (50 mg. kg-1). Furthermore, the concentration of photolyzed PBBs (2 micrometers) required to displace 50% of the specifically-bound [3H] TCDD from its high-affinity cytosolic Ah receptor was approximately 150 times lower than that required for fireMaster BP-6 (300 micrometers), as measured by sucrose density gradient centrifugation analysis. The results suggest that the photolysis of the commercial PBB mixture yields products which possess increased biologic activity.

  4. Transcriptional regulation of the tyrosine hydroxylase gene by glucocorticoid and cyclic AMP

    SciTech Connect

    Lewis, E.J.; Harrington, C.A.; Chikaraishi, D.M.

    1987-06-01

    Glucocorticoid and cyclic AMP increase tyrosine hydroxylase (TH) activity and mRNA levels in pheochromocytoma cultures. The transcriptional activity of the TH gene, as measured by nuclear run-on assay, is also increased when cultures are treated with the synthetic glucocorticoid dexamethasone or agents that increase intracellular cyclic AMP, such as forskolin and 8-BrcAMP. Both inducers effect transcriptional changes within 10 min after treatment and are maximal after 30 min for forskolin and after 60 min for dexamethasone. The 5' flanking sequences of the TH gene were fused to the bacterial gene chloramphenicol acetyltransferase (CAT), and the hybrid gene was transfected into pheochromocytoma cultures and GH/sub 4/ pituitary cells. In both cell lines, a region of the TH gene containing bases -272 to +27 conferred induction of CAT by cyclic AMP, but not by glucocorticoid. The same results were found when a region of the TH gene containing -773 to + 27 was used. Thus, the sequences required for induction of TH by cyclic AMP are contained within 272 bases of 5' flanking sequence, but sequences sufficient for glucocorticoid regulation are not contained with 773 bases.

  5. Cinnamate-4-hydroxylase expression in Arabidopsis. Regulation in response to development and the environment.

    PubMed Central

    Bell-Lelong, D A; Cusumano, J C; Meyer, K; Chapple, C

    1997-01-01

    Cinnamate-4-hydroxylase (C4H) is the first Cyt P450-dependent monooxygenase of the phenylpropanoid pathway. To study the expression of this gene in Arabidopsis thaliana, a C4H cDNA clone from the Arabidopsis expressed sequence tag database was identified and used to isolate its corresponding genomic clone. The entire C4H coding sequence plus 2.9 kb of its promoter were isolated on a 5.4-kb HindIII fragment of this cosmid. Inspection of the promoter sequence revealed the presence of a number of putative regulatory motifs previously identified in the promoters of other phenylpropanoid pathway genes. The expression of C4H was analyzed by RNA blot hybridization analysis and in transgenic Arabidopsis carrying a C4H-beta-glucuronidase transcriptional fusion. C4H message accumulation was light-dependent, but was detectable even in dark-grown seedlings. Consistent with these data, C4H mRNA was accumulated to light-grown levels in etiolated det1-1 mutant seedlings. C4H is widely expressed in various Arabidopsis tissues, particularly in roots and cells undergoing lignification. The C4H-driven beta-glucuronidase expression accurately reflected the tissue-specificity and wound-inducibility of the C4H promoter indicated by RNA blot hybridization analysis. A modest increase in C4H expression was observed in the tt8 mutant of Arabidopsis. PMID:9085570

  6. Human dopamine {beta}-hydroxylase locus and the chromosome 9q34 region in alcoholism

    SciTech Connect

    Parsian. A.; Suarez, B.K.; Hampe, C.

    1994-09-01

    Human dopamine {beta}-hydroxylase (DBH) is responsible for conversion of dopamine to norepinephrine in catecholamine neurons. Potential inhibitors of this enzyme do exist, but they are generally not effective in vivo in reducing tissue concentrations of catecholamines. The gene for DBH has been localized to 9q34 by linkage analysis and in situ hybridization. Recently there have been reports indicating a suggestive evidence of linkage between DNA markers in 9q34 region and alcoholism. In order to test for this suggestive linkage, we have genotyped a sample of 134 subjects with alcoholism, 30 alcoholic families (n=302) and 92 normal controls. The alcoholic subjects are probands of multiple incidence families. The normal controls are an epidemiologically ascertained samples of middle-aged, unrelated individuals. The two groups were matched for sex and ethnic background. The markers used in this study were dinucleotide repeats in the DBH gene, and two highly informative (CA) markers (D9S64, D9S66) flanking the DBH gene. A preliminary affected-sib-pair analysis was carried out under two diagnostic schemes. Regardless of whether `probable` alcoholics are classified as unaffected (t=0.63) or affected (t=1.50), these data do not reveal a significant excess in DBH marker sharing among affected-sib-pairs. However, the comparison of the DBH marker allele frequencies between the unrelated alcoholic panel and the unrelated normal control panel was significant at the p=0.04 level.

  7. Prolyl hydroxylase 2: a novel regulator of β2 -adrenoceptor internalization.

    PubMed

    Yan, Biao; Huo, Zhaoxia; Liu, Ying; Lin, Xiaoping; Li, Jun; Peng, Luying; Zhao, Hong; Zhou, Zhao-Nian; Liang, Xingqun; Liu, Yi; Zhu, Weidong; Liang, Dandan; Li, Li; Sun, Yunfu; Cui, Jianmin; Chen, Yi-Han

    2011-12-01

    Adrenergic receptor (AR)-mediated signalling is modulated by oxygen levels. Prolyl hydroxylases (PHDs) are crucial for intracellular oxygen sensing and organism survival. However, it remains to be clarified whether or how PHDs are involved in the regulation of β(2) -adrenoceptor (β(2) -AR) signalling. Here we show that PHD2 can modulate the rate of β(2) -AR internalization through interactions with β-arrestin 2. PHD2 hydroxylates β-arrestin 2 at the proline (Pro)(176), Pro(179) and Pro(181) sites, which retards the recruitment of β-arrestin 2 to the plasma membrane and inhibits subsequent co-internalization with β(2) -AR into the cytosol. β(2) -AR internalization is critical to control the temporal and spatial aspects of β(2) -AR signalling. Identifying novel regulators of β(2) -AR internalization will enable us to develop new strategies to manipulate receptor signalling and provide potential targets for drug development in the prevention and treatment of diseases associated with β(2) -AR signalling dysregulation.

  8. Differential expression of tyrosine hydroxylase mRNA in the developing rat mesencephalon.

    PubMed

    Solberg, Y; Pollack, Y; Silverman, W F

    1992-12-01

    1. With respect to the mesostriatal projection, the mesencephalon is composed of two dopaminergic (DA) cell populations, called dorsal tier and ventral tier. Strong evidence suggests differences in both the spatial and the temporal sequence of the innervation of the striatum between the two groups, with the ventral tier neurons innervating striatal patches prenatally and dorsal tier cells innervating striatal matrix postnatally. 2. Using in situ hybridization, we have examined the expression of the gene coding for tyrosine hydroxylase (TH) in mesencephalic DA neurons with respect to their postnatal development. Two ontogenic patterns of expression were observed: (a) dorsal tier neurons of the medial mesencephalon exhibited a sharp increase in expression beginning after birth, peaking on day 14, then decreasing and, finally, stabilizing; and (b) ventral tier neurons and dorsal tier cells from the lateral and the medial-dorsal mesencephalon showed only a slight increase in TH mRNA, reaching a plateau at P10. 3. The time course of the observed increase in TH gene expression in the first group, generally parallels the innervation of their target cells in the striatal matrix, suggesting that TH gene expression in these cells may be influenced by their postsynaptic cells or by the innervation process.

  9. Evaluation of impact of steroid replacement treatment on bone health in children with 21-hydroxylase deficiency.

    PubMed

    Delvecchio, M; Soldano, L; Lonero, A; Ventura, A; Giordano, P; Cavallo, L; Grano, M; Brunetti, G; Faienza, M F

    2015-04-01

    There are conflicting data regarding the potential impact of chronic glucocorticoid (GC) therapy on the bone mineral density of patients with congenital adrenal hyperplasia (CAH). Previous studies performed by dual-energy X-ray absorptiometry reported conflicting results. The purpose of this study was to assess the impact of chronic GC replacement treatment in children with classical and non classical CAH due to 21-hydroxylase deficiency (21-OHD) by quantitative ultrasonometry (QUS), an easy, cheap, and radiation-free technique. The study population consisted of nineteen 21-OHD patients (nine males) on lifelong GC treatment. Anthropometric, hormonal, and treatment data were recorded for each patient, and bone quality was assessed by QUS measurements. QUS findings (amplitude-dependent speed of sound and bone transmission time) were normal in 21-OHD patients and did not correlate with duration of treatment, daily, total, and yearly hydrocortisone dose. Furthermore, no significant correlation was found between QUS findings and 17α-hydroxy progesterone, Δ4-androstenedione, and testosterone levels. In conclusion, our results provide reassurance that currently used replacement doses of GC do not have a major impact on bone in patients with CAH. QUS seems to be a reliable tool for screening of bone health in children with 21-OHD.

  10. Inhibition of prolyl hydroxylase domain-containing protein downregulates vascular angiotensin II type 1 receptor.

    PubMed

    Matsuura, Hirohide; Ichiki, Toshihiro; Ikeda, Jiro; Takeda, Kotaro; Miyazaki, Ryohei; Hashimoto, Toru; Narabayashi, Eriko; Kitamoto, Shiro; Tokunou, Tomotake; Sunagawa, Kenji

    2011-09-01

    Inhibition of prolyl hydroxylase domain-containing protein (PHD) by hypoxia stabilizes hypoxia-inducible factor 1 and increases the expression of target genes, such as vascular endothelial growth factor. Although the systemic renin-angiotensin system is activated by hypoxia, the role of PHD in the regulation of the renin-angiotensin system remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin II type 1 receptor (AT(1)R). Hypoxia, cobalt chloride, and dimethyloxalylglycine, all known to inhibit PHD, reduced AT(1)R expression in vascular smooth muscle cells. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT(1)R expression. Cobalt chloride diminished angiotensin II-induced extracellular signal-regulated kinase phosphorylation. Cobalt chloride decreased AT(1)R mRNA through transcriptional and posttranscriptional mechanisms. Oral administration of cobalt chloride (14 mg/kg per day) to C57BL/6J mice receiving angiotensin II infusion (490 ng/kg per minute) for 4 weeks significantly attenuated perivascular fibrosis of the coronary arteries without affecting blood pressure level. These data suggest that PHD inhibition may be beneficial for the treatment of cardiovascular diseases by inhibiting renin-angiotensin system via AT(1)R downregulation.

  11. Tryptophan hydroxylase Is Required for Eye Melanogenesis in the Planarian Schmidtea mediterranea

    PubMed Central

    Lambrus, Bramwell G.; Cochet-Escartin, Olivier; Gao, Jiarong; Newmark, Phillip A.; Collins, Eva-Maria S.; Collins, James J.

    2015-01-01

    Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian Schmidtea mediterranea. We characterize the tryptophan hydroxylase (tph) gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that tph is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in S. mediterranea, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in tph knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP), or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems. PMID:26017970

  12. Cloning, expression and purification of isoflavone-2'-hydroxylase from Astragalus membranaceus Bge. Var. mongolicus (Bge.) Hsiao.

    PubMed

    Chen, Jing; Yuan, Hui; Zhang, Lin; Pan, Haiyun; Xu, Rongyan; Zhong, Yang; Chen, Jiakuan; Nan, Peng

    2015-03-01

    Plant cytochrome P450 enzymes play vital roles in the biosynthesis of plant secondary metabolites, including phenylpropanoids and phytoalexins. Isoflavone-2'-hydroxylase (AmI2'H) from Astragalus membranaceus Bge. Var. mongolicus (Bge.) Hsiao is a membrane protein and an eukaryotic cytochrome P450 enzyme involved in isoflavonoid biosynthesis. We cloned the AmI2'H gene by employing RACE methods and modified the gene sequence to facilitate protein expression and increase protein solubility. Two vectors, pET-28a(+) and pCW ori(+), were used to express AmI2'H in Escherichia coli. The expression efficiency and purity of target protein were analyzed and demonstrated that pET-28a(+) vector containing the AmI2'H gene could produce larger amounts of target proteins with higher purity than pCWori(+). The purified proteins were identified as AmI2'H by LC-ESI-MS/MS analysis and their proper folding was assessed by CO difference spectrum.

  13. Loss of Epithelial Hypoxia-Inducible Factor Prolyl Hydroxylase 2 Accelerates Skin Wound Healing in Mice

    PubMed Central

    Kalucka, Joanna; Ettinger, Andreas; Franke, Kristin; Mamlouk, Soulafa; Singh, Rashim Pal; Farhat, Katja; Muschter, Antje; Olbrich, Susanne; Breier, Georg; Katschinski, Dörthe M.; Huttner, Wieland; Weidemann, Alexander

    2013-01-01

    Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β3-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds. PMID:23798557

  14. Novel enhancement mechanism of tyrosine hydroxylase enzymatic activity by nitric oxide through S-nitrosylation

    PubMed Central

    Wang, Yuanyuan; Sung, Chun Chau; Chung, Kenny K. K.

    2017-01-01

    Tyrosine hydroxylase (TH) is a rate-limiting step enzyme in the synthesis of catecholamines. Catecholamines function both as hormone and neurotransmitters in the peripheral and central nervous systems, therefore TH’s expression and enzymatic activity is tightly regulated by various mechanisms. Several post-translational modifications have been shown to regulate TH’s enzymatic activity such as phosphorylation, nitration and S-glutathionylation. While phosphorylation at N-terminal of TH can activate its enzymatic activity, nitration and S-glutathionylation can inactivate TH. In this study, we found that TH can also be S-nitrosylated by nitric oxide (NO). S-nitrosylation is a reversible modification of cysteine (cys) residue in protein and is known to be an emerging signaling mechanism mediated by NO. We found that TH can be S-nitrosylated at cys 279 and TH S-nitrosylation enhances its enzymatic activity both in vitro and in vivo. These results provide a novel mechanism of how NO can modulate TH’s enzymatic activity through S-nitrosylation. PMID:28287127

  15. Prolyl hydroxylase domain-2 (PHD2) inhibition may be a better therapeutic strategy in renal anemia.

    PubMed

    Soni, Hitesh

    2014-05-01

    Recombinant human erythropoietin (rHuEPO) has revolutionized the life of dialysis patients with anemia of chronic kidney disease (CKD). Newer erythropoietin analogues with improved profile have been introduced recently. However, there are many concerns such as safety, economy and patient compliance with these newer rHuEPo analogues. Small molecules aimed to inhibit prolyl hydroxylase domain-2 (PHD2) may prevent degradation of hypoxia inducible factor-2 (HIF2) which leads to endogenous erythropoietin production. This therapeutic intervention may not only overcome the patient compliance and economic burden but also possibly overcome the safety issues related to rHuEPO and its analogues. Moreover, PHD2 inhibitors may increase the endogenous circulating iron availability via suppression of hepcidin, a master regulator of iron homeostasis which further reduces the need for exogenous intravenous iron administration for effective erythropoiesis in renal anemia patients. In conclusion, small molecule PHD2 inhibitors may have better therapeutic efficacy and potential to address clinical concerns associated with rHuEPO and rHuEPO mimetic peptides.

  16. Hepatic Cholesterol-25-Hydroxylase Overexpression Improves Systemic Insulin Sensitivity in Mice

    PubMed Central

    Noebauer, Britta; Jais, Alexander; Todoric, Jelena; Gossens, Klaus; Sutterlüty-Fall, Hedwig

    2017-01-01

    Obesity is a major risk factor for several diseases including diabetes, heart disease, and some forms of cancer and due to its rapidly increasing prevalence it has become one of the biggest problems medicine is facing today. All the more surprising, a substantial percentage of obese patients are metabolically healthy when classified based on insulin resistance and systemic inflammation. Oxysterols are naturally occurring molecules that play important role in various metabolic and inflammatory processes and their levels are elevated in patients suffering from obesity and diabetes. 25-Hydroxycholesterol (25-OHC) is produced in cells from cholesterol by the enzyme cholesterol 25-hydroxylase (Ch25h) and is involved in lipid metabolism, inflammatory processes, and cell proliferation. Here, we investigated the role of hepatic Ch25h in the transition from metabolically healthy obesity to insulin resistance and diabetes. Using several different experimental approaches, we demonstrated the significance of Ch25h on the border of “healthy” and “diseased” states of obesity. Adenovirus-mediated Ch25h overexpression in mice improved glucose tolerance and insulin sensitivity and lowered HOMA-IR. Our data suggest that low hepatic Ch25h levels could be considered a risk marker for unhealthy obesity. PMID:28299341

  17. Additive effects of serotonin transporter and tryptophan hydroxylase-2 gene variation on emotional processing.

    PubMed

    Herrmann, Martin J; Huter, Theresa; Müller, Frauke; Mühlberger, Andreas; Pauli, Paul; Reif, Andreas; Renner, Tobias; Canli, Turhan; Fallgatter, Andreas J; Lesch, Klaus-Peter

    2007-05-01

    Prior studies reported that functional variants of both the serotonin transporter (5-HTT) and tryptophan hydroxylase-2 genes (TPH2), 2 key regulators of the serotonergic signaling pathway, modulate amygdala activation during emotional processing. We addressed the question whether these 2 gene variants modulate each other, using an emotional picture-processing task. Specifically, we measured event-related potentials (ERPs) during a passive emotional picture perception task, focusing on ERPs for the early posterior negativity (EPN) around 240 ms and for the slow wave starting at 315 ms. We found evidence for increased neural activity at 240 ms in individuals who carried 1 or 2 copies of the low-expression short variant of the 5-HTT. Carriers of T variant of the TPH2 also showed a tendency toward increased neural activity at 240 ms. Moreover, we observed an additive effect of both genotypes for EPN, with highest neural activity to emotional stimuli in individuals carrying combination of both short variant of 5-HTT and T variant of TPH2. Our results indicate that both the 5-HTT and the TPH2 genotypes modulate the sensory encoding of affective stimuli during early steps of visual processing and reveal additive effects of 2 genes in the serotonergic control of emotion regulation.

  18. Alpha 2-adrenergic receptors influence tyrosine hydroxylase activity in retinal dopamine neurons.

    PubMed

    Iuvone, P M; Rauch, A L

    1983-12-12

    Dopamine (DA) is a putative neurotransmitter in a population of interneurons in the mammalian retina that are activated by photic stimulation. Pharmacological studies were conducted to determine if alpha 2-adrenergic receptors influence the activity of retinal tyrosine hydroxylase (TH), a biochemical indicator of changes in the activity of the DA-containing neurons. TH activity was low in dark-adapted retinas and high in light-exposed retinas. Systemic administration of the alpha 2-adrenoceptor antagonists, yohimbine and piperoxane, to dark-adapted rats significantly stimulated TH activity. This effect was apparently mediated locally within the retina because the response could also be elicited by direct injection of yohimbine into the vitreous. The dose-response relationships for the effects of alpha 2-adrenoceptor antagonists on retinal TH activity were similar to those for the effects on brain noradrenergic neurons, where alpha 2-adrenoceptors have been shown to be involved in the autoregulation of neuronal activity. Clonidine, an alpha 2-adrenoceptor agonist, had no effect when administered alone to dark-adapted rats, but it attenuated the stimulatory effect of yohimbine. In contrast, clonidine decreased TH activity of light-exposed retinas, an effect that was reversed by yohimbine. These observations suggest that alpha 2-adrenoceptors influence the activity of retinal DA-containing neurons.

  19. Notch Downregulation and Extramedullary Erythrocytosis in Hypoxia-Inducible Factor Prolyl 4-Hydroxylase 2-Deficient Mice.

    PubMed

    Myllymäki, Mikko N M; Määttä, Jenni; Dimova, Elitsa Y; Izzi, Valerio; Väisänen, Timo; Myllyharju, Johanna; Koivunen, Peppi; Serpi, Raisa

    2017-01-15

    Erythrocytosis is driven mainly by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2) (PHD2/EGLN1), the major downregulator of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable of extramedullary erythropoiesis. We studied HIF-P4H-2-deficient (Hif-p4h-2(gt/gt)) mice, which show slightly induced erythropoiesis upon aging despite nonincreased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased erythroid burst-forming unit (BFU-E) growth, and the mice were protected against anemia. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligand genes Jag1, Jag2, and Dll1 and target Hes1 became downregulated upon aging HIF-2α dependently. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIFα stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.

  20. Hyperplasia of pulmonary neuroepithelial bodies (NEB) in lungs of prolyl hydroxylase -1(PHD-1) deficient mice.

    PubMed

    Pan, Jie; Yeger, Herman; Ratcliffe, Peter; Bishop, Tammie; Cutz, Ernest

    2012-01-01

    Pulmonary NEB, widely distributed within the airway mucosa of mammalian lungs, are presumed hypoxia sensitive airway O(2) sensors responding to changes in airway gas concentration. NEB cell hyperplasia has been reported after exposure to chronic hypoxia and in a variety of paediatric and adult lung disorders. Prolyl hydroxylases (PHD 1-3) regulate the stability of hypoxia-inducible factors (HIF's) in an O(2)-dependent manner and function as intrinsic oxygen sensors. To determine a possible role of PHD-1in NEB cells we have quantitated NEB's in lungs of neonatal (P2) and adult (2 months) PHD-1-deficient mice and compared them to wild type (WT) control mice. Lung tissues fixed in formalin and embedded in paraffin were processed for immunoperoxidase method and frozen sections for multilabel immunoflourescence using antibodies for NEB markers synaptophysin, synaptic vesicle protein 2 and the peptide CGRP. The frequency and size of NEB in lungs of PHD-1 deficient neonatal mice (P2) and at 2 months was increased significantly compared to WT controls (p < 0.01). The present data suggests an important role for PHD enzymes in NEB cell biology deserving further studies. Since the PHD-1 deficient mouse appears to be the first animal model showing NEB cell hyperplasia it may be useful for studies of NEB physiology and pathobiology.