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Sample records for cmp-n-acetylneuraminic acid hydroxylase

  1. Inactivation of CMP-N-acetylneuraminic acid hydroxylase occurred prior to brain expansion during human evolution

    PubMed Central

    Chou, Hsun-Hua; Hayakawa, Toshiyuki; Diaz, Sandra; Krings, Matthias; Indriati, Etty; Leakey, Meave; Paabo, Svante; Satta, Yoko; Takahata, Naoyuki; Varki, Ajit

    2002-01-01

    Humans are genetically deficient in the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an Alu-mediated inactivating mutation of the gene encoding the enzyme CMP-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase (CMAH). This mutation occurred after our last common ancestor with bonobos and chimpanzees, and before the origin of present-day humans. Here, we take multiple approaches to estimate the timing of this mutation in relationship to human evolutionary history. First, we have developed a method to extract and identify sialic acids from bones and bony fossils. Two Neandertal fossils studied had clearly detectable Neu5Ac but no Neu5Gc, indicating that the CMAH mutation predated the common ancestor of humans and Neandertals, ≈0.5–0.6 million years ago (mya). Second, we date the insertion event of the inactivating human-specific sahAluY element that replaced the ancestral AluSq element found adjacent to exon 6 of the CMAH gene in the chimpanzee genome. Assuming Alu source genes based on a phylogenetic tree of human-specific Alu elements, we estimate the sahAluY insertion time at ≈2.7 mya. Third, we apply molecular clock analysis to chimpanzee and other great ape CMAH genes and the corresponding human pseudogene to estimate an inactivation time of ≈2.8 mya. Taken together, these studies indicate that the CMAH gene was inactivated shortly before the time when brain expansion began in humankind's ancestry, ≈2.1–2.2 mya. In this regard, it is of interest that although Neu5Gc is the major sialic acid in most organs of the chimpanzee, its expression is selectively down-regulated in the brain, for as yet unknown reasons. PMID:12192086

  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. 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.

  4. 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.

  5. Characterization and Solubilization of Kaurenoic Acid Hydroxylase from Gibberella fujikuroi.

    PubMed Central

    Jennings, J. C.; Coolbaugh, R. C.; Nakata, D. A.; West, C. A.

    1993-01-01

    A key step in gibberellin biosynthesis is the conversion of ent-kaurenoic acid to ent-7[alpha]-hydroxykaurenoic acid, mediated by the enzyme kaurenoic acid hydroxylase. A cell-free system obtained from Gibberella fujikuroi (Saw.) Wr. was used to characterize kaurenoic acid hydroxylase activity. Microsomal preparations from disrupted fungal cells, in the presence of O2 and NADPH, converted [17-14C]ent-kaurenoic acid to oxidation products that were separated by high-performance liquid chromatography and identified as ent-7[alpha]-hydroxykaurenoic acid and gibberellin A14 by combined gas chromatography-mass spectrometry. Flavin adenine dinucleotide and the chloride salts of several monovalent cations stimulated the conversion of ent-kaurenoic acid to these products, whereas CO and a number of known inhibitors of cytochrome P-450-dependent reactions, including paclobutrazol, tetcyclacis, BAS 111.W, flurprimidol, triarimol, metyrapone, and 1-phenylimida-zole, significantly reduced kaurenoic acid hydroxylase activity. Kaurenoic acid hydroxylase was solubilized from fungal microsomes by treatment with 1 M KCl. The properties of the enzyme noted above suggest that kaurenoic acid hydroxylase from G. fujikuroi is a cytochrome P-450-dependent monooxygenase. PMID:12231743

  6. Expression of sialic acid and polysialic acid in serogroup B Neisseria meningitidis: divergent transcription of biosynthesis and transport operons through a common promoter region.

    PubMed Central

    Swartley, J S; Ahn, J H; Liu, L J; Kahler, C M; Stephens, D S

    1996-01-01

    We studied capsule-defective (Cap-) serogroup B meningococcal mutants created through Tn916 or omega-fragment mutagenesis. The Cap- phenotypes were the results of insertions in three of four linked genes (synX, synC, and synD) involved in CMP-N-acetylneuraminic acid and polysialic acid capsule biosynthesis, and in ctrA the first of four linked genes involved in capsule membrane transport. Mutations in the CMP-N-acetylneuraminic acid biosynthesis genes synX and synC caused defects in lipooligosaccharide sialylation but not mutations in the putative (alpha2 -> 8)-linked polysialyltransferase (synD) or in ctrA. Reverse transcriptase PCR studies indicated that the four biosynthesis genes (synX to -D) and the capsule transport genes (ctr to -D) were separately transcribed as operons. The operons were separated by a 134-bp intergenic region. Primer extension of synX and ctrA demonstrated that transcription of the operons was divergently initiated from adjacent start sites present in the intergenic region. Both transcriptional start sites were preceded by a perfect -10 Pribnow promoter binding region. The synX to -D, but not the ctrA to -D, transcriptional start site was preceded by a sequence bearing strong homology to the consensus sigma 70 -35 promoter binding sequence. Both promoters showed transcriptional activity when cloned behind a lacZ reporter gene in Escherichia coli. Our results confirm the intrinsic relationship between polysialic acid capsule biosynthesis and lipooligosaccharide sialylation pathways in group B Neisseria meningitidis. Our study also suggests that the intergenic region separating the synX to -D and ctrA to -D operons is an important control point for the regulation of group B capsule expression through coordinated transcriptional regulation of the synX to -D and drA to -D promoters. PMID:8763931

  7. 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

  8. Chlamydia pneumoniae encodes a functional aromatic amino acid hydroxylase

    PubMed Central

    Abromaitis, Stephanie; Hefty, P. Scott; Stephens, Richard S.

    2010-01-01

    Chlamydia pneumoniae is a community-acquired respiratory pathogen that has been associated with the development of atherosclerosis. Analysis of the C. pneumoniae genome identified a gene (Cpn1046) homologous to eukaryotic aromatic amino acid hydroxylases. Aromatic amino acid hydroxylases (AroAA-H) hydroxylate phenylalanine, tyrosine, and tryptophan into tyrosine, dihydroxyphenylalanine (L-DOPA), and 5-hydroxytryptophan, respectively. Sequence analysis of Cpn1046 demonstrated that residues essential for AroAA-H enzymatic function are conserved and that a subset of Chlamydia species contain an AroAA-H homolog. The chlamydial AroAA-H are transcriptionally linked to a putative bacterial membrane transport protein. We determined that recombinant Cpn1046 is able to hydroxylate phenylalanine, tyrosine, and tryptophan with roughly equivalent activity for all three substrates. Cpn1046 is expressed within 24 h of infection, allowing C. pneumoniae to hydroxylae host stores of aromatic amino acids during the period of logarithmic bacterial growth. From these results we can conclude that C. pneumoniae, as well as a subset of other Chlamydia species, encode an AroAA-H that is able to use all three aromatic amino acids as substrates. The maintenance of this gene within a number of Chlamydia suggests that the enzyme may have an important role in shaping the metabolism or overall pathogenesis of these bacteria. PMID:19141112

  9. Chlamydia pneumoniae encodes a functional aromatic amino acid hydroxylase.

    PubMed

    Abromaitis, Stephanie; Hefty, P Scott; Stephens, Richard S

    2009-03-01

    Chlamydia pneumoniae is a community-acquired respiratory pathogen that has been associated with the development of atherosclerosis. Analysis of the C. pneumoniae genome identified a gene (Cpn1046) homologous to eukaryotic aromatic amino acid hydroxylases (AroAA-Hs). AroAA-Hs hydroxylate phenylalanine, tyrosine, and tryptophan into tyrosine, dihydroxyphenylalanine, and 5-hydroxytryptophan, respectively. Sequence analysis of Cpn1046 demonstrated that residues essential for AroAA-H enzymatic function are conserved and that a subset of Chlamydia species contain an AroAA-H homolog. The chlamydial AroAA-Hs are transcriptionally linked to a putative bacterial membrane transport protein. We determined that recombinant Cpn1046 is able to hydroxylate phenylalanine, tyrosine, and tryptophan with roughly equivalent activity for all three substrates. Cpn1046 is expressed within 24 h of infection, allowing C. pneumoniae to hydroxylate host stores of aromatic amino acids during the period of logarithmic bacterial growth. From these results we can conclude that C. pneumoniae, as well as a subset of other Chlamydia species, encode an AroAA-H that is able to use all three aromatic amino acids as substrates. The maintenance of this gene within a number of Chlamydia suggests that the enzyme may have an important role in shaping the metabolism or overall pathogenesis of these bacteria. PMID:19141112

  10. 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.

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

    PubMed

    Schalk, M; Croteau, R

    2000-10-24

    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

  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. In Vitro Inhibition of Chick Embryo Lysyl Hydroxylase by Homogentisic Acid

    PubMed Central

    Murray, John C.; Lindberg, Kenneth A.; Pinnell, Sheldon R.

    1977-01-01

    Homogentisic acid inhibits the in vitro activity of chick embryo lysyl hydroxylase, a microsomal enzyme which catalyzes the transformation of certain lysyl residues in collagen to hydroxylysine. Chick embryo lysyl hydroxylase activity was measured as specific tritium release as tritium water from a [4,5-3H]lysine-labeled unhydroxylated collagen substrate prepared from chick calvaria. Kinetic studies revealed a linear, noncompetitive type of inhibition with respect to collagen substrate with a Ki of 120-180 μM. The inhibition by homogentisic acid was reversible in that enzyme activity could be restored after dialysis of preincubated mixtures of homogentisic acid with enzyme or substrate. The inhibition by homogentisic acid was competitive with respect to ascorbic acid, and the addition of reducing agents, such as ascorbic acid or 1,4-dithiothreitol, protected lysyl hydroxylase activity from homogentisic acid inhibition. In organ cultures of embryonic chick calvaria, biosynthesis of hydroxylysine-derived intermolecular collagen cross-links was inhibited in a dose-dependent manner by 0.5-5 mM homogentisic acid. Because homogentisic acid inhibits the formation of hydroxylysine in a cell-free assay and in organ cultures, this compound must pass into the cells of calvaria to inhibit intracellular hydroxylysine formation and subsequently to diminish the reducible intermolecular cross-links of the newly synthesized collagen. We propose that the inhibition of lysyl hydroxylase and the resulting hydroxylsine-deficient, structurally modified collagen may be clinically significant in the defective connective tissue found in alkaptonuric patients. PMID:405402

  15. (+)-Abscisic Acid 8′-Hydroxylase Is a Cytochrome P450 Monooxygenase1

    PubMed Central

    Krochko, Joan E.; Abrams, Garth D.; Loewen, Mary K.; Abrams, Suzanne R.; Cutler, Adrian J.

    1998-01-01

    Abscisic acid (ABA) 8′-hydroxylase catalyzes the first step in the oxidative degradation of (+)-ABA. The development of a robust in vitro assay has now permitted detailed examination and characterization of this enzyme. Although several factors (buffer, cofactor, and source tissue) were critical in developing the assay, the most important of these was the identification of a tissue displaying high amounts of in vivo enzyme activity (A.J. Cutler, T.M. Squires, M.K. Loewen, J.J. Balsevich [1997] J Exp Bot 48: 1787–1795). (+)-ABA 8′-hydroxylase is an integral membrane protein that is localized to the microsomal fraction in suspension-cultured maize (Zea mays) cells. (+)-ABA metabolism requires both NADPH and molecular oxygen. NADH was not an effective cofactor, although there was substantial stimulation of activity (synergism) when it was included at rate-limiting NADPH concentrations. The metabolism of (+)-ABA was progressively inhibited at O2 concentrations less than 10% (v/v) and was very low (less than 5% of control) under N2. (+)-ABA 8′-hydroxylase activity was inhibited by tetcyclacis (50% inhibition at 10−6 m), cytochrome c (oxidized form), and CO. The CO inhibition was reversible by light from several regions of the visible spectrum, but most efficiently by blue and amber light. These data strongly support the contention that (+)-ABA 8′-hydroxylase is a cytochrome P450 monooxygenase. PMID:9808729

  16. Cholesterol 7{alpha}-hydroxylase is phosphorylated at multiple amino acids

    SciTech Connect

    Stroup, D. . E-mail: dstroup1@kent.edu; Ramsaran, J.R.

    2005-04-15

    The activity of cholesterol 7{alpha}-hydroxylase (gpCYP7A1), the rate limiting enzyme in bile acid synthesis, has been postulated to be regulated by phosphorylation/dephosphorylation. This study has found that several kinase activators rapidly reduce the amount of bile acid produced by the human hepatoma cell line, HepG2, and that gpCYP7A1 from HepG2 cell extracts eluted in the phosphoprotein fraction of FeIII columns. After incubating the HepG2 cells with radioactive orthophosphate, the band identified as gpCYP7Al on immunoblots was strongly labeled. Recombinant gpCYP7A was expressed as 6x HIS fusion polypeptides and subjected to kinase assays. The locations of phosphorylation were mapped further by screening synthetic peptides against AMP-activated protein kinase (AMPK), c-Jun N-terminal kinase, protein kinase A, and a panel of nine protein kinase C isoforms. AMPK, also known as 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase, phosphorylated cholesterol 7{alpha}-hydroxylase, suggesting a potential mechanism of coordination of cholesterol synthesis and degradation.

  17. WRINKLED1 Rescues Feedback Inhibition of Fatty Acid Synthesis in Hydroxylase-Expressing Seeds.

    PubMed

    Adhikari, Neil D; Bates, Philip D; Browse, John

    2016-05-01

    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

  18. 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

  19. 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...

  20. 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.

  1. Determination of microsomal lauric acid hydroxylase activity by HPLC with flow-through radiochemical quantitation

    SciTech Connect

    Romano, M.C.; Straub, K.M.; Yodis, L.A.P.; Eckardt, R.D.; Newton, J.F.

    1988-04-01

    An assay for the microsomal hydroxylation of lauric acid (LA), based on HPLC with flow-through radiochemical detection, has been developed. Conditions were optimized for resolution and quantitation of three microsomal metabolites of /sup 14/C-LA, one of which has not been reported previously as a metabolite of LA in mammalian microsomal incubations. These products, 12-(omega)-hydroxy-LA, 11-(omega-1)-hydroxy-LA, and a novel metabolite, 10-(omega-2)-hydroxy-LA, were isolated by HPLC and identified by gas chromatography/mass spectrometry. In the presence of NADPH, the formation of all three metabolites was linear with time and microsomal protein concentration. Hydrogen peroxide also supported the microsomal metabolism of LA, although the ratio of metabolites was substantially different than that produced by NADPH-supported microsomes. Several biochemical probes (metyrapone, ..cap alpha..-naphthoflavone, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, and 10-undecynoic acid) were used to dissociate the three LA hydroxylase activities. These experiments suggest that the site-specific hydroxylation (omega-, (omega-1), (omega-2)-) of LA may be catalyzed by different isozymes of cytochrome P-450.

  2. 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. PMID:22459802

  3. Cholesterol and bile acids regulate cholesterol 7 alpha-hydroxylase expression at the transcriptional level in culture and in transgenic mice.

    PubMed

    Ramirez, M I; Karaoglu, D; Haro, D; Barillas, C; Bashirzadeh, R; Gil, G

    1994-04-01

    Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is the rate-limiting enzyme in bile acid biosynthesis. It is subject to a feedback control, whereby high levels of bile acids suppress its activity, and cholesterol exerts a positive control. It has been suggested that posttranscriptional control plays a major part in that regulation. We have studied the mechanisms by which cholesterol and bile acids regulate expression of the 7 alpha-hydroxylase gene and found it to be solely at the transcriptional level by using two different approaches. First, using a tissue culture system, we localized a liver-specific enhancer located 7 kb upstream of the transcriptional initiation site. We also showed that low-density lipoprotein mediates transcriptional activation of chimeric genes, containing either the 7 alpha-hydroxylase or the albumin enhancer in front of the 7 alpha-hydroxylase proximal promoter, to the same extent as the in vivo cholesterol-mediated regulation of 7 alpha-hydroxylase mRNA. In a second approach, using transgenic mice, we have found that expression of an albumin enhancer-7 alpha-hydroxylase-lacZ fusion gene is restricted to the liver and is regulated by cholesterol and bile acids in a manner quantitatively similar to that of the endogenous gene. We also found, that a liver-specific enhancer is necessary for expression of the rat 7 alpha-hydroxylase gene, in agreement with the tissue culture experiments. Together, these results demonstrate that cholesterol and bile acids regulate the expression of the 7 alpha-hydroxylase gene solely at the transcriptional level. PMID:8139578

  4. Reassessment of the Role of Aromatic Amino Acid Hydroxylases and the Effect of Infection by Toxoplasma gondii on Host Dopamine

    PubMed Central

    Wang, Zi T.; Harmon, Steve; O'Malley, Karen L.

    2014-01-01

    Toxoplasma gondii infection has been described previously to cause infected mice to lose their fear of cat urine. This behavioral manipulation has been proposed to involve alterations of host dopamine pathways due to parasite-encoded aromatic amino acid hydroxylases. Here, we report successful knockout and complementation of the aromatic amino acid hydroxylase AAH2 gene, with no observable phenotype in parasite growth or differentiation in vitro and in vivo. Additionally, expression levels of the two aromatic amino acid hydroxylases were negligible both in tachyzoites and in bradyzoites. Finally, we were unable to confirm previously described effects of parasite infection on host dopamine either in vitro or in vivo, even when AAH2 was overexpressed using the BAG1 promoter. Together, these data indicate that AAH enzymes in the parasite do not cause global or regional alterations of dopamine in the host brain, although they may affect this pathway locally. Additionally, our findings suggest alternative roles for the AHH enzymes in T. gondii, since AAH1 is essential for growth in nondopaminergic cells. PMID:25547791

  5. Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE.

    PubMed

    Van de Wouwer, Dorien; Vanholme, Ruben; Decou, Raphaël; Goeminne, Geert; Audenaert, Dominique; Nguyen, Long; Höfer, René; Pesquet, Edouard; Vanholme, Bartel; Boerjan, Wout

    2016-09-01

    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

  6. Chlorophenol hydroxylases encoded by plasmid pJP4 differentially contribute to chlorophenoxyacetic acid degradation.

    PubMed

    Ledger, T; Pieper, D H; González, B

    2006-04-01

    Phenoxyalkanoic compounds are used worldwide as herbicides. Cupriavidus necator JMP134(pJP4) catabolizes 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), using tfd functions carried on plasmid pJP4. TfdA cleaves the ether bonds of these herbicides to produce 2,4-dichlorophenol (2,4-DCP) and 4-chloro-2-methylphenol (MCP), respectively. These intermediates can be degraded by two chlorophenol hydroxylases encoded by the tfdB(I) and tfdB(II) genes to produce the respective chlorocatechols. We studied the specific contribution of each of the TfdB enzymes to the 2,4-D/MCPA degradation pathway. To accomplish this, the tfdB(I) and tfdB(II) genes were independently inactivated, and growth on each chlorophenoxyacetate and total chlorophenol hydroxylase activity were measured for the mutant strains. The phenotype of these mutants shows that both TfdB enzymes are used for growth on 2,4-D or MCPA but that TfdB(I) contributes to a significantly higher extent than TfdB(II). Both enzymes showed similar specificity profiles, with 2,4-DCP, MCP, and 4-chlorophenol being the best substrates. An accumulation of chlorophenol was found to inhibit chlorophenoxyacetate degradation, and inactivation of the tfdB genes enhanced the toxic effect of 2,4-DCP on C. necator cells. Furthermore, increased chlorophenol production by overexpression of TfdA also had a negative effect on 2,4-D degradation by C. necator JMP134 and by a different host, Burkholderia xenovorans LB400, harboring plasmid pJP4. The results of this work indicate that codification and expression of the two tfdB genes in pJP4 are important to avoid toxic accumulations of chlorophenols during phenoxyacetic acid degradation and that a balance between chlorophenol-producing and chlorophenol-consuming reactions is necessary for growth on these compounds. PMID:16597983

  7. Induction of Benzoic Acid 2-Hydroxylase in Virus-Inoculated Tobacco.

    PubMed Central

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

    1993-01-01

    Salicylic acid (SA) plays an important role in the induction of plant resistance to pathogens. An accompanying article (N. Yalpani, J. Leon, M.A. Lawton, I. Raskin [1993] Plant Physiol 103: 315-321) 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 (Nicotiana tabacum L. cv Xanthi-nc) 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-1 g-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[deg]C. TMV induction of BA2H activity and SA accumulation were inhibited when inoculated tobacco plants were incubated at 32[deg]C. However, when inoculated plants were incubated for 4 d at 32[deg]C and then transferred to 24[deg]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[deg]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. PMID:12231939

  8. A Conserved Acidic Residue in Phenylalanine Hydroxylase Contributes to Cofactor Affinity and Catalysis

    PubMed Central

    2015-01-01

    The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may not be essential for holding the bridging water molecules together, because many of these waters are retained even in the Ala mutant. However, interactions via the bridging waters contribute to cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of

  9. Stereospecificity of fatty acid 2-hydroxylase and differential functions of 2-hydroxy fatty acid enantiomers[S

    PubMed Central

    Guo, Lin; Zhang, Xu; Zhou, Dequan; Okunade, Adewole L.; Su, Xiong

    2012-01-01

    FA 2-hydroxylase (FA2H) is an NAD(P)H-dependent enzyme that initiates FA α oxidation and is also responsible for the biosynthesis of 2-hydroxy FA (2-OH FA)-containing sphingolipids in mammalian cells. The 2-OH FA is chiral due to the asymmetric carbon bearing the hydroxyl group. Our current study performed stereochemistry investigation and showed that FA2H is stereospecific for the production of (R)-enantiomers. FA2H knockdown in adipocytes increases diffusional mobility of raft-associated lipids, leading to reduced GLUT4 protein level, glucose uptake, and lipogenesis. The effects caused by FA2H knockdown were reversed by treatment with exogenous (R)-2-hydroxy palmitic acid, but not with the (S)-enantiomer. Further analysis of sphingolipids demonstrated that the (R)-enantiomer is enriched in hexosylceramide whereas the (S)-enantiomer is preferentially incorporated into ceramide, suggesting that the observed differential effects are in part due to synthesis of sphingolipids containing different 2-OH FA enantiomers. These results may help clarify the mechanisms underlying the recently identified diseases associated with FA2H mutations in humans and may lead to potential pharmaceutical and dietary treatments. This study also provides critical information to help study functions of 2-OH FA enantiomers in FA α oxidation and possibly other sphingolipid-independent pathways. PMID:22517924

  10. 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.

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

    PubMed Central

    2016-01-01

    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. PMID:26252467

  12. Lipophilic modification enhances anti-colitic properties of rosmarinic acid by potentiating its HIF-prolyl hydroxylases inhibitory activity.

    PubMed

    Jeong, Seongkeun; Park, Huijeong; Hong, Sungchae; Yum, Soohwan; Kim, Wooseong; Jung, Yunjin

    2015-01-15

    Inhibition of hypoxia inducible factor-prolyl hydroxylase-2 (HPH), leading to activation of hypoxia inducible factor (HIF)-1 is a potential therapeutic strategy for the treatment of colitis. Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid is a naturally occurring polyphenolic compound with two catechols, a or inhibition of HPH. To improve accessibility of highly hydrophilic RA to HPH, an intracellular target, RA was chemically modified to decrease hydrophilicity. Of the less-hydrophilic derivatives, rosmarinic acid methyl ester (RAME) most potently inhibited HPH. Accordingly, RAME prevented hydroxylation of HIF-1α and consequently stabilized HIF-1α protein in cells. RAME inhibition of HPH and induction of HIF-1α were diminished by elevated doses of the required factors of HPH, 2-ketoglutarate and ascorbate. RAME induction of HIF-1α led to activation of an ulcer healing pathway, HIF-1-vascular endothelial growth factor (VEGF), in human colon carcinoma cells. RAME administered rectally ameliorated TNBS-induced rat colitis and substantially decreased the levels of pro-inflammatory mediators in the inflamed colonic tissue. In parallel with the cellular effects of RAME, RAME up-regulated HIF-1α and VEGF in the inflamed colonic tissue. Thus, lipophilic modification of RA improves its ability to inhibit HPH, leading to activation of the HIF-1-VEGF pathway. RAME, a lipophilic RA derivative, may exert anti-colitic effects via activation of the ulcer healing pathway. PMID:25483211

  13. Molecular Docking Study of Catecholamines and [4-(Propan-2-yl) Phenyl]Carbamic acid with Tyrosine Hydroxylase

    PubMed Central

    Parveen, Zahida; Nawaz, Muhammad Sulaman; Shakil, Shazi; Greig, Nigel H.; Kamal, Mohammad A.

    2016-01-01

    Parkinson’s disease is a major age-related neurodegenerative disorder. As the classical disease-related motor symptoms are associated with the loss of dopamine-generating cells within the substantia nigra, tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines has become an important target in the development of Parkinson’s disease drug candidates, with the focus to augment TH levels or its activity. By contrast, TH inhibitors are of relevance in the treatment of conditions associated with catecholamine over-production, as occurs in pheochromocytomas. To aid characterizing new drug candidates, a molecular docking study of catecholamines and a novel hypothetical compound [4-(propan-2-yl) phenyl]carbamic acid (PPCA) with TH is described. Docking was performed using Autodock4.2 and results were analyzed using Chimera1.5.2. All the studied ligands were found to bind within a deep narrow groove lined with polar aromatic and acidic residues within TH. Our results corroborated a ‘hexa interacting amino acids unit’ located in this deep narrow groove crucial to the interaction of PPCA and the studied catecholamines with TH, whereby the ‘His361-His336 dyad’ was found to be even more crucial to these binding interactions. PPCA displayed a binding interaction with human TH that was comparable to the original TH substrate, L-tyrosine. Hence PPCA may warrant in vitro and in vivo characterization with TH to assess its potential as a candidate therapeutic. PMID:22583429

  14. Molecular cloning of the. alpha. -subunit of human prolyl 4-hydroxylase: The complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts

    SciTech Connect

    Helaakoski, T.; Vuori, K.; Myllylae, R.; Kivirikko, K.I.; Pihlajaniemi, T. )

    1989-06-01

    Prolyl 4-hydroxylase an {alpha}{sub 2}{beta}{sub 2} tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. The authors report here on the isolation of cDNA clones encoding the {alpha}-subunit of the enzyme from human tumor HT-1080, placenta, and fibroblast cDNA libraries. Eight overlapping clones covering almost all of the corresponding 3,000-nucleotide mRNA, including all the coding sequences, were characterized. These clones encode a polypeptide of 517 amino acid residues and a signal peptide of 17 amino acids. Previous characterization of cDNA clones for the {beta}-subunit of prolyl 4-hydroxylase has indicated that its C terminus has the amino acid sequence Lys-Asp-Gly-Leu, which, it has been suggested, is necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The {alpha}-subunit does not have this C-terminal sequence, and thus one function of the {beta}-subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle. Southern blot analyses of human genomic DNA with a cDNA probe for the {alpha}-subunit suggested the presence of only one gene encoding the two types of mRNA, which appear to result from mutually exclusive alternative splicing of primary transcripts of one gene.

  15. Inductions of the fatty acid 2-hydroxylase (FA2H) gene by Δ9-tetrahydrocannabinol in human breast cancer cells

    PubMed Central

    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

    2014-01-01

    To investigate gene(s) being regulated by Δ9-tetrahydrocannabinol (Δ9-THC), we performed DNA microarray analysis of human breast cancer MDA-MB-231 cells, which are poorly differentiated breast cancer cells, treated with Δ9-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 Δ9-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 Δ9-THC-regulated gene, and that Δ9-THC induces differentiation signal(s) in poorly differentiated MDA-MB-231 cells. PMID:23535410

  16. Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs

    PubMed Central

    MIYAGAWA, Shuji; MATSUNARI, Hitomi; WATANABE, Masahito; NAKANO, Kazuaki; UMEYAMA, Kazuhiro; SAKAI, Rieko; TAKAYANAGI, Shuko; TAKEISHI, Toki; FUKUDA, Tooru; YASHIMA, Sayaka; MAEDA, Akira; EGUCHI, Hiroshi; OKUYAMA, Hiroomi; NAGAYA, Masaki; NAGASHIMA, Hiroshi

    2015-01-01

    Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs. PMID:26227017

  17. Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.

    PubMed

    Miyagawa, Shuji; Matsunari, Hitomi; Watanabe, Masahito; Nakano, Kazuaki; Umeyama, Kazuhiro; Sakai, Rieko; Takayanagi, Shuko; Takeishi, Toki; Fukuda, Tooru; Yashima, Sayaka; Maeda, Akira; Eguchi, Hiroshi; Okuyama, Hiroomi; Nagaya, Masaki; Nagashima, Hiroshi

    2015-01-01

    Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs. PMID:26227017

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Substrate Specificity and Ligand Interactions of CYP26A1, the Human Liver Retinoic Acid Hydroxylase

    PubMed Central

    Thatcher, Jayne E.; Buttrick, Brian; Shaffer, Scott A.; Shimshoni, Jakob A.; Goodlett, David R.; Nelson, Wendel L.

    2011-01-01

    All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. atRA is also used as a drug, and synthetic atRA analogs and inhibitors of retinoic acid (RA) metabolism have been developed. The hepatic clearance of atRA is mediated primarily by CYP26A1, but design of CYP26A1 inhibitors is hindered by lack of information on CYP26A1 structure and structure-activity relationships of its ligands. The aim of this study was to identify the primary metabolites of atRA formed by CYP26A1 and to characterize the ligand selectivity and ligand interactions of CYP26A1. On the basis of high-resolution tandem mass spectrometry data, four metabolites formed from atRA by CYP26A1 were identified as 4-OH-RA, 4-oxo-RA, 16-OH-RA and 18-OH-RA. 9-cis-RA and 13-cis-RA were also substrates of CYP26A1. Forty-two compounds with diverse structural properties were tested for CYP26A1 inhibition using 9-cis-RA as a probe, and IC50 values for 10 inhibitors were determined. The imidazole- and triazole-containing inhibitors [S-(R*,R*)]-N-[4-[2-(dimethylamino)-1-(1H-imidazole-1-yl)propyl]-phenyl]2-benzothiazolamine (R116010) and (R)-N-[4-[2-ethyl-1-(1H-1,2,4-triazol-1-yl)butyl]phenyl]-2-benzothiazolamine (R115866) were the most potent inhibitors of CYP26A1 with IC50 values of 4.3 and 5.1 nM, respectively. Liarozole and ketoconazole were significantly less potent with IC50 values of 2100 and 550 nM, respectively. The retinoic acid receptor (RAR) γ agonist CD1530 was as potent an inhibitor of CYP26A1 as ketoconazole with an IC50 of 530 nM, whereas the RARα and RARβ agonists tested did not significantly inhibit CYP26A1. The pan-RAR agonist 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid and the peroxisome proliferator-activated receptor ligands rosiglitazone and pioglitazone inhibited CYP26A1 with IC50 values of 3.7, 4.2, and 8.6 μM, respectively. These data demonstrate that CYP26A1 has high ligand selectivity but accepts structurally related nuclear

  4. Sialylation Facilitates the Maturation of Mammalian Sperm and Affects Its Survival in Female Uterus.

    PubMed

    Ma, Xue; Pan, Qian; Feng, Ying; Choudhury, Biswa P; Ma, Qianhong; Gagneux, Pascal; Ma, Fang

    2016-06-01

    Establishment of adequate levels of sialylation is crucial for sperm survival and function after insemination; however, the mechanism for the addition of the sperm sialome has not been identified. Here, we report evidence for several different mechanisms that contribute to the establishment of the mature sperm sialome. Directly quantifying the source of the nucleotide sugar CMP-beta-N-acetylneuraminic acid in epididymal fluid indicates that transsialylation occurs in the upper epididymis. Western blots for the low-molecular-mass sialoglycoprotein (around 20-50 kDa) in C57BL/6 mice epididymal fluid reflect that additional sialome could be obtained by glycosylphosphatidylinositol-anchored sialoglycopeptide incorporation during epididymal transit in the caput of the epididymis. Additionally, we found that in Cmah (CMP-N-acetylneuraminic acid hydroxylase)-/- transgenic mice, epididymal sperm obtained sialylated-CD52 from seminal vesicle fluid (SVF). Finally, we used Gfp (green fluorescent protein)+/+ mouse sperm to test the role of sialylation on sperm for protection from female leukocyte attack. There is very low phagocytosis of the epididymal sperm when compared to that of sperm coincubated with SVF. Treating sperm with Arthrobacter ureafaciens sialidase (AUS) increased phagocytosis even further. Our results highlight the different mechanisms of increasing sialylation, which lead to the formation of the mature sperm sialome, as well as reveal the sialome's function in sperm survival within the female genital tract. PMID:27075617

  5. A cytotoxic humanized anti-ganglioside antibody produced in a murine cell line defective of N-glycolylated-glycoconjugates.

    PubMed

    Fernández-Marrero, Yuniel; Roque-Navarro, Lourdes; Hernández, Tays; Dorvignit, Denise; Molina-Pérez, Marively; González, Addys; Sosa, Katya; López-Requena, Alejandro; Pérez, Rolando; de Acosta, Cristina Mateo

    2011-12-01

    Gangliosides containing the N-glycolyl (NGc) form of sialic acid are tumor-associated antigens and promising candidates for cancer therapy. We previously generated the murine 14F7 monoclonal antibody (mAb), specific for the N-glycolyl-GM3 ganglioside (NGcGM3), which induced an oncosis-like type of cell death on malignant cell lines expressing this antigen and recognized breast carcinoma by immunoscintigraphy in cancer patients. As humanization is expected to enhance its use for human cancer therapy, herein we describe the design and generation of two humanized versions of the 14F7 mAb by disrupting potential human T cell epitopes on its variable region. No differences in antigen reactivity or cytotoxic properties were detected among the variants tested and with respect to the chimeric counterpart. Humanized 14F7 genes were transfected into the NGcGM3-expressing NS0 cell line. Therefore, in the industrial scaling-up of the transfectoma in serum-free medium, cell viability was lost due to the cytotoxic effect of the secreted antibody. This shortcoming was solved by knocking down the CMP-N-acetylneuraminic acid hydroxylase enzyme, thus impairing the synthesis of NGc-glycoconjugates. Humanized 14F7 mAb is of potential value for the therapy of NGcGM3-expressing tumors. PMID:21802167

  6. Incorporation of N-acetylneuraminic acid into Haemophilus somnus lipooligosaccharide (LOS): enhancement of resistance to serum and reduction of LOS antibody binding.

    PubMed

    Inzana, Thomas J; Glindemann, Gretchen; Cox, Andrew D; Wakarchuk, Warren; Howard, Michael D

    2002-09-01

    Haemophilus somnus isolates from cases of thrombotic meningoencephalitis, pneumonia, and other disease sites are capable of undergoing a high rate of phase variation in the oligosaccharide component of their lipooligosaccharides (LOS). In contrast, the LOS of commensal strains isolated from the normal reproductive tract phase vary little or not at all. In addition, the LOS of H. somnus shares conserved epitopes with LOS from Neisseria gonorrhoeae, Haemophilus influenzae, and other species that can incorporate sialic acid into their LOS. We now report that growth of disease isolates of H. somnus with CMP-N-acetylneuraminic acid (CMP-NeuAc) or NeuAc added to the medium resulted in incorporation of NeuAc into the LOS. However, NeuAc was not incorporated into the LOS of commensal isolates and one disease isolate following growth in medium containing CMP-NeuAc or NeuAc. Sialylated LOS was detected by an increase in the molecular size or an increase in the amount of the largest-molecular-size LOS electrophoretic bands, which disappeared following treatment with neuraminidase. Sialylated LOS could also be detected by reactivity with Limax flavus agglutinin lectin, which is specific for sialylated species, by dot blot assay; this reactivity was also reversed by neuraminidase treatment. H. somnus strain 2336 LOS was found to contain some sialic acid when grown in medium lacking CMP-NeuAc or NeuAc, although supplementation enhanced NeuAc incorporation. In contrast strain 738, an LOS phase variant of strain 2336, was less extensively sialylated when the growth medium was supplemented with CMP-NeuAc or NeuAc, as determined by electrophoretic profiles and electrospray mass spectrometry. The sialyltransferase of H. somnus strain 738 was confirmed to preferentially sialylate the Gal(beta)-(1-3)-GlcNAc component of the lacto-N-tetraose structure by capillary electrophoresis assay. Enhanced sialylation of the strain 2336 LOS inhibited the binding of monoclonal antibodies to LOS by

  7. 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.

  8. Arabidopsis Sphingolipid Fatty Acid 2-Hydroxylases (AtFAH1 and AtFAH2) Are Functionally Differentiated in Fatty Acid 2-Hydroxylation and Stress Responses1[OA

    PubMed Central

    Nagano, Minoru; Takahara, Kentaro; Fujimoto, Masaru; Tsutsumi, Nobuhiro; Uchimiya, Hirofumi; Kawai-Yamada, Maki

    2012-01-01

    2-Hydroxy fatty acids (2-HFAs) are predominantly present in sphingolipids and have important physicochemical and physiological functions in eukaryotic cells. Recent studies from our group demonstrated that sphingolipid fatty acid 2-hydroxylase (FAH) is required for the function of Arabidopsis (Arabidopsis thaliana) Bax inhibitor-1 (AtBI-1), which is an endoplasmic reticulum membrane-localized cell death suppressor. However, little is known about the function of two Arabidopsis FAH homologs (AtFAH1 and AtFAH2), and it remains unclear whether 2-HFAs participate in cell death regulation. In this study, we found that both AtFAH1 and AtFAH2 had FAH activity, and the interaction with Arabidopsis cytochrome b5 was needed for the sufficient activity. 2-HFA analysis of AtFAH1 knockdown lines and atfah2 mutant showed that AtFAH1 mainly 2-hydroxylated very-long-chain fatty acid (VLCFA), whereas AtFAH2 selectively 2-hydroxylated palmitic acid in Arabidopsis. In addition, 2-HFAs were related to resistance to oxidative stress, and AtFAH1 or 2-hydroxy VLCFA showed particularly strong responses to oxidative stress. Furthermore, AtFAH1 interacted with AtBI-1 via cytochrome b5 more preferentially than AtFAH2. Our results suggest that AtFAH1 and AtFAH2 are functionally different FAHs, and that AtFAH1 or 2-hydroxy VLCFA is a key factor in AtBI-1-mediated cell death suppression. PMID:22635113

  9. One-Pot Production of l-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

    PubMed Central

    Nakano, Masashi; Kino, Kuniki

    2015-01-01

    We developed a novel process for efficient synthesis of l-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of l-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize l-THA. By using these recombinant enzymes, l-THA was obtained by l-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. l-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased l-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the l-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, l-THA was successfully obtained from l-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of l-THA. PMID:25795668

  10. Tyrosine hydroxylase and tryptophan hydroxylase do not form heterotetramers.

    PubMed

    Mockus, S M; Yohrling, G J; Vrana, K E

    1998-02-01

    Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) both contain a C-terminal tetramerization domain composed of a leucine heptad repeat embedded within a 4,3-hydrophobic repeat. Previous mutagenesis experiments and X-ray crystallographic studies have demonstrated that these repeats are required for tetramer assembly of the hydroxylase enzymes via coiled-coil interactions. The specificity of these particular C-terminal intersubunit binding motifs was investigated by determining if TH and TPH can form heterotetramers when coexpressed in bacteria. Bacterial cells were contransformed with TH and TPH expression plasmids under kanamycin and ampicillin selection, respectively. Immunoprecipitation of induced bacterial supernatants with a TPH monoclonal antibody demonstrated that, unlike the human TH isoforms, TH and TPH do not form heterotetramers. The data suggest that specificity of oligomerization of the aromatic amino acid hydroxylases may be partially determined by polar amino acids interspersed within the coiled-coil. This finding should be influential in the development of eukaryotic expression systems and ultimately in gene therapy approaches. PMID:9589369

  11. Refined regio- and stereoselective hydroxylation of L-pipecolic acid by protein engineering of L-proline cis-4-hydroxylase based on the X-ray crystal structure.

    PubMed

    Koketsu, Kento; Shomura, Yasuhito; Moriwaki, Kei; Hayashi, Mikiro; Mitsuhashi, Satoshi; Hara, Ryotaro; Kino, Kuniki; Higuchi, Yoshiki

    2015-04-17

    Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe(2+)/α-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of L-proline into hydroxy-L-prolines (Hyps). L-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium meliloti and Mesorhizobium loti catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of L-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of L-Pro and L-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip. PMID:25171735

  12. Intersubunit binding domains within tyrosine hydroxylase and tryptophan hydroxylase.

    PubMed

    Yohrling, G J; Jiang, G C; Mockus, S M; Vrana, K E

    2000-08-01

    Tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT) belongs to the aromatic amino acid hydroxylase superfamily, which includes phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH). The crystal structures for both PAH and TH have been reported, but a crystallographic model of TPH remains elusive. For this reason, we have utilized the information presented in the TH crystal structure in combination with primary sequence alignments to design point mutations in potential structural domains of the TPH protein. Mutation of a TH salt bridge (K170E) was sufficient to alter enzyme macromolecular assembly. We found that the disruption of the cognate intersubunit dimerization salt bridge (K111-E223) in TPH, however, did not affect the macromolecular assembly of TPH. Enzyme peaks representing only tetramers were observed with size exclusion chromatography. By contrast, a single-point mutation within the tetramerization domain of TPH (L435A) was sufficient to disrupt the normal homotetrameric assembly of TPH. These studies indicate that, although the proposed salt bridge dimerization interface of TH is conserved in TPH, this hypothetical TPH intersubunit binding domain, K111-E223, is not required for the proper macromolecular assembly of the protein. However, leucine 435 within the tetramerization domain is necessary for the proper macromolecular assembly of TPH. PMID:10900078

  13. Identification of amino acids important for the catalytic activity of the collagen glucosyltransferase associated with the multifunctional lysyl hydroxylase 3 (LH3).

    PubMed

    Wang, Chunguang; Risteli, Maija; Heikkinen, Jari; Hussa, Anna-Kaisa; Uitto, Lahja; Myllyla, Raili

    2002-05-24

    Collagen glucosyltransferase (GGT) activity has recently been shown to be associated with human lysyl hydroxylase (LH) isoform 3 (LH3) (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., Myllylä, R. (2000) J. Biol. Chem. 275, 36158-36163). The LH and GGT activities of the multifunctional LH3 protein modify lysyl residues in collagens posttranslationally to form hydroxylysyl and glucosylgalactosyl hydroxylysyl residues respectively. We now report that in the nematode, Caenorhabditis elegans, where only one ortholog is found for lysyl hydroxylase, the LH and GGT activities are also associated with the same gene product. The aim of the present studies is the identification of amino acids important for the catalytic activity of GGT. Our data indicate that the GGT active site is separate from the carboxyl-terminal LH active site of human LH3, the amino acids important for the GGT activity being located at the amino-terminal part of the molecule. Site-directed mutagenesis of a conserved cysteine at position 144 to isoleucine and a leucine at position 208 to isoleucine caused a marked reduction in GGT activity. These amino acids were conserved in C. elegans LH and mammalian LH3, but not in LH1 or LH2, which lack GGT activity. The data also reveal a DXD-like motif in LH3 characteristic of many glycosyltransferases and the mutagenesis of aspartates of this motif eliminated the GGT activity. Reduction in GGT activity was not accompanied by a change in the LH activity of the molecule. Thus GGT activity can be manipulated independently of LH activity in LH3. These data provide the information needed to design knock-out studies for investigation of the function of glucosylgalactosyl hydroxylysyl residues of collagens in vivo. PMID:11896059

  14. Virus-induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid-7-hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis.

    PubMed

    Salim, Vonny; Yu, Fang; Altarejos, Joaquín; De Luca, Vincenzo

    2013-12-01

    Iridoids are a major group of biologically active molecules that are present in thousands of plant species, and one versatile iridoid, secologanin, is a precursor for the assembly of thousands of monoterpenoid indole alkaloids (MIAs) as well as a number of quinoline alkaloids. This study uses bioinformatics to screen large databases of annotated transcripts from various MIA-producing plant species to select candidate genes that may be involved in iridoid biosynthesis. Virus-induced gene silencing of the selected genes combined with metabolite analyses of silenced plants was then used to identify the 7-deoxyloganic acid 7-hydroxylase (CrDL7H) that is involved in the 3rd to last step in secologanin biosynthesis. Silencing of CrDL7H reduced secologanin levels by at least 70%, and increased the levels of 7-deoxyloganic acid to over 4 mg g(-1) fresh leaf weight compared to control plants in which this iridoid is not detected. Functional expression of this CrDL7H in yeast confirmed its biochemical activity, and substrate specificity studies showed its preference for 7-deoxyloganic acid over other closely related substrates. Together, these results suggest that hydroxylation precedes carboxy-O-methylation in the secologanin pathway in Catharanthus roseus. PMID:24103035

  15. Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family.

    PubMed

    Shanklin, John; Whittle, Edward

    2003-06-19

    Pseudomonas oleovorans alkane omega-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases. No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains. To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB. These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation. These data provide the first experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site. PMID:12804773

  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. PMID:23832359

  17. 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

  18. Overexpression of Cholesterol 7α-hydroxylase promotes hepatic bile acid synthesis and secretion and maintains cholesterol homeostasis

    PubMed Central

    Li, Tiangang; Matozel, Michelle; Boehme, Shannon; Kong, Bo; Nilsson, Lisa-Mari; Guo, Grace; Ellis, Ewa; Chiang, John Y. L.

    2011-01-01

    Summary We reported previously that mice overexpressing Cyp7a1 (Cyp7a1-tg) are protected against high fat diet-induced hypercholesterolemia, obesity and insulin resistance (1). Here we investigated the underlying mechanism of bile acid signaling in maintaining cholesterol homeostasis in Cyp7a1-tg mice. Cyp7a1-tg mice had 2-fold higher Cyp7a1 activity and bile acid pool than wild type mice. Gallbladder bile acid composition changed from predominantly cholic acid (57%) in wild type to chenodeoxycholic acid (54%) in Cyp7a1-tg mice. Cyp7a1-tg mice had higher biliary and fecal cholesterol and bile acid secretion rates than wild type mice. Surprisingly, hepatic de novo cholesterol synthesis was markedly induced in Cyp7a1-tg mice but intestine fractional cholesterol absorption in Cyp7a1-tg mice remained the same as wild type mice despite increased intestine bile acids. Interestingly, hepatic but not intestinal expression of several cholesterol (ABCG5/G8, SR-B1) and bile acid (ABCB11) transporters were significantly induced in Cyp7a1-tg mice. Treatment of mouse or human hepatocytes with a farnesoid X receptor (FXR) agonist GW4064 or bile acids induced hepatic Abcg5/g8 expression. A functional FXR binding site was identified in the Abcg5 gene promoter. Study of tissue-specific Fxr knockout mice demonstrated that loss of the Fxr gene in the liver attenuated bile acid induction of hepatic Abcg5/g8 and gallbladder cholesterol content, suggesting a role of FXR in the regulation of cholesterol transport. In summary, this study revealed a new mechanism by which increased Cyp7a1 activity expands the hydrophobic bile acid pool, stimulating hepatic cholesterol synthesis and biliary cholesterol secretion without increasing intestinal cholesterol absorption. This study demonstrated that Cyp7a1 plays a critical role in maintaining cholesterol homeostasis and underscores the importance of bile acid signaling in regulating overall cholesterol homeostasis. PMID:21319191

  19. 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

  20. Computational study of human tyrosine hydroxylase mutants to uphold [4-(Propan-2-yl) Phenyl]Carbamic acid as a potential inhibitor.

    PubMed

    Nawaz, Muhammad S; Parveen, Zahida; Wang, Liyong; Rashid, Sajid; Fatmi, Muhammad Q; Kamal, Mohammad A

    2014-01-01

    Neurodegenerative diseases that afflict nervous system are characterized by progressive nervous system dysfunction and associated with the one-set of many diseases like Segawa's syndrome (recessive form), autosomal recessive L-dopa-responsive dystonia, L-dopa non-responsive dystonia or progressive early-onset encephalopathy and recessive L-dopa-responsive parkinsonism. It has been reported that a number of mutations in coding regions, splice sites and promoter regions of tyrosine hydroxylase (TH) are associated with many such diseases. TH is responsible for catalyzing the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine. This reaction is considered as rate-limiting step in the biosynthesis of catecholamines, dopamine, norepinephrine and epinephrine, which has made TH an important target for drug development. In our previous study using comparative molecular docking approach, it was concluded that [4-(Propan-2-yl) Phenyl]Carbamic acid (PPCA) may serve as a potential inhibitor. By further extending, our focus is to determine the binding affinities of PPCA and mutated TH. 3D structures of mutated TH were predicted and subjected to molecular docking studies. PPCA was found to bind in the deep narrow groove lined with polar and aromatic amino acids in 14 out of 17 mutants under study (R202H, L205P, H215Y, G216S, T245P, F278P, T283M, R297W, R306H, C328F, A345V, L356M, T368M, Q381K, P461L, T463M and D467G). Our results corroborate efficient binding of PPCA with normal and mutated TH, indicating that PPCA might be a strong therapeutic candidate for the management of Parkinson's disease and other related disorders. It may be a valuable target for evaluation in preclinical models. PMID:25230230

  1. Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups

    PubMed Central

    Bergfeld, Anne K.; Pearce, Oliver M. T.; Diaz, Sandra L.; Pham, Tho; Varki, Ajit

    2012-01-01

    The two major mammalian sialic acids are N-acetylneuraminic acid and N-glycolylneuraminic acid (Neu5Gc). The only known biosynthetic pathway generating Neu5Gc is the conversion of CMP-N-acetylneuraminic acid into CMP-Neu5Gc, which is catalyzed by the CMP-Neu5Ac hydroxylase enzyme. Given the irreversible nature of this reaction, there must be pathways for elimination or degradation of Neu5Gc, which would allow animal cells to adjust Neu5Gc levels to their needs. Although humans are incapable of synthesizing Neu5Gc due to an inactivated CMAH gene, exogenous Neu5Gc from dietary sources can be metabolically incorporated into tissues in the face of an anti-Neu5Gc antibody response. However, the metabolic turnover of Neu5Gc, which apparently prevents human cells from continued accumulation of this immunoreactive sialic acid, has not yet been elucidated. In this study, we show that pre-loaded Neu5Gc is eliminated from human cells over time, and we propose a conceivable Neu5Gc-degrading pathway based on the well studied metabolism of N-acetylhexosamines. We demonstrate that murine tissue cytosolic extracts harbor the enzymatic machinery to sequentially convert Neu5Gc into N-glycolylmannosamine, N-glycolylglucosamine, and N-glycolylglucosamine 6-phosphate, whereupon irreversible de-N-glycolylation of the latter results in the ubiquitous metabolites glycolate and glucosamine 6-phosphate. We substantiate this finding by demonstrating activity of recombinant human enzymes in vitro and by studying the fate of radiolabeled pathway intermediates in cultured human cells, suggesting that this pathway likely occurs in vivo. Finally, we demonstrate that the proposed degradative pathway is partially reversible, showing that N-glycolylmannosamine and N-glycolylglucosamine (but not glycolate) can serve as precursors for biosynthesis of endogenous Neu5Gc. PMID:22692205

  2. Multiple mechanisms regulate circadian expression of the gene for cholesterol 7alpha-hydroxylase (Cyp7a), a key enzyme in hepatic bile acid biosynthesis.

    PubMed

    Noshiro, Mitsuhide; Usui, Emiko; Kawamoto, Takeshi; Kubo, Hiroshi; Fujimoto, Katsumi; Furukawa, Masae; Honma, Sato; Makishima, Makoto; Honma, Ken-ichi; Kato, Yukio

    2007-08-01

    Cholesterol 7alpha-hydroxylase (CYP7A) and sterol 12alpha-hydroxylase (CYP8B) in bile acid biosynthesis and 3-hydroxyl-3-methylglutaryl CoA reductase (HMGCR) in cholesterol biosynthesis are the key enzymes in hepatic metabolic pathways, and their transcripts exhibit circadian expression profiles in rodent liver. The authors determined transcript levels of these enzymes and the regulatory factors for Cyp7a--including Dbp, Dec2, E4bp4, Hnf4alpha, Pparalpha, Lxralpha, Rev-erbalpha, and Rev-erbbeta--in the liver of wild-type and homozygous Clock mutant mice (Clock/Clock) and examined the effects of these transcription factors on the transcription activities of Cyp7a. The expression profile of the Cyp7a transcript in wild-type mice showed a strong circadian rhythm in both the 12L:12D light-dark cycle and constant darkness, and that in Clock/Clock also exhibited a circadian rhythm at an enhanced level with a lower amplitude, although its protein level became arrhythmic at a high level. The expression profile of Cyp8b mRNA in wild-type mice showed a shifted circadian rhythm from that of Cyp7a, becoming arrhythmic in Clock/Clock at an expression level comparable to that of wild-type mice. The expression profile of Hmgcr mRNA also lost its strong circadian rhythm in Clock/Clock , showing an expression level comparable to that of wild-type mice. The expressions of Dbp, Dec2, Rev-erbalpha, and Rev-erb beta--potent regulators for Cyp7a expression--were abolished or became arrhythmic in Clock/Clock, while other regulators for Cyp7a-Lxralpha, Hnf4alpha, Pparalpha, and E4bp4--had either less affected or enhanced expression in Clock/Clock. In luciferase reporter assays, REV-ERBalpha/beta, DBP, LXRalpha, and HNF4alpha increased the promoter activity of Cyp7a, whereas DEC2 abolished the transcription from the Cyp7a promoter: E4BP4 and PPARalpha were moderate negative regulators. Furthermore, knockdown of REV-ERBalpha/beta with siRNA suppressed Cyp7a transcript levels, and in the

  3. Carboxyl terminal deletion analysis of tryptophan hydroxylase.

    PubMed

    Mockus, S M; Kumer, S C; Vrana, K E

    1997-10-17

    Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the synthesis of serotonin and participates (in a non-rate-limiting fashion) in melatonin biosynthesis. In rabbit, TPH exists as a tetramer of four identical 51007 dalton (444 amino acids) protein subunits. An intersubunit binding domain responsible for tetramer formation of TPH was identified by assessing the role of a carboxyl terminal leucine heptad and 4-3 hydrophobic repeat. These repeats are conserved in all of the aromatic amino acid hydroxylases and have been shown to be required for the assembly of tyrosine hydroxylase tetramers. Polymerase chain reaction was utilized to create three TPH carboxyl terminal deletions (C delta8, C delta12 and C delta17) that sequentially remove members of the leucine heptad and 4-3 hydrophobic repeat. Each deletion and full-length recombinant TPH was expressed in bacteria to obtain soluble enzyme extracts for subsequent activity and structural analysis. It was found that removal of 8, 12 or 17 amino acids from the carboxyl terminus of TPH did not significantly alter enzymatic activity when compared to full-length recombinant TPH. However, the macromolecular structure of the deletions was dramatically affected as determined by dimeric and monomeric profiles on size exclusion chromatography. It can be concluded that amino acids 428-444 (the C-terminal 17 amino acids) comprise an intersubunit binding domain that is required for tetramer formation of TPH, but that tetramer assembly is not essential for full enzymatic activity. PMID:9392522

  4. Tyrosine hydroxylase- and/or aromatic L-amino acid decarboxylase-expressing neurons in the rat arcuate nucleus: ontogenesis and functional significance.

    PubMed

    Ugrumov, M; Melnikova, V; Ershov, P; Balan, I; Calas, A

    2002-07-01

    This study has evaluated in vivo, ex vivo and in vitro the ontogenesis and functional significance of the neurons of the arcuate nucleus (AN) expressing either individual enzymes of dopamine (DA) synthesis, tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC) as well as both of them in rats from the 17th embryonic day (E) till adulthood. Immunocytochemistry, image analysis, confocal microscopy, high performance liquid chromatography with electrochemical detection and radioimmunoassay were used to solve this problem. Monoenzymatic TH-containing neurons were initially observed on E18 located in the ventrolateral AN whereas the neurons expressing only AADC or both AADC and TH first appeared on E20 in the dorsomedial AN. On E21, the monoenzymatic TH- or AADC-expressing neurons comprised more than 99% of the whole neuron population expressing the DA-synthesizing enzymes. In spite of an extremely small number (<1%) of the neurons expressing both enzymes (DArgic neurons), the dissected AN (ex vivo) and its primary cell culture (in vitro) contained a surprisingly high amount of DA and L-dihydroxyphenylalanine (L-DOPA) which were released in response to membrane depolarization. Furthermore, DA production in the AN of fetuses occurred to be sufficient to provide an inhibitory control of prolactin secretion, as in adults. The above data suggest that DA could be synthesized, at least in the AN of fetuses, by monoenzymatic neurons containing either TH or AADC, in co-operation. This hypothesis may be extended to adult animals as their AN contained the same populations of the neurons expressing DA-synthesizing enzymes as in fetuses though the proportion of true DArgic neurons increased up to 38%. During ontogenesis, the monoenzymatic TH- and AADC-containing neurons established axosomatic and axo-axonal junctions that might facilitate the L-DOPA transport from the former to the latter. Moreover, the monoenzymatic AADC-expressing neurons project their axons to

  5. Novel Antibodies Reactive with Sialyl Lewis X in Both Humans and Mice Define Its Critical Role in Leukocyte Trafficking and Contact Hypersensitivity Responses*

    PubMed Central

    Matsumura, Ryuji; Hirakawa, Jotaro; Sato, Kaori; Ikeda, Toshiaki; Nagai, Motoe; Fukuda, Minoru; Imai, Yasuyuki; Kawashima, Hiroto

    2015-01-01

    Sialyl Lewis X (sLex) antigen functions as a common carbohydrate determinant recognized by all three members of the selectin family. However, its expression and function in mice remain undefined due to the poor reactivity of conventional anti-sLex monoclonal antibodies (mAbs) with mouse tissues. Here, we developed novel anti-sLex mAbs, termed F1 and F2, which react well with both human and mouse sLex, by immunizing fucosyltransferase (FucT)-IV and FucT-VII doubly deficient mice with 6-sulfo-sLex-expressing cells transiently transfected with an expression vector encoding CMP-N-acetylneuraminic acid hydroxylase. F1 and F2 specifically bound both the N-acetyl and the N-glycolyl forms of sLex as well as 6-sulfo-sLex, a major ligand for L-selectin expressed in high endothelial venules, and efficiently blocked physiological lymphocyte homing to lymph nodes in mice. Importantly, both of the mAbs inhibited contact hypersensitivity responses not only when administered in the L-selectin-dependent sensitization phase but also when administered in the elicitation phase in mice. When administered in the latter phase, F1 and F2 efficiently blocked rolling of mouse leukocytes along blood vessels expressing P- and E-selectin in the auricular skin in vivo. Consistent with these findings, the mAbs blocked P- and E-selectin-dependent leukocyte rolling in a flow chamber assay. Taken together, these results indicate that novel anti-sLex mAbs reactive with both human and mouse tissues, with the blocking ability against leukocyte trafficking mediated by all three selectins, have been established. These mAbs should be useful in determining the role of sLex antigen under physiological and pathological conditions. PMID:25944902

  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. PMID:26937021

  7. The regulatory domain of human tryptophan hydroxylase 1 forms a stable dimer.

    PubMed

    Zhang, Shengnan; Hinck, Cynthia S; Fitzpatrick, Paul F

    2016-08-01

    The three eukaryotic aromatic amino acid hydroxylases phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase have essentially identical catalytic domains and discrete regulatory domains. The regulatory domains of phenylalanine hydroxylase form ACT domain dimers when phenylalanine is bound to an allosteric site. In contrast the regulatory domains of tyrosine hydroxylase form a stable ACT dimer that does not bind the amino acid substrate. The regulatory domain of isoform 1 of human tryptophan hydroxylase was expressed and purified; mutagenesis of Cys64 was required to prevent formation of disulfide-linked dimers. The resulting protein behaved as a dimer upon gel filtration and in analytical ultracentrifugation. The sw value of the protein was unchanged from 2.7 to 35 μM, a concentration range over which the regulatory domain of phenylalanine hydroxylase forms both monomers and dimers, consistent with the regulatory domain of tryptophan hydroxylase 1 forming a stable dimer stable that does not undergo a monomer-dimer equilibrium. Addition of phenylalanine, a good substrate for the enzyme, had no effect on the sw value, consistent with there being no allosteric site for the amino acid substrate. PMID:27255998

  8. Zebrafish tyrosine hydroxylase 2 gene encodes tryptophan hydroxylase.

    PubMed

    Ren, Guiqi; Li, Song; Zhong, Hanbing; Lin, Shuo

    2013-08-01

    The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons. PMID:23754283

  9. 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

  10. Rapid deoxyribonucleic acid analysis by allele-specific polymerase chain reaction for detection of mutations in the steroid 21-hydroxylase gene

    SciTech Connect

    Wilson, R.C.; Wei, J.Q.; Cheng, K.C.

    1995-05-01

    Rapid DNA analysis based on allele-specific polymerase chain reaction (PCR) using mutation site-specific primers was developed to detect mutations in the CYP21 gene known to cause steroid 21-hydroxylase deficiency. In contrast to the previous method, in which PCR of genomic DNA was followed by dot blot analysis with radio active probes and multiple rounds of stripping and reprobing for each of the 8 most common mutation sites, the results using this new method were immediately visualized after the PCR run by ethidium bromide-stained agarose gel electrophoresis. Using allele-specific PCR, mutation(s) were identified on 148 affected chromosomes out of 160 tested. Although mutation(s) were identified on only one chromosome of 11 of these patients, their parents showed a consistent pattern on DNA analysis. The only exception was that in one family, in which the parents each had a detectable mutation, a mutation was detected on only one allele of the patient. Most likely there is a mutation in the patient`s other allele that could have arisen de novo or was inherited from the parent and was not evident in the transmitting parent`s phenotype. When compared with the dot blot procedure, allele-specific PCR is more rapid, less labor-intensive, and avoids the use of radioactivity. 26 refs., 3 figs., 2 tabs.

  11. 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. PMID:25140701

  12. Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson's disease.

    PubMed

    Azzouz, Mimoun; Martin-Rendon, Enca; Barber, Robert D; Mitrophanous, Kyriacos A; Carter, Emma E; Rohll, Jonathan B; Kingsman, Susan M; Kingsman, Alan J; Mazarakis, Nicholas D

    2002-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra. This loss leads to complete dopamine depletion in the striatum and severe motor impairment. It has been demonstrated previously that a lentiviral vector system based on equine infectious anemia virus (EIAV) gives rise to highly efficient and sustained transduction of neurons in the rat brain. Therefore, a dopamine replacement strategy using EIAV has been investigated as a treatment in the 6-hydroxydopamine (6-OHDA) animal model of PD. A self-inactivating EIAV minimal lentiviral vector that expresses tyrosine hydroxylase (TH), aromatic amino acid dopa decarboxylase (AADC), and GTP cyclohydrolase 1 (CH1) in a single transcription unit has been generated. In cultured striatal neurons transduced with this vector, TH, AADC, and CH1 proteins can all be detected. After stereotactic delivery into the dopamine-denervated striatum of the 6-OHDA-lesioned rat, sustained expression of each enzyme and effective production of catecholamines were detected, resulting in significant reduction of apomorphine-induced motor asymmetry compared with control animals (p < 0.003). Expression of each enzyme in the striatum was observed for up to 5 months after injection. These data indicate that the delivery of three catecholaminergic synthetic enzymes by a single lentiviral vector can achieve functional improvement and thus open the potential for the use of this vector for gene therapy of late-stage PD patients. PMID:12451130

  13. Streptomyces coelicolor A3(2) CYP102 Protein, a Novel Fatty Acid Hydroxylase Encoded as a Heme Domain without an N-Terminal Redox Partner▿

    PubMed Central

    Lamb, David C.; Lei, Li; Zhao, Bin; Yuan, Hang; Jackson, Colin J.; Warrilow, Andrew G. S.; Skaug, Tove; Dyson, Paul J.; Dawson, Eric S.; Kelly, Steven L.; Hachey, David L.; Waterman, Michael R.

    2010-01-01

    The gene from Streptomyces coelicolor A3(2) encoding CYP102B1, a recently discovered CYP102 subfamily which exists solely as a single P450 heme domain, has been cloned, expressed in Escherichia coli, purified, characterized, and compared to its fusion protein family members. Purified reconstitution metabolism experiments with spinach ferredoxin, ferredoxin reductase, and NADPH revealed differences in the regio- and stereoselective metabolism of arachidonic acid compared to that of CYP102A1, exclusively producing 11,12-epoxyeicosa-5,8,14-trienoic acid in addition to the shared metabolites 18-hydroxy arachidonic acid and 14,15-epoxyeicosa-5,8,11-trienoic acid. Consequently, in order to elucidate the physiological function of CYP102B1, transposon mutagenesis was used to generate an S. coelicolor A3(2) strain lacking CYP102B1 activity and the phenotype was assessed. PMID:20097805

  14. Δ9-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 Central

    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

    2014-01-01

    We recently reported that Δ9-tetrahydrocannabinol (Δ9-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 Δ9-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-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 Δ9-THC mediated increase in FA2H. In support of this hypothesis, we herein demonstrated that; (i) Δ9 THC activated the basal transcriptional activity of PPARα in a concentration-dependent manner, (ii) the concomitant up-regulation of PPARα/FA2H was caused by Δ9-THC, (iii) PA could activate PPARα after the PPARα expression plasmid was introduced, and (iv) the Δ9-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 Δ9 THC up-regulation of FA2H in MDA-MB-231 cells. PMID:25291031

  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

    2014-12-01

    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. PMID:25291031

  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. A chimeric tyrosine/tryptophan hydroxylase. The tyrosine hydroxylase regulatory domain serves to stabilize enzyme activity.

    PubMed

    Mockus, S M; Kumer, S C; Vrana, K E

    1997-08-01

    The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t1/2 of 84 min at 37 degrees C, TPH was much less stable (t1/2 = 28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by a t1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain. PMID:9356925

  18. CYP704B1 Is a Long-Chain Fatty Acid ω-Hydroxylase Essential for Sporopollenin Synthesis in Pollen of Arabidopsis1[W][OA

    PubMed Central

    Dobritsa, Anna A.; Shrestha, Jay; Morant, Marc; Pinot, Franck; Matsuno, Michiyo; Swanson, Robert; Møller, Birger Lindberg; Preuss, Daphne

    2009-01-01

    Sporopollenin is the major component of the outer pollen wall (exine). Fatty acid derivatives and phenolics are thought to be its monomeric building blocks, but the precise structure, biosynthetic route, and genetics of sporopollenin are poorly understood. Based on a phenotypic mutant screen in Arabidopsis (Arabidopsis thaliana), we identified a cytochrome P450, designated CYP704B1, as being essential for exine development. CYP704B1 is expressed in the developing anthers. Mutations in CYP704B1 result in impaired pollen walls that lack a normal exine layer and exhibit a characteristic striped surface, termed zebra phenotype. Heterologous expression of CYP704B1 in yeast cells demonstrated that it catalyzes ω-hydroxylation of long-chain fatty acids, implicating these molecules in sporopollenin synthesis. Recently, an anther-specific cytochrome P450, denoted CYP703A2, that catalyzes in-chain hydroxylation of lauric acid was also shown to be involved in sporopollenin synthesis. This shows that different classes of hydroxylated fatty acids serve as essential compounds for sporopollenin formation. The genetic relationships between CYP704B1, CYP703A2, and another exine gene, MALE STERILITY2, which encodes a fatty acyl reductase, were explored. Mutations in all three genes resulted in pollen with remarkably similar zebra phenotypes, distinct from those of other known exine mutants. The double and triple mutant combinations did not result in the appearance of novel phenotypes or enhancement of single mutant phenotypes. This implies that each of the three genes is required to provide an indispensable subset of fatty acid-derived components within the sporopollenin biosynthesis framework. PMID:19700560

  19. CYP704B1 is a long-chain fatty acid omega-hydroxylase essential for sporopollenin synthesis in pollen of Arabidopsis.

    PubMed

    Dobritsa, Anna A; Shrestha, Jay; Morant, Marc; Pinot, Franck; Matsuno, Michiyo; Swanson, Robert; Møller, Birger Lindberg; Preuss, Daphne

    2009-10-01

    Sporopollenin is the major component of the outer pollen wall (exine). Fatty acid derivatives and phenolics are thought to be its monomeric building blocks, but the precise structure, biosynthetic route, and genetics of sporopollenin are poorly understood. Based on a phenotypic mutant screen in Arabidopsis (Arabidopsis thaliana), we identified a cytochrome P450, designated CYP704B1, as being essential for exine development. CYP704B1 is expressed in the developing anthers. Mutations in CYP704B1 result in impaired pollen walls that lack a normal exine layer and exhibit a characteristic striped surface, termed zebra phenotype. Heterologous expression of CYP704B1 in yeast cells demonstrated that it catalyzes omega-hydroxylation of long-chain fatty acids, implicating these molecules in sporopollenin synthesis. Recently, an anther-specific cytochrome P450, denoted CYP703A2, that catalyzes in-chain hydroxylation of lauric acid was also shown to be involved in sporopollenin synthesis. This shows that different classes of hydroxylated fatty acids serve as essential compounds for sporopollenin formation. The genetic relationships between CYP704B1, CYP703A2, and another exine gene, MALE STERILITY2, which encodes a fatty acyl reductase, were explored. Mutations in all three genes resulted in pollen with remarkably similar zebra phenotypes, distinct from those of other known exine mutants. The double and triple mutant combinations did not result in the appearance of novel phenotypes or enhancement of single mutant phenotypes. This implies that each of the three genes is required to provide an indispensable subset of fatty acid-derived components within the sporopollenin biosynthesis framework. PMID:19700560

  20. Clofibrate-induced cytochrome P450-lauric acid omega hydroxylase(P450LA omega):purification, cDNA cloning, sequence and regulation

    SciTech Connect

    Hardwick, J.P.; Song, B.J.; Gonzalez, F.J.

    1986-05-01

    A cytochrome P450 that hydroxylates lauric acid at the 12 position (P450LA omega) was isolated from liver microsomes of clofibrate treated rats. P450LA omega was immunologically distinct from P450s a,b,c,d,e,f,g,h,j,PB1, and PCN1. Polyclonal antibody against P450LA omega was utilized to screen a gt11 cDNA library. A clone (pP450LA omega), was isolated and its sequence determined. The P450LA omega mRNA is a minimum 2387 nts in length and codes for a P450 of Mr.58,222 daltons. This protein shares less than 35% amino acid similarity with P450s b,c,d,e,f,PB1, and PCN1; however, it does contain a hydrophobic amino terminal peptide and a conserved sequence surrounding the Cys residue at position 456, which is similar to other microsomal P450s. P450LA omega is present at high levels in untreated rat kidney and is induced by clofibrate in both kidney and liver. This induction is the result of an accumulation of mRNA through a rapid transcriptional activation of the P450LA gene. Southern blotting data suggest the presence of 2 or 3 genes in the P450LA omega family. This P450 gene family may be associated with arachidonic acid and prostraglandin metabolism in kidney and other tissues.

  1. CYP98A22, a phenolic ester 3’-hydroxylase specialized in the synthesis of chlorogenic acid, as a new tool for enhancing the furanocoumarin concentration in Ruta graveolens

    PubMed Central

    2012-01-01

    Background Furanocoumarins are molecules with proven therapeutic properties and are produced in only a small number of medicinal plant species such as Ruta graveolens. In vivo, these molecules play a protective role against phytophageous insect attack. Furanocoumarins are members of the phenylpropanoids family, and their biosynthetic pathway is initiated from p-coumaroyl coA. The enzymes belonging to the CYP98A cytochrome P450 family have been widely described as being aromatic meta-hydroxylases of various substrates, such as p-coumaroyl ester derivatives, and are involved in the synthesis of coumarins such as scopoletin. In furanocoumarin-producing plants, these enzymes catalyze the step directly downstream of the junction with the furanocoumarin biosynthetic pathway and might indirectly impact their synthesis. Results In this work, we describe the cloning and functional characterization of the first CYP98A encoding gene isolated from R. graveolens. Using Nicotiana benthamiana as a heterologous expression system, we have demonstrated that this enzyme adds a 3-OH to p-coumaroyl ester derivatives but is more efficient to convert p-coumaroyl quinate into chlorogenic acid than to metabolize p-coumaroyl shikimate. Plants exposed to UV-B stress showed an enhanced expression level of the corresponding gene. The R. graveolens cyp98a22 open reading frame and the orthologous Arabidopsis thaliana cyp98a3 open reading frame were overexpressed in stable transgenic Ruta plants. Both plant series were analyzed for their production of scopoletin and furanocoumarin. A detailed analysis indicates that both genes enhance the production of furanocoumarins but that CYP98A22, unlike CYP98A3, doesn’t affect the synthesis of scopoletin. Conclusions The overexpression of CYP98A22 positively impacts the concentration of furanocoumarins in R. graveolens. This gene is therefore a valuable tool to engineer plants with improved therapeutical values that might also be more resistant to

  2. Structural studies of dopamine. beta. -hydroxylase

    SciTech Connect

    Papadopoulos, N.J.

    1985-01-01

    Dopamine ..beta..-hydroxylase catalyzes the conversion of dopamine to norepinephrine, a ..beta..-hydroxylation reaction, utilizing ascorbic acid as reducing agent and molecular oxygen as cosubstrate. Modifications of the previously published purification procedure for D..beta..H have produced findings which show that (1) enzyme is inactivated by ascorbate autooxidation during the isolation procedure, (2) active as well as inactive D..beta..H co-purify throughout the entire purification procedure and (3) beef liver catalase totally protects against this time dependent inactivation. The stoichiometry of copper binding to the active sites of D..beta..H has been investigated using /sup 19/F-NMR and radioactive binding experiments. The data unequivocally show that homogeneous D..beta..H (isolated in the presence of catalase) specifically binds up to approx.8 copper atoms per enzyme tetramer. Distance determinations done using NMR relaxation rate theory show that anion activators of the catalytic reaction are bound at a fairly far distance from the Cu/sup 2 +/ centers. Spin-echo electron paramagnetic resonance spectroscopy indicates that at least one, possibly two, histidines are bound as equatorial ligands to each Cu/sup 2 +/ ion. The combined data indicate that highly purified dopamine ..beta..-hydroxylase contains a 2 copper atom active site, composed of magnetically non-interacting metal centers. Active site components are distant from the Cu/sup 2 +/ centers, suggesting a possible movement of active site residues or components after reduction of enzyme bound copper in order to achieve the insertion of 1 atom of oxygen into the benzylic C-H bond of dopamine.

  3. Therapeutic potential of the inhibition of the retinoic acid hydroxylases CYP26A1 and CYP26B1 by xenobiotics.

    PubMed

    Nelson, Cara H; Buttrick, Brian R; Isoherranen, Nina

    2013-01-01

    Retinoic acid (RA), the active metabolite of vitamin A, is an important endogenous signaling molecule regulating cell cycle and maintenance of epithelia. RA isomers are also used as drugs to treat various cancers and dermatological diseases. However, the therapeutic uses of RA isomers are limited due to side effects such as teratogenicity and resistance to treatment emerging mainly from autoinduction of RA metabolism. To improve the therapeutic usefulness of retinoids, RA metabolism blocking agents (RAMBAs) have been developed. These inhibitors generally target the cytochrome P450 (CYP) enzymes because RA clearance is predominantly mediated by P450s. Since the initial identification of inhibitors of RA metabolism, CYP26 enzymes have been characterized as the main enzymes responsible for RA clearance. This makes CYP26 enzymes an attractive target for the development of novel therapeutics for cancer and dermatological conditions. The basic principle of development of CYP26 inhibitors is that endogenous RA concentrations will be increased in the presence of a CYP26 inhibitor, thus, potentiating the activity of endogenous RA in a cell-type specific manner. This will reduce side effects compared to administration of RA and allow for more targeted therapy. In clinical trials, inhibitors of RA metabolism have been effective in treatment of psoriasis and other dermatological conditions as well as in some cancers. However, no CYP26 inhibitor has yet been approved for clinical use. This review summarizes the history of development of RAMBAs, the clinical and preclinical studies with the various structural series and the available knowledge of structure activity relationships of CYP26 inhibitors. PMID:23688132

  4. 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.

  5. 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.

  6. A Pulsed EPR Study of Amino Acid and Tetrahydropterin Binding in a Tyrosine Hydroxylase Nitric Oxide Complex: Evidence for Substrate Rearrangements in Formation of the Oxygen-Reactive Complex

    PubMed Central

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

    2013-01-01

    Tyrosine hydroxylase is a non-heme 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. PMID:24168553

  7. 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.

  8. Genetics Home Reference: 21-hydroxylase deficiency

    MedlinePlus

    ... deficiency is an inherited disorder that affects the adrenal glands . The adrenal glands are located on top of the kidneys and ... body. In people with 21-hydroxylase deficiency , the adrenal glands produce excess androgens, which are male sex hormones. ...

  9. 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

    2015-01-01

    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

  10. 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

  11. 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 Central

    Edson, Katheryne Z.; Rettie, Allan E.

    2014-01-01

    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. PMID:23688133

  12. 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.

  13. Phenylacetylene reversibly inhibits the phenol hydroxylase of Pseudomonas sp. CF600 at high concentrations but is oxidized at lower concentrations.

    PubMed

    Kagle, Jeanne; Hay, Anthony G

    2006-09-01

    Alkynes are mechanism-based inhibitors of several bacterial monooxygenases, including the soluble methane monooxygenase (sMMO) of Methylococcus capsulatus and the toluene o-monooxygenase (TOM) of Burkholderia cepacia G4. In this paper, we investigated the inhibition of the phenol hydroxylase of Pseudomonas sp. CF600 by the alkyne phenylacetylene. Growth of CF600 on phenol and phenol hydroxylase activity were inhibited by phenylacetylene concentrations greater than 1.0 mM. Unlike other alkynes, which irreversibly inhibit a number of monooxygenases, inhibition of phenol hydroxylase by phenylacetylene was reversible, as demonstrated by the ability of washed cells to regain phenol hydroxylase activity. Additionally, phenylacetylene was metabolized by phenol-grown cells, yielding a yellow meta-ring fission product which absorbed light maximally at 412 nm. Phenol-grown CF600 transformed phenylacetylene to hydroxyphenylacetylene and 2-hydroxy-6-oxo-octa-2,4-dien-7-ynoic acid as detected by gas chromatography--mass spectroscopy and high-performance liquid chromatography (HPLC), respectively, while neither a derivative of CF600 with a non-functional phenol hydroxylase nor wild-type CF600 grown on acetate transformed phenylacetylene. These results demonstrate that the phenol hydroxylase of CF600 has broader substrate specificity than previously reported. They also suggest that phenylacetylene acts as a competitive inhibitor rather than as a mechanism-based inhibitor of this phenol hydroxylase. PMID:16485115

  14. Dopamine beta-hydroxylase deficiency

    PubMed Central

    Senard, Jean-Michel; Rouet, Philippe

    2006-01-01

    Dopamine beta-hydroxylase (DβH) deficiency is a very rare form of primary autonomic failure characterized by a complete absence of noradrenaline and adrenaline in plasma together with increased dopamine plasma levels. The prevalence of DβH deficiency is unknown. Only a limited number of cases with this disease have been reported. DβH deficiency is mainly characterized by cardiovascular disorders and severe orthostatic hypotension. First symptoms often start during a complicated perinatal period with hypotension, muscle hypotonia, hypothermia and hypoglycemia. Children with DβH deficiency exhibit reduced ability to exercise because of blood pressure inadaptation with exertion and syncope. Symptoms usually worsen progressively during late adolescence and early adulthood with severe orthostatic hypotension, eyelid ptosis, nasal stuffiness and sexual disorders. Limitation in standing tolerance, limited ability to exercise and traumatic morbidity related to falls and syncope may represent later evolution. The syndrome is caused by heterogeneous molecular alterations of the DBH gene and is inherited in an autosomal recessive manner. Restoration of plasma noradrenaline to the normal range can be achieved by therapy with the synthetic precursor of noradrenaline, L-threo-dihydroxyphenylserine (DOPS). Oral administration of 100 to 500 mg DOPS, twice or three times daily, increases blood pressure and reverses the orthostatic intolerance. PMID:16722595

  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. PMID:24631264

  16. 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

  17. Human Skin Aryl Hydrocarbon Hydroxylase

    PubMed Central

    Bickers, David R.; Kappas, Attallah

    1978-01-01

    Coal tar products, which are widely used in treating dermatologic disease, contain numerous polycyclic aromatic hydrocarbons, including 3,4-benzo[a]pyrene (BP). BP is among the most potent environmental chemical carcinogens and is known to evoke tumors in the skin of experimental animals and perhaps also of man. In this study the effect of cutaneous application of coal tar solution (U. S. Pharmacopeia) on aryl hydrocarbon hydroxylase (AHH) activity in the skin of patients usually treated with this drug was investigated. AHH, a cytochrome P-450 dependent carcinogen-metabolizing enzyme appears to play an important role in the activation of polycyclic hydrocarbons into reactive moieties that can bind to DNA and that may directly induce cancer. Application of coal tar solution to human skin caused a two to five-fold induction of cutaneous AHH in nine subjects. In further studies, the incubation of human skin with coal tar solution in vitro also caused variable induction of cutaneous AHH. Maximum responses in both systems occurred after 24 h and enzyme activity in vitro was time- and tissue- and substrate-concentration dependent. Studies in experimental animals showed that topical application of coal tar solution caused induction of AHH in skin and, after percutaneous absorption, in liver as well. Assay of several defined constituents of coal tar for AHH induction showed that BP was the most potent inducer of AHH tested. These studies indicate that topical application of coal tar solution in doses ordinarily used in treating dermatologic disease causes induction of AHH in human skin and suggest that such induced enzymatic activity could relate to carcinogenic responses to this agent in skin or, after percutaneous absorption, in other tissues as well. PMID:711851

  18. Are Striatal Tyrosine Hydroxylase Interneurons Dopaminergic?

    PubMed Central

    Xenias, Harry S.; Ibáñez-Sandoval, Osvaldo; Koós, Tibor

    2015-01-01

    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. PMID:25904808

  19. Genetics Home Reference: dopamine beta-hydroxylase deficiency

    MedlinePlus

    ... CONGENITAL Sources for This Page Cubells JF, Zabetian CP. Human genetics of plasma dopamine beta-hydroxylase activity: ... GeneReview: Dopamine Beta-Hydroxylase Deficiency Kim CH, Zabetian CP, Cubells JF, Cho S, Biaggioni I, Cohen BM, Robertson ...

  20. 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. PMID:21981961

  1. Primary structure, tissue distribution, and chromosomal localization of a novel isoform of lysyl hydroxylase (lysyl hydroxylase 3)

    PubMed

    Valtavaara, M; Szpirer, C; Szpirer, J; Myllylä, R

    1998-05-22

    We report characterization of a novel isoform of lysyl hydroxylase (lysyl hydroxylase 3, LH3). The cDNA clones encode a polypeptide of 738 amino acids, including a signal peptide. The amino acid sequence has a high overall identity with LH1 and LH2, the isoforms characterized earlier. Conserved regions are present in the carboxyl-terminal portion of the isoforms and also in the central part of the molecules. Histidine and asparagine residues, which are conserved in the other isoforms and are known to be required for enzymatic activity, are also conserved in the novel isoform. The gene for LH3 (PLOD3) has been assigned to human chromosome 7q36 and rat chromosome 12. Gene expression of LH3 is highly regulated in adult human tissues. A strong hybridization signal, corresponding to an mRNA 2.75 kilobases in size, is obtained in heart, placenta and pancreas on multiple tissue RNA blots. Expression of the cDNA in vitro results in the synthesis of a protein that hydroxylates lysyl residues in collagenous sequences in a non-triple helical conformation. PMID:9582318

  2. Prolyl hydroxylase-2 inhibits liver tumor cell proliferation and cyclin D1 expression in a hydroxylase-dependent manner.

    PubMed

    Tao, Yifeng; Lin, Feng; Li, Ruidong; Shen, Jie; Wang, Zhengxin

    2016-08-01

    Prolyl hydroxylase 2 is a key regulator of hypoxia-inducible factor 1 alpha protein, and has previously been implicated as a tumor suppressor in various cancers. However, the function of prolyl hydroxylase 2 in liver cancer has yet to be elucidated. Characterization of prolyl hydroxylase 2 function and related mechanisms in liver cancer may enable the development of targeted therapy. Here we found that prolyl hydroxylase 2 overexpression in human hepatocellular carcinoma cancer cell lines inhibited cell proliferation, while prolyl hydroxylase 2 knockdown enhanced cell proliferation. Further analyses revealed that the prolyl hydroxylase 2-mediated inhibition of cell proliferation was due to a cell cycle arrest at the G1/S transition. Moreover, the block in cell cycle was facilitated by negative regulation of cyclin D1, a process dependent on the hydroxylase activity of prolyl hydroxylase 2. Using an in vivo xenograft mouse model, we found that the overexpression of prolyl hydroxylase 2 led to a reduction in tumor size. Evaluation of paired human liver cancer patient samples revealed that prolyl hydroxylase 2 protein levels were significantly reduced in 6 of the 10 cancer tissues as compared to their respective normal tissue controls. Furthermore, elevated expression of prolyl hydroxylase 2 was associated with significantly prolonged survival in patients with liver cancer. These results suggest that prolyl hydroxylase 2 plays an important tumor suppressive role in liver cancer and may prove to be of prognostic and therapeutic value. PMID:27307407

  3. Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger.

    PubMed

    Matsuda, J; Okabe, S; Hashimoto, T; Yamada, Y

    1991-05-25

    Roots of several solanaceous plants produce anticholinergic alkaloids, hyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11), catalyzes hydroxylation of hyoscyamine in the biosynthetic pathway leading to scopolamine. We report here on the isolation of cDNA clones encoding the hydroxylase from a cDNA library made from mRNA of the cultured roots of Hyoscyamus niger. The library was screened with three synthetic oligonucleotides that encode amino acid sequences of internal peptide fragments of the purified hydroxylase. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame that encodes 344 amino acids (Mr = 38,999). All 12 internal peptide fragments determined in the purified enzyme were found in the amino acid sequence deduced from the cDNA. With computer-aided comparison to other proteins we found that the hydroxylase is homologous to two synthases involved in the biosynthesis of beta-lactam antibiotics in some microorganisms and the gene products of tomato pTOM13 cDNA and maize A2 locus which had been proposed to catalyze oxidative reactions in the biosynthesis of ethylene and anthocyan, respectively. RNA blotting hybridization showed that mRNA of the hydroxylase is abundant in cultured roots and present in plant roots, but absent in leaves, stems, and cultured cells of H. niger. PMID:2033047

  4. Tryptophan hydroxylase-1 regulates immune tolerance and inflammation.

    PubMed

    Nowak, Elizabeth C; de Vries, Victor C; Wasiuk, Anna; Ahonen, Cory; Bennett, Kathryn A; Le Mercier, Isabelle; Ha, Dae-Gon; Noelle, Randolph J

    2012-10-22

    Nutrient deprivation based on the loss of essential amino acids by catabolic enzymes in the microenvironment is a critical means to control inflammatory responses and immune tolerance. Here we report the novel finding that Tph-1 (tryptophan hydroxylase-1), a synthase which catalyses the conversion of tryptophan to serotonin and exhausts tryptophan, is a potent regulator of immunity. In models of skin allograft tolerance, tumor growth, and experimental autoimmune encephalomyelitis, Tph-1 deficiency breaks allograft tolerance, induces tumor remission, and intensifies neuroinflammation, respectively. All of these effects of Tph-1 deficiency are independent of its downstream product serotonin. Because mast cells (MCs) appear to be the major source of Tph-1 and restoration of Tph-1 in the MC compartment in vivo compensates for the defect, these experiments introduce a fundamentally new mechanism of MC-mediated immune suppression that broadly impacts multiple arms of immunity. PMID:23008335

  5. Mechanism of salicylate hydroxylase-catalyzed decarboxylation.

    PubMed

    Suzuki, K; Katagiri, M

    1981-02-13

    Salicylate hydroxylase (salicylate, NADH: oxygen oxidoreductase (1-hydroxylating, decarboxylating), EC 1.14.13.1) in Pseudomonas putida catalyzed hydroxylation of the substrate analogue, salicylaldehyde, to form catechol and formate with stoichiometric consumption of NADH and O2. Consequently, a study of primary product derived from the carboxyl group of the authentic substrate, salicylate, was undertaken. The experimental results revealed that CO2 not H2CO3, was produced first. PMID:7213760

  6. 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.

  7. 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

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

    PubMed

    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

  9. 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

  10. Reconstitution of cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata EHRH.).

    PubMed Central

    Petersen, M; Seitz, H U

    1988-01-01

    Cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata) was solubilized from microsomal membranes with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonic acid). Cytochrome P-450 was separated from NADPH: cytochrome c (P-450) reductase by ion-exchange chromatography on DEAE-Sephacel. NADPH:cytochrome c (P-450) reductase was further purified by affinity chromatography on 2',5'-ADP-Sepharose 4B. This procedure resulted in a 248-fold purification of the enzyme; on SDS/polyacrylamide-gel electrophoresis after silver staining, only one band, corresponding to a molecular mass of 80 kDa, was present. The digitoxin 12 beta-hydroxylase activity could be reconstituted by incubating partially purified cytochrome P-450 and NADPH:cytochrome c (P-450) reductase together with naturally occurring microsomal lipids and flavin nucleotides. This procedure yielded about 10% of the original amount of digitoxin 12 beta-hydroxylase. PMID:3137929

  11. 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. PMID:9367532

  12. Coexpression of Tyrosine Hydroxylase, GTP Cyclohydrolase I, Aromatic Amino Acid Decarboxylase, and Vesicular Monoamine Transporter 2 from a Helper Virus-Free Herpes Simplex Virus Type 1 Vector Supports High-Level, Long-Term Biochemical and Behavioral Correction of a Rat Model of Parkinson’s Disease

    PubMed Central

    SUN, MEI; KONG, LINGXIN; WANG, XIAODAN; HOLMES, COURTNEY; GAO, QINGSHENG; ZHANG, GUO-RONG; PFEILSCHIFTER, JOSEF; GOLDSTEIN, DAVID S.; GELLER, ALFRED I.

    2006-01-01

    Parkinson’s disease is due to the selective loss of nigrostriatal dopaminergic neurons. Consequently, many therapeutic strategies have focused on restoring striatal dopamine levels, including direct gene transfer to striatal cells, using viral vectors that express specific dopamine biosynthetic enzymes. The central hypothesis of this study is that coexpression of four dopamine biosynthetic and transporter genes in striatal neurons can support the efficient production and regulated, vesicular release of dopamine: tyrosine hydroxylase (TH) converts tyrosine to l-3,4-dihydroxyphenylalanine (l -DOPA), GTP cyclohydrolase I (GTP CH I) is the rate-limiting enzyme in the biosynthesis of the cofactor for TH, aromatic amino acid decarboxylase (AADC) converts l -DOPA to dopamine, and a vesicular monoamine transporter (VMAT-2) transports dopamine into synaptic vesicles, thereby supporting regulated, vesicular release of dopamine and relieving feedback inhibition of TH by dopamine. Helper virus-free herpes simplex virus type 1 vectors that coexpress the three dopamine biosynthetic enzymes (TH, GTP CH I, and AADC; 3-gene-vector) or these three dopamine biosynthetic enzymes and the vesicular monoamine transporter (TH, GTP CH I, AADC, and VMAT-2; 4-gene-vector) were compared. Both vectors supported production of dopamine in cultured fibroblasts. These vectors were microinjected into the striatum of 6-hydroxydopamine-lesioned rats. These vectors carry a modified neurofilament gene promoter, and γ-aminobutyric acid (GABA)-ergic neuron-specific gene expression was maintained for 14 months after gene transfer. The 4-gene-vector supported higher levels of correction of apomorphine-induced rotational behavior than did the 3-gene-vector, and this correction was maintained for 6 months. Proximal to the injection sites, the 4-gene-vector, but not the 3-gene-vector, supported extracellular levels of dopamine and dihydroxyphenylacetic acid (DOPAC) that were similar to those observed in

  13. Cloning, functional expression, and characterization of CYP709C1, the first sub-terminal hydroxylase of long chain fatty acid in plants. Induction by chemicals and methyl jasmonate.

    PubMed

    Kandel, Sylvie; Morant, Marc; Benveniste, Irène; Blée, Elizabeth; Werck-Reichhart, Danièle; Pinot, Franck

    2005-10-28

    We cloned and characterized CYP709C1, a new plant cytochrome P450 belonging to the P450 family, that so far has no identified function except for clustering with a fatty acid metabolizing clade of P450 enzymes. We showed here that CYP709C1 is capable of hydroxylating fatty acids at the omega-1 and omega-2 positions. This work was performed after recoding and heterologous expression of a full-length cDNA isolated from a wheat cDNA library in an engineered yeast strain. Investigation on substrate specificity indicates that CYP709C1 metabolizes different fatty acids varying in their chain length (C12 to C18) and unsaturation. CYP709C1 is the first identified plant cytochrome P450 that can catalyze sub-terminal hydroxylation of C18 fatty acids. cis-9,10-Epoxystearic acid is metabolized with the highest efficiency, i.e. K((m)(app)) of 8 microM and V(max(app)) of 328 nmol/min/nmol P450. This, together with the fact that wheat possesses a microsomal peroxygenase able to synthesize this compound from oleic acid, strongly suggests that it is a physiological substrate. Hydroxylated fatty acids are implicated in plant defense events. We postulated that CYP709C1 could be involved in plant defense by producing such compounds. This receives support from the observation that (i) sub-terminal hydroxylation of 9,10-epoxystearic acid is induced (15-fold after 3 h) in microsomes of wheat seedlings treated with the stress hormone methyl jasmonate and (ii) CYP709C1 is enhanced at the transcriptional level by this treatment. CYP709C1 transcript also accumulated after treatment with a combination of the safener naphthalic acid anhydride and phenobarbital. This indicates a possible detoxifying function for CYP709C1 that we discussed. PMID:16120613

  14. 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

  15. 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...

  16. Structural characterization of human cholesterol 7α-hydroxylase

    PubMed Central

    Tempel, Wolfram; Grabovec, Irina; MacKenzie, Farrell; Dichenko, Yaroslav V.; Usanov, Sergey A.; Gilep, Andrei A.; Park, Hee-Won; Strushkevich, Natallia

    2014-01-01

    Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B′ helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand. PMID:24927729

  17. Structural characterization of human cholesterol 7α-hydroxylase.

    PubMed

    Tempel, Wolfram; Grabovec, Irina; MacKenzie, Farrell; Dichenko, Yaroslav V; Usanov, Sergey A; Gilep, Andrei A; Park, Hee-Won; Strushkevich, Natallia

    2014-09-01

    Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B' helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand. PMID:24927729

  18. High-level expression of rat PC12 tyrosine hydroxylase cDNA in Escherichia coli: purification and characterization of the cloned enzyme.

    PubMed Central

    Wang, Y H; Citron, B A; Ribeiro, P; Kaufman, S

    1991-01-01

    A rat cDNA containing the complete coding sequence for rat tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) was isolated from a rat PC12 cDNA library and subcloned in a bacterial expression plasmid, and large amounts of functional enzyme were produced in Escherichia coli. The recombinant enzyme was purified approximately 20-fold to a final specific activity of 1.8 mumol/min per mg of protein, with a yield of 30%. As much as 1 mg of pure protein could be obtained from 1 g of wet bacterial cells. The purified hydroxylase was shown to be homogeneous by denaturing polyacrylamide electrophoresis and isoelectric focusing. Amino acid analysis of the N terminus (25 residues) revealed 100% identity with rat PC12 tyrosine hydroxylase, as deduced from its cDNA sequence. Several of the kinetic properties of the recombinant enzyme resembled those of the native PC12 hydroxylase. However, in contrast to the native enzyme, the purified recombinant hydroxylase was shown to be in an activated form. Phosphorylation with cAMP-dependent protein kinase resulted in stoichiometric incorporation of phosphate, but the kinetic profile of the recombinant enzyme was unaffected. Several clues to these differences are considered that may provide insight into the structural features important to the regulation of tyrosine hydroxylase. Images PMID:1681542

  19. The isolation and properties of phenylalanine hydroxylase from human liver

    PubMed Central

    Woo, Savio L. C.; Gillam, Shirley Su; Woolf, Louis I.

    1974-01-01

    Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The Km values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria. PMID:4854919

  20. Polymorphic variation in the 11beta-hydroxylase gene associates with reduced 11-hydroxylase efficiency.

    PubMed

    Barr, Marianne; MacKenzie, Scott M; Friel, Elaine C; Holloway, Christine D; Wilkinson, Donna M; Brain, Nick J R; Ingram, Mary C; Fraser, Robert; Brown, Morris; Samani, Nilesh J; Caulfield, Mark; Munroe, Patricia B; Farrall, Martin; Webster, John; Clayton, David; Dominiczak, Anna F; Connell, John M C; Davies, Eleanor

    2007-01-01

    The -344 C/T and intron 2 conversion variants in the CYP11B2 gene, encoding aldosterone synthase, have been associated with markers of impaired 11beta-hydroxylase activity. We hypothesize that this association is because of variations in the adjacent 11beta-hydroxylase gene (CYP11B1) and arises through linkage disequilibrium between CYP11B1 and CYP11B2. The pattern of variation across the entire CYP11B locus was determined by sequencing 26 normotensive subjects stratified by and homozygous for the -344 and intron conversion variants. Eighty-three variants associated with -344 and intron conversion were identified. Haplotype analysis revealed 4 common haplotypes, accounting for 68% of chromosomes, confirming strong linkage disequilibrium across the region. Two novel CYP11B1 polymorphisms upstream of the coding region (-1889 G/T and -1859 A/G) were identified as contributing to the common haplotypes. Given the potential for such mutations to affect transcriptional regulation of CYP11B1, these were analyzed further. A total of 512 hypertensive subjects from the British Genetics of Hypertension Study population were genotyped for these polymorphisms. A significant association was identified between the -1889 polymorphism and urinary tetrahydrodeoxycortisol/total cortisol metabolite ratio, indicating reduced 11beta-hydroxylase efficiency. A similar pattern was observed for the -1859 polymorphism, but this did not achieve statistical significance. Functional studies in vitro using luciferase reporter gene constructs show that these polymorphisms significantly alter the transcriptional response of CYP11B1 to stimulation by adrenocorticotropic hormone or forskolin. This study strongly suggests that the impaired 11beta-hydroxylase efficiency associated previously with the CYP11B2 -344 and intron conversion variants is because of linkage with these newly identified polymorphisms in CYP11B1. PMID:17075029

  1. Mimivirus Collagen Is Modified by Bifunctional Lysyl Hydroxylase and Glycosyltransferase Enzyme*

    PubMed Central

    Luther, Kelvin B.; Hülsmeier, Andreas J.; Schegg, Belinda; Deuber, Stefan A.; Raoult, Didier; Hennet, Thierry

    2011-01-01

    Collagens, the most abundant proteins in animals, are modified by hydroxylation of proline and lysine residues and by glycosylation of hydroxylysine. Dedicated prolyl hydroxylase, lysyl hydroxylase, and collagen glycosyltransferase enzymes localized in the endoplasmic reticulum mediate these modifications prior to the formation of the collagen triple helix. Whereas collagen-like proteins have been described in some fungi, bacteria, and viruses, the post-translational machinery modifying collagens has never been described outside of animals. We demonstrate that the L230 open reading frame of the giant virus Acanthamoeba polyphaga mimivirus encodes an enzyme that has distinct lysyl hydroxylase and collagen glycosyltransferase domains. We show that mimivirus L230 is capable of hydroxylating lysine and glycosylating the resulting hydroxylysine residues in a native mimivirus collagen acceptor substrate. Whereas in animals from sponges to humans the transfer of galactose to hydroxylysine in collagen is conserved, the mimivirus L230 enzyme transfers glucose to hydroxylysine, thereby defining a novel type of collagen glycosylation in nature. The presence of hydroxylysine in mimivirus proteins was confirmed by amino acid analysis of mimivirus recovered from A. polyphaga cultures. This work shows for the first time that collagen post-translational modifications are not confined to the domains of life. The utilization of glucose instead of the galactose found throughout animals as well as a bifunctional enzyme rather than two separate enzymes may represent a parallel evolutionary track in collagen biology. These results suggest that giant viruses may have contributed to the evolution of collagen biology. PMID:22045808

  2. 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

  3. Organization and evolution of the rat tyrosine hydroxylase gene

    SciTech Connect

    Brown, E.R.; Coker, G.T. III; O'Malley, K.L.

    1987-08-11

    This report describes the organization of the rat tyrosine hydroxylase (TH) gene and compares its structure with the human phenylalanine hydroxylase gene. Both genes are single copy and contain 13 exons separated by 12 introns. Remarkably, the positions of 10 out 12 intron/exon boundaries are identical for the two genes. These results support the idea that these hydroxylases genes are members of a gene family which has a common evolutionary origin. The authors predict that this ancestral gene would have encoded exons similar to those of TH prior to evolutionary drift to other members of this gene family.

  4. Purification and characterization of the ncgl2923 -encoded 3-hydroxybenzoate 6-hydroxylase from Corynebacterium glutamicum.

    PubMed

    Yang, Yi-Fan; Zhang, Jun-Jie; Wang, Song-He; Zhou, Ning-Yi

    2010-12-01

    Corynebacterium glutamicum ATCC 13032 metabolizes 3-hydroxybenzoate via gentisate. We have now characterized the ncgl2923 -encoded 3-hydroxybenzoate 6-hydroxylase involved in the initial step of 3-hydroxybenzoate catabolism by this strain, a first 3-hydroxybenzoate 6-hydroxylase molecularly and biochemically characterized from a Gram-positive strain. The ncg12923 gene from Corynebacterium glutamicum ATCC 13032 was shown to encode 3-hydroxybenzoate 6-hydroxylase, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Ncgl2923 was expressed with an N-terminal six-His tag and purified to apparent homogeneity by Ni²(+)-nitrilotriacetic acid affinity chromatography. The purified H₆-Ncgl2923 showed a single band at apparent molecular mass of 49 kDa on a sodium dodecyl sulfate polyacrylamide gel electrophoresis and was found to be most likely a trimer as determined by gel filtration chromatography. It had a specific activity of 6.92 ± 0.39 U mg⁻¹ against 3-hydroxybenzoate and with a K(m) value of 53.4 ± 4.7 μM using NADH as a cofactor. The product formed from the 3-hydroxybenzoate hydroxylation catalyzed by H₆-Ncgl2923 was identified by high-performance liquid chromatography as gentisate, a ring-cleavage substrate in the microbial aromatic degradation. The enzyme exhibited a maximum activity at pH 7.5 in phosphate buffer, and adding flavin adenine dinucleotide to a final concentration of 15 μM would enhance the activity by three-fold. Although this enzyme shares no more than 33% identity with any of reported 3-hydroxybenzoate 6-hydroxylases from Gram-negative bacterial strains, there is little difference in subunit sizes and biochemical characteristics between them. PMID:20806251

  5. Selective Inhibition of Collagen Prolyl 4-Hydroxylase in Human Cells

    PubMed Central

    Vasta, James D.; Andersen, Kristen A.; Deck, Kathryn M.; Nizzi, Christopher P.; Eisenstein, Richard S.; Raines, Ronald T.

    2016-01-01

    Collagen is the most abundant protein in animals. Its overproduction is associated with fibrosis and cancer metastasis. The stability of collagen relies on post-translational modifications, the most prevalent being the hydroxylation of collagen strands by collagen prolyl 4-hydroxylases (CP4Hs). Catalysis by CP4Hs enlists an iron cofactor to convert proline residues to 4 hydroxyproline residues, which are essential for the conformational stability of mature collagen. Ethyl 3,4-dihydroxybenzoate (EDHB) is commonly used as a “P4H” inhibitor in cells, but suffers from low potency, poor selectivity, and off-target effects that cause iron deficiency. Dicarboxylates of 2,2′-bipyridine are among the most potent known CP4H inhibitors but suffer from a high affinity for free iron. A screen of biheteroaryl compounds revealed that replacing one pyridyl group with a thiazole moiety retains potency and enhances selectivity. A diester of 2 (5-carboxythiazol-2-yl)pyridine-5-carboxylic acid is bioavailable to human cells and inhibits collagen biosynthesis at concentrations that neither cause general toxicity nor disrupt iron homeostasis. These data anoint a potent and selective probe for CP4H and a potential lead for the development of a new class of antifibrotic and antimetastatic agents. PMID:26535807

  6. Androgen biosynthesis in the stomach: expression of cytochrome P450 17 alpha-hydroxylase/17,20-lyase messenger ribonucleic acid and protein, and metabolism of pregnenolone and progesterone by parietal cells of the rat gastric mucosa.

    PubMed

    Le Goascogne, C; Sananès, N; Eychenne, B; Gouézou, M; Baulieu, E E; Robel, P

    1995-04-01

    Dehydroepiandrosterone (DHEA) and its conjugates persist in the rat brain, for up to 1 month after ablation of both adrenals and gonads. Since DHEA synthesis in brain from pregnenolone (PREG) was excluded, we have considered other tissular sources including the digestive tract. In situ hybridization with specific oligonucleotide probes showed that the parietal cells of the gastric mucosa, contrary to other cell types, strongly expressed P450(17) alpha messenger RNA. Expression of the enzyme in the parietal cells was confirmed by immunocytochemistry with specific antibodies. An intense reaction was observed in the stomach of adult males and of cyclic or pregnant females. Access to food did not influence the intensity of immunostaining. It appeared at postnatal days 16-21, then the number of positive cells increased rapidly and leveled off at adult age. Parietal cells were released by pronase digestion of everted stomachs from adult male and female rats and were purified by density gradient centrifugation on Nycodenz. 5 x 10(4) to 1.6 x 10(6) cells were incubated with either 1 microM 14C-PREG or 14C-progesterone (14C-PROG) at 37 C under 95% O2-5% CO2, for 10-180 min. PREG was converted to 17-OH PREG and to androstenediol, whereas PROG was converted to 17-OH PROG and to testosterone. Only minute amounts of either DHEA or androstenedione, respectively, were detected at any incubation time, indicating their fast conversion to the corresponding 17 beta-hydroxysteroids. 3H-25-OH cholesterol was not metabolized to 3H-PREG, and 14C-PREG was not converted to 14C-PROG, in accordance with negative immunocytochemical results with antibodies to cytochrome P450scc and 3 beta-hydroxysteroid dehydrogenase delta 5-->4-isomerase (3 beta-HSD). In conclusion, the parietal cells, which are known as the source of gastric acid secretion, can synthesize testosterone from PROG and androstenediol from PREG. The physiological relevance of such conversions remains to be established. PMID

  7. Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1.

    PubMed

    Saa, Laura; Jaureguibeitia, Arrate; Largo, Eneko; Llama, María J; Serra, Juan L

    2010-03-01

    Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His(6)PheA1 and His(6)PheA2 were purified and its catalytic activity characterized. His(6)PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His(6)PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His(6)PheA1 and His(6)PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction. PMID:19787347

  8. Tyrosine hydroxylase deficiency with severe clinical course.

    PubMed

    Zafeiriou, D I; Willemsen, M A; Verbeek, M M; Vargiami, E; Ververi, A; Wevers, R

    2009-05-01

    Tyrosine hydroxylase (TH) deficiency is a rare autosomal recessive disorder mapped to chromosome 11p15.5. Its clinical expression varies with presentations as dopa-responsive dystonia (recessive Segawa's disease), dopa-responsive infantile parkinsonism, dopa-responsive spastic paraplegia, progressive infantile encephalopathy or dopa-non-responsive dystonia. We describe a 7-year-old boy with progressive infantile encephalopathy and non-responsiveness to dopamine. The patient demonstrated generalized hypotonia, pyramidal tract dysfunction and temperature instability after the second month of life. Dystonia, tremor and oculogyric crises complicated the clinical picture during the following months. Neurotransmitter analysis in CSF disclosed almost undetectable levels of HVA and MHPG, whereas serum prolactin was profoundly increased. Subsequent molecular analysis revealed homozygosity for a missense mutation (c.707T>C) in the TH gene. l-Dopa therapy in both high and low doses resulted in massive hyperkinesias, while substitution with selegiline exerted only a mild beneficial effect. Today, at the age of 7 years, the patient demonstrates severe developmental retardation with marked trunkal hypotonia, hypokinesia and occasionally dystonic and/or hyperkinetic crises. He is the third Greek patient with TH deficiency to be reported. Since all three patients carry the same pathogenetic mutation, a founder effect is suspected. PMID:19282209

  9. Neonatal mass screening for 21-hydroxylase deficiency

    PubMed Central

    Tajima, Toshihiro; Fukushi, Masaru

    2016-01-01

    Abstract. Congenital adrenal hyperplasia(CAH)due to 21-hydroxylase deficiency (21-OHD) is an inherited autosomal recessive disorder. Its incidence is 1 in 10,000 to 20,000 worldwide. This disease shows phenotypic differences, and it is divided into three forms i.e., the salt wasting (SW), simple virilizing (SV), and nonclassic (NC) forms. The most severe form of SW manifests in the first months of life with life-threatening adrenal insufficiency, leading to death. To prevent death by adrenal insufficiency in neonates with the SW form and wrong gender assignment of 46,XX female patients with SW and SV, neonatal mass screening of 21-OHD is performed in several countries including Japan. However, the positive predictive value (PPV) remains low, especially in preterm infants. To reduce the false positive rate and increase the PPV, liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) as a second-tier test may be useful. In this review, the current knowledge on neonatal mass screening of 21-OHD is summarized. PMID:26865749

  10. Release kinetics of prolyl hydroxylase inhibitors from collagen barrier membranes.

    PubMed

    Hamid, Omar; Pensch, Manuela; Agis, Hermann

    2015-03-01

    Collagen barrier membranes are used in guided tissue regeneration to support healing. This strategy, however, relies on the healing capacity of the tissue. Pharmacological inhibitors of prolyl hydroxylases can support regeneration by enhancing angiogenesis and are therefore a promising tool for periodontology. Here we evaluate the release kinetics of the prolyl hydroxylase inhibitors dimethyloxalylglycine and L-mimosine from collagen barrier membranes. Dimethyloxalylglycine and L-mimosine were lyophilized onto the collagen barrier membranes. The morphology of the collagen barrier membranes was analysed using scanning electron microscopy. The release of prolyl hydroxylase inhibitors was assessed by colorimetric and spectroscopic methods. Their ability to induce a cellular response was assessed in bioassays with gingival and periodontal ligament fibroblasts based on vascular endothelial growth factor production, proliferation, and metabolic activity of the cells. We found that loading of collagen barrier membranes with prolyl hydroxylase inhibitors did not change the overall membrane morphology. Assessment of the release kinetics by direct measurements and based on vascular endothelial growth factor production showed that supernatants obtained from the collagen barrier membranes in the first 6 hours had a sufficient level of prolyl hydroxylase inhibitors to induce vascular endothelial growth factor production. A similar kinetic was found when cell proliferation was assessed. Changes in metabolic activity did not reach the level of significance in the MTT assay. In conclusion, collagen barrier membranes can release prolyl hydroxylase inhibitors thereby increasing the pro-angiogenic capacity of periodontal cells in vitro. These findings provide the basis for preclinical studies to evaluate the regenerative capacity of prolyl hydroxylase inhibitors in periodontology and oral surgery. PMID:25326176

  11. Complete genomic structure of mouse lysyl hydroxylase 2 and lysyl hydroxylase 3/collagen glucosyltransferase.

    PubMed

    Ruotsalainen, H; Vanhatupa, S; Tampio, M; Sipilä, L; Valtavaara, M; Myllylä, R

    2001-04-01

    Lysyl hydroxylase is an enzyme involved in collagen biosynthesis, catalyzing the hydroxylation of lysyl residues as a post-translational event. Three isoforms have been characterized so far (LH1, LH2, LH3). Our recent findings indicate that LH3 possesses, not only lysyl hydroxylase activity, but also galactosylhydroxylysyl glucosyltransferase activity [Heikkinen et al., J. Biol. Chem. 275 (2000) 36158-36163]. We report here the characterization of mouse LH2 (Plod2) and LH3/glucosyltransferase (Plod3) genes. Plod2 spans approximately 50 kb of the genomic DNA, and is organized in 20 exons, one of the exons being alternatively spliced in the RNA processing. Plod3 spans approximately 10 kb of the genomic DNA, and contains 19 exons. Analysis of the 5' flanking region with many transcription start sites reveals the lack of a TATAA box in both genes. Sequence analysis indicated many retroposon-like elements within the Plod3 gene. A comparison was carried out among the LH1, LH2 and LH3 gene structures characterized so far from different species. PMID:11334715

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

    MedlinePlus

    ... nerves ) and difficulties with the muscles that control eye movement. Affected individuals may have a loss of sharp ... look in the same direction (strabismus), rapid involuntary eye movements (nystagmus), or difficulty moving the eyes intentionally (supranuclear ...

  13. A case of 17 alpha-hydroxylase deficiency

    PubMed Central

    Kim, Sung Mee

    2015-01-01

    17α-hydroxylase and 17,20-lyase are enzymes encoded by the CYP17A1 gene and are required for the synthesis of sex steroids and cortisol. In 17α-hydroxylase deficiency, there are low blood levels of estrogens, androgens, and cortisol, and resultant compensatory increases in adrenocorticotrophic hormone that stimulate the production of 11-deoxycorticosterone and corticosterone. In turn, the excessive levels of mineralocorticoids lead to volume expansion and hypertension. Females with 17α-hydroxylase deficiency are characterized by primary amenorrhea and delayed puberty, with accompanying hypertension. Affected males usually have female external genitalia, a blind vagina, and intra-abdominal testes. The treatment of this disorder is centered on glucocorticoid and sex steroid replacement. In patients with 17α-hydroxylase deficiency who are being raised as females, estrogen should be supplemented, while genetically female patients with a uterus should also receive progesterone supplementation. Here, we report a case of a 21-year-old female with 17α-hydroxylase deficiency who had received inadequate treatment for a prolonged period of time. We also include a brief review of the recent literature on this disorder. PMID:26161337

  14. New biaryl-chalcone derivatives of pregnenolone via Suzuki-Miyaura cross-coupling reaction. Synthesis, CYP17 hydroxylase inhibition activity, QSAR, and molecular docking study.

    PubMed

    Al-Masoudi, Najim A; Kadhim, Rawaa A; Abdul-Rida, Nabeel A; Saeed, Bahjat A; Engel, Matthias

    2015-09-01

    A new class of steroids is being synthesized for its ability to prevent intratumoral androgen production by inhibiting the activity of CYP17 hydroxylase enzyme. The scheme involved the synthesis of chalcone derivative of pregnenolone 5 which was further modified to the corresponding biaryl-chalcone pregnenolone analogs 16-25 using Suzuki-Miyaura cross-coupling reaction. The synthesized compounds were tested for activity using human CYP17α hydroxylase expressed in Escherichia coli. Compounds 21 was the most active inhibitor in this series, with IC50 values of 0.61μM and selectivity profile of 88.7% inhibition of hydroxylase enzyme. Molecular docking study of 21 was performed and showed the hydrogen bonds and hydrophobic interaction with the amino acid residues of the active site of CYP17. PMID:26051784

  15. Advances in the molecular characterization of tryptophan hydroxylase.

    PubMed

    Mockus, S M; Vrana, K E

    1998-06-01

    The neurotransmitter serotonin has been implicated in numerous physiological functions and pathophysiological disorders. The hydroxylation of the aromatic amino acid tryptophan is rate-limiting in the synthesis of serotonin. Tryptophan hydroxylase (TPH), as the rate-limiting enzyme, determines the concentrations of serotonin in vivo. Relative serotonin concentrations are clearly important in neural transmission, but serotonin has also been reported to function as a local antioxidant. Identification of the mechanisms regulating TPH activity has been hindered by its low levels in tissues and the instability of the enzyme. Several TPH expression systems have been developed to circumvent these problems. In addition, eukaryotic expressions systems are currently being developed and represent a new avenue of research for identifying TPH regulatory mechanisms. Recombinant DNA technology has enabled the synthesis of TPH deletions, chimeras, and point mutations that have served as tools for identifying structural and functional domains within TPH. Notably, the experiments have proven long-held hypotheses that TPH is organized into N-terminal regulatory and C-terminal catalytic domains, that serine-58 is a site for PKA-mediated phosphorylation, and that a C-terminal leucine zipper is involved in formation of the tetrameric holoenzyme. Several new findings have also emerged regarding regulation of TPH activity by posttranslational phosphorylation, kinetic inhibition, and covalent modification. Inhibition of TPH by L-DOPA may have implications for depression in Parkinson's disease (PD) patients. In addition, TPH inactivation by nitric oxide may be involved in amphetamine-induced toxicity. These regulatory concepts, in conjunction with new systems for studying TPH activity, are the focus of this article. PMID:9770640

  16. Nonflowering plants possess a unique folate-dependent phenylalanine hydroxylase that is localized in chloroplasts.

    PubMed

    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

    2010-10-01

    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

  17. 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

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

    DOE PAGESBeta

    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,more » we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis.« less

  19. Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

    PubMed

    Csernetics, Árpád; Tóth, Eszter; Farkas, Anita; Nagy, Gábor; Bencsik, Ottó; Vágvölgyi, Csaba; Papp, Tamás

    2015-02-01

    Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene. PMID:25504221

  20. Identification and functional characterization of leukotriene B4 20-hydroxylase of human polymorphonuclear leukocytes.

    PubMed Central

    Soberman, R J; Harper, T W; Murphy, R C; Austen, K F

    1985-01-01

    A single reaction product was formed during the incubation of 1.5 microM (5S,12R)-dihydroxy-6,14-cis-8,10-trans-[3H]icosatetraenoic acid (leukotriene B4, LTB4) for 30 min at 37 degrees C in 10 mM potassium phosphate buffer (pH 7.5) with 100 microM NADPH and the 150,000 X g supernatant of sonicated human polymorphonuclear leukocytes (PMN). The reaction product exhibited the same mobility on reversed-phase HPLC (RP-HPLC) and TLC as standard 20-hydroxy-LTB4 (20-OH-LTB4). When the omega-oxidation product of [3H]LTB4 was eluted from a Sep-Pak, resolved by RP-HPLC, and analyzed by GC/MS, its structure was determined to be solely 20-OH-LTB4. The Km of the 20-hydroxylase for [3H]LTB4 at its optimal pH of 7.5 was 0.22 +/- 0.08 microM (mean +/- SD, n = 4) and the Vmax was 48 +/- 11 pmol/min X mg of protein (mean +/- SD, n = 4). When the concentration of [3H]LTB4 was fixed at 1.5 microM, the Km for NADPH was 1.01 +/- 0.59 microM (mean +/- SD, n = 3). The location in the 150,000 X g supernatant of the LTB4 20-hydroxylase distinguishes it from the cytochrome P-450 system of liver, lung, and kidney microsomes and from the NADPH oxidase-cytochrome b-245 system of the human PMN. The LTB4 20-hydroxylase is either a unique cytochrome P-450 or other monooxygenase. PMID:2986111

  1. Regulation of calf renal 25-hydroxyvitamin D-hydroxylase activities by calcium-regulating hormones.

    PubMed

    Engstrom, G W; Goff, J P; Horst, R L; Reinhardt, T A

    1987-11-01

    Parathyroid hormone and 1,25-dihydroxyvitamin D3 had opposite effects on calf renal 25-hydroxyvitamin D3 24-, 23-, and 1 alpha-hydroxylase activities. Parathyroid hormone administration increased renal 25-hydroxyvitamin D3-1 alpha-hydroxylase activity 7-fold while 25-hydroxyvitamin D3-23- and 24-hydroxylase activities were essentially the same as controls. Administration of 1,25-dihydroxyvitamin D3 increased 25-hydroxyvitamin D3-23-hydroxylase and 24-hydroxylase activities 4-fold and decreased 25-hydroxyvitamin D3-1 alpha-hydroxylase activity to undetectable concentrations. Vitamin D deficiency increased 25-hydroxyvitamin D3-1 alpha -hydroxylase activity 13-fold, and 25-hydroxyvitamin D3-23-hydroxylase and 24-hydroxylase activities were undetectable. These results confirm previous reports with regard to control of renal 25-hydroxyvitamin D3-24-hydroxylase and 1 alpha -hydroxylase in other species and represent new findings relative to the control of 25-hydroxyvitamin D3-23-hydroxylase. Plasma P was lower and 1,25-dihydroxyvitamin D3 higher in calves treated with parathyroid hormone, and Ca and 1,25-dihydroxyvitamin D3 were lower in the vitamin D-deficient calves. 1,25-Dihydroxyvitamin D3-treated calves had higher plasma P and lower Mg than controls. Further studies using this calf model should lead to better understanding of Ca-regulating hormones control of vitamin D metabolism. PMID:3693631

  2. 6-Hydroxy-3-Succinoylpyridine Hydroxylase Catalyzes a Central Step of Nicotine Degradation in Agrobacterium tumefaciens S33

    PubMed Central

    Huang, Haiyan; Wang, Shuning

    2014-01-01

    Nicotine is a main alkaloid in tobacco and is also the primary toxic compound in tobacco wastes. It can be degraded by bacteria via either pyridine pathway or pyrrolidine pathway. Previously, a fused pathway of the pyridine pathway and the pyrrolidine pathway was proposed for nicotine degradation by Agrobacterium tumefaciens S33, in which 6-hydroxy-3-succinoylpyridine (HSP) is a key intermediate connecting the two pathways. We report here the purification and properties of an NADH-dependent HSP hydroxylase from A. tumefaciens S33. The 90-kDa homodimeric flavoprotein catalyzed the oxidative decarboxylation of HSP to 2,5-dihydroxypyridine (2,5-DHP) in the presence of NADH and FAD at pH 8.0 at a specific rate of about 18.8±1.85 µmol min−1 mg protein−1. Its gene was identified by searching the N-terminal amino acid residues of the purified protein against the genome draft of the bacterium. It encodes a protein composed of 391 amino acids with 62% identity to HSP hydroxylase (HspB) from Pseudomonas putida S16, which degrades nicotine via the pyrrolidine pathway. Considering the application potential of 2,5-DHP in agriculture and medicine, we developed a route to transform HSP into 2,5-DHP with recombinant HSP hydroxylase and an NADH-regenerating system (formate, NAD+ and formate dehydrogenase), via which around 0.53±0.03 mM 2,5-DHP was produced from 0.76±0.01 mM HSP with a molar conversion as 69.7%. This study presents the biochemical properties of the key enzyme HSP hydroxylase which is involved in the fused nicotine degradation pathway of the pyridine and pyrrolidine pathways and a new green route to biochemically synthesize functionalized 2,5-DHP. PMID:25054198

  3. Structure and Mechanism of a Viral Collagen Prolyl Hydroxylase

    PubMed Central

    2015-01-01

    The Fe(II)- and 2-oxoglutarate (2-OG)-dependent dioxygenases comprise a large and diverse enzyme superfamily the members of which have multiple physiological roles. Despite this diversity, these enzymes share a common chemical mechanism and a core structural fold, a double-stranded β-helix (DSBH), as well as conserved active site residues. The prolyl hydroxylases are members of this large superfamily. Prolyl hydroxylases are involved in collagen biosynthesis and oxygen sensing in mammalian cells. Structural–mechanistic studies with prolyl hydroxylases have broader implications for understanding mechanisms in the Fe(II)- and 2-OG-dependent dioxygenase superfamily. Here, we describe crystal structures of an N-terminally truncated viral collagen prolyl hydroxylase (vCPH). The crystal structure shows that vCPH contains the conserved DSBH motif and iron binding active site residues of 2-OG oxygenases. Molecular dynamics simulations are used to delineate structural changes in vCPH upon binding its substrate. Kinetic investigations are used to report on reaction cycle intermediates and compare them to the closest homologues of vCPH. The study highlights the utility of vCPH as a model enzyme for broader mechanistic analysis of Fe(II)- and 2-OG-dependent dioxygenases, including those of biomedical interest. PMID:26368022

  4. Structural Effects on Arthrobacter Methylene Hydroxylase Activity1

    PubMed Central

    Hayasaka, Steven; Klein, D. A.

    1971-01-01

    Arthrobacter 4-44-2 (ATCC 25581), capable of subterminal oxidation of n-hexadecane to 2-, 3-, and 4-alcoholic and ketonic products, was examined for the ability of this methylene hydroxylase capability to be induced and repressed and for structural relationships influencing methylene function oxidation. Induction was best carried out by use of n-alkanes from 10 to 16 carbons in length and was especially strong with methylcyclohexane among cyclic compounds tested. Induction was not observed with several related alcohols, 1-unsaturated compounds, or methoxy and ethoxy compounds tested. After induction, n-alkanes 14 and 16 carbons in length were transformed to the corresponding internal oxidation products; however, no activity was observed with even-carbon alkanes of shorter chain length. Hexadecene-1 and all alcohols tested, including cyclododecanol, were transformed to corresponding ketonic or aldehydic products. Cyclic compounds tested, including cyclododecane, were not oxidized by induced cells, suggesting that a methyl group plays a role in orientation of the substrate for the methylene hydroxylation but that the methyl function was not as critical after completion of the hydroxylation step regardless of structural configuration. Acetate strongly repressed induction of n-hexadecane methylene hydroxylase activity. Inducibility of methylene hydroxylase activity was confirmed by use of cell-free systems with methylcyclohexane as an inducer. A stimulation of methylene hydroxylase activity by addition of reduced pyridine nucleotides and ferrous ion was indicated. PMID:5139534

  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. Serum Dopamine Beta Hydroxylase and Maltreatment in Psychiatrically Hospitalized Boys.

    ERIC Educational Resources Information Center

    Galvin, Matthew; And Others

    1995-01-01

    Males (ages 7 to 17) in a psychiatric hospital were studied while off psychoactive medication to determine how serum dopamine beta hydroxylase (DBH) activity varies with childhood maltreatment experiences. Lowest DBH levels were found in boys maltreated before 72 months of age or with the principal diagnosis of conduct disorder solitary aggressive…

  7. Recommendations for the nutrition management of phenylalanine hydroxylase deficiency

    PubMed Central

    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.

    2014-01-01

    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. Genet Med 16 2, 121–131. PMID:24385075

  8. 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. PMID:24385075

  9. Long-term effect of RU24722 on tyrosine hydroxylase in the rat locus coeruleus: differential effects of two enantiomeric forms.

    PubMed

    Bourde, O; Schmitt, P; Robert, F; Richard, F; Carbonnele, A C; Thal, C; Pujol, J F

    1993-12-01

    RU24722, as a racemic mixture, has been found to act on neuronal activity and the long-term regulation of tyrosine hydroxylase in the locus coeruleus of the rat. In this study, the effects of two enantiomeric derivatives of RU24722 (3 alpha and 16 alpha forms), as compared to the racemic form itself, are studied. The short-term effect was estimated 20 min after treatment by measuring variations in 3,4-dihydroxyphenylacetic acid content in the locus coeruleus. The long-term effect was determined by evaluating tyrosine hydroxylase protein concentration in the locus coeruleus 3 days after a single injection. Comparison of actions of both enantiomers showed that the 16 alpha form was 3-fold more potent in eliciting tyrosine hydroxylase protein elevations at three days, whereas the 3 alpha isomer increased 3,4-dihydroxyphenylacetic acid content 2-fold more in the short-term. These results seem to show that the 16 alpha configuration is crucial for the long term regulation of tyrosine hydroxylase protein elicited by RU24722 within the locus coeruleus. PMID:7904207

  10. Structural Characterization of the Catalytic Sites of Mononuclear Nonheme Fe Hydroxylases Using ²H-ESEEM.

    PubMed

    McCracken, John

    2015-01-01

    Aromatic amino acid hydroxylases are members of a larger group of enzymes that use a mononuclear nonheme Fe center to catalyze a variety of thermodynamically challenging reactions in which O2 is used in the oxidative transformation of substrates. The hydroxylase enzymes are catalytically active in the ferrous oxidation state and are high-spin. To render the catalytic site EPR-active, we have used nitric oxide (NO) as a surrogate for substrate O2 to form an S=3/2 paramagnetic center. While the continuous-wave (cw)-EPR spectra of NO-enzyme adducts are rather generic, they provide electron spin echo envelope modulation (ESEEM) data that are rich with structural information derived from ligand hyperfine couplings. This chapter will focus on (2)H-ESEEM spectroscopy, an approach that we have taken for assigning these spectra and harvesting the unique information on Fe(II) coordination chemistry that they provide. While these spectroscopic measurements are routine, an emphasis will be placed on the analysis of cw-EPR and (2)H-ESEEM data using an unconstrained nonlinear optimization approach. These analysis methods are based on simple custom "scripts" that run in the MATLAB environment and that use EasySpin, a public-domain EPR simulation package, as their calculation engine. The examples provided here use a strategy that can be adapted for the treatment of most EPR measurements. PMID:26478489

  11. Characterization of a Novel Phenol Hydroxylase in Indoles Biotranformation from a Strain Arthrobacter sp. W1

    PubMed Central

    Li, Xinliang; Zhang, Xuwang; Zhou, Jiti

    2012-01-01

    Background Indigoids, as popular dyes, can be produced by microbial strains or enzymes catalysis. However, the new valuable products with their transformation mechanisms, especially inter-conversion among the intermediates and products have not been clearly identified yet. Therefore, it is necessary to investigate novel microbial catalytic processes for indigoids production systematically. Findings A phenol hydroxylase gene cluster (4,606 bp) from Arthrobacter sp. W1 (PHw1) was obtained. This cluster contains six components in the order of KLMNOP, which exhibit relatively low sequence identities (37–72%) with known genes. It was suggested that indole and all the tested indole derivatives except for 3-methylindole were transformed to various substituted indigoid pigments, and the predominant color products derived from indoles were identified by spectrum analysis. One new purple product from indole, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one, should be proposed as the dimerization of isatin and 7-hydroxylindole at the C-2 and C-6 positions. Tunnel entrance and docking studies were used to predict the important amino acids for indoles biotransformation, which were further proved by site-directed mutagenesis. Conclusions/Significance We showed that the phenol hydroxylase from genus Arthrobacter could transform indoles to indigoids with new chemical compounds being produced. Our work should show high insights into understanding the mechanism of indigoids bio-production. PMID:23028517

  12. 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

  13. Methylfolate modulates potassium evoked neuro-secretion: evidence for a role at the pteridine cofactor level of tyrosine 3-hydroxylase.

    PubMed

    Lucock, M D; Green, M; Levene, M I

    1995-06-01

    We have previously shown that 5-methyltetrahydrofolate influences neuro-secretion. The present study more precisely characterises the processes involved and considers one probable site of action. Focusing on the tyrosine-noradrenalin axis in cerebellum we showed 5-methyltetrahydrofolate causes a significant reduction in the apparent K+ evoked secretion of noradrenalin to only 12.9% of control release. Evidence supports the idea that this could actually be due to increased synthesis leading to; depletion of reserves, possibly through leakage, exocytotic inhibition via activation of presynaptic receptors or end product inhibition by noradrenalin at the pteridine cofactor level of tyrosine hydroxylase: a) concomitant decreased measurement of perfusate and intracellular tyrosine with released noradrenalin following 5-methyltetrahydrofolate treatment supports the idea of increased transmitter turn over; b) kinetic studies indicate that at saturating concentrations of tyrosine and in the presence of an inhibitor of L-DOPA decarboxylase, 5-methyltetrahydrofolate partially duplicates the rate limiting behaviour of a synthetic pteridine cofactor--DL,2-amino-4-hydroxy-6,7,dimethyltetrahydropteridine. We debate whether, in vivo, CSF 5-methyltetrahydrofolate might interact at the tetrahydrobiopterin cofactor level of tyrosine hydroxylase and other aromatic amino-acid hydroxylases. PMID:7566370

  14. 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

  15. Haplotypes of the steroid 21-hydroxylase gene region encoding mild steroid 21-hydroxylase deficiency.

    PubMed Central

    Haglund-Stengler, B; Martin Ritzén, E; Gustafsson, J; Luthman, H

    1991-01-01

    Haplotypes of the complement 4 (C4) and steroid 21-hydroxylase [21-OHase; steroid hydrogen-donor: oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10] repeated gene complex were studied in nine families with at least one member affected with a mild form of 21-OHase deficiency. DNA probes from different parts of the repeated C4/21-OHase unit were used to follow the segregation of hybridization patterns in the families. Ten structurally distinct haplotypes of the C4/21-OHase gene region were identified, and the encoded phenotype was assigned to 34 of the 36 C4/21-OHase haplotypes. Four structurally different haplotypes with three C4/21-OHase repeat units were found. Eight of the nine haplotypes found with triplications of the C4/21-OHase repeat unit encoded the mild form of 21-OHase deficiency, whereas one particular triplicated haplotype encoded a severe form of the disease. In one case the mild form of 21-OHase deficiency was encoded by a haplotype with a single C4/21-OHase repeat unit. Mild 21-OHase deficiency was predicted in a patient by the presence of a triplicated haplotype. The finding of deranged 21-OHase genes on all triplicated C4/21-OHase haplotypes indicate that most of these common haplotypes carry mutated 21-OHase genes, and thus may cause functional polymorphism of general importance in the population. PMID:1924294

  16. Biofilm Formation by Neisseria gonorrhoeae

    PubMed Central

    Greiner, L. L.; Edwards, J. L.; Shao, J.; Rabinak, C.; Entz, D.; Apicella, M. A.

    2005-01-01

    Studies were performed in continuous-flow chambers to determine whether Neisseria gonorrhoeae could form a biofilm. Under these growth conditions, N. gonorrhoeae formed a biofilm with or without the addition of 10 μM sodium nitrite to the perfusion medium. Microscopic analysis of a 4-day growth of N. gonorrhoeae strain 1291 revealed evidence of a biofilm with organisms embedded in matrix, which was interlaced with water channels. N. gonorrhoeae strains MS11 and FA1090 were found to also form biofilms under the same growth conditions. Cryofield emission scanning electron microscopy and transmission electron microscopy confirmed that organisms were embedded in a continuous matrix with membranous structures spanning the biofilm. These studies also demonstrated that N. gonorrhoeae has the capability to form a matrix in the presence and absence of CMP-N-acetylneuraminic acid (CMP-Neu5Ac). Studies with monoclonal antibody 6B4 and the lectins soy bean agglutinin and Maackia amurensis indicated that the predominate terminal sugars in the biofilm matrix formed a lactosamine when the biofilm was grown in the absence of CMP-Neu5Ac and sialyllactosamine in the presence of CMP-Neu5Ac. N. gonorrhoeae strain 1291 formed a biofilm on primary urethral epithelial cells and cervical cells in culture without loss of viability of the epithelial cell layer. Our studies demonstrated that N. gonorrhoeae can form biofilms in continuous-flow chambers and on living cells. Studies of these biofilms may have implications for understanding asymptomatic gonococcal infection. PMID:15784536

  17. Amino-terminal analysis of tryptophan hydroxylase: protein kinase phosphorylation occurs at serine-58.

    PubMed

    Kumer, S C; Mockus, S M; Rucker, P J; Vrana, K E

    1997-10-01

    Tryptophan hydroxylase (TPH) catalyzes the rate-limiting and committed step in serotonin biosynthesis. Within this enzyme, two distinct domains have been hypothesized to exist, an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. In the present experiments, the functional boundary between the putative domains was defined using deletion mutagenesis. A full-length cDNA clone for rabbit TPH was engineered for expression in bacteria. Five amino-terminal deletions were constructed using PCR, i.e., Ndelta50, Ndelta60, Ndelta90, Ndelta106, and Ndelta116 (referring to the number of amino acids deleted from the amino terminus). Enzymatic activity was determined for each mutant after expression in bacteria. Whereas deletion of 116 amino acids (Ndelta116) abolished enzyme activity, all of the other amino-terminal deletions exhibited increased specific activity relative to the recombinant wild-type TPH. The ability of the cyclic AMP-dependent protein kinase (PKA) to phosphorylate members of the deletion series was also examined. Deletion of the first 60 amino-terminal residues abolished the ability of the enzyme to serve as a substrate for PKA, yet the native and Ndelta50 enzymes were phosphorylated. Moreover, a serine-58 point mutant (S58A) was not phosphorylated by PKA. In conclusion, the first 106 amino acids comprise a regulatory domain that is phosphorylated by PKA at serine-58. In addition, the boundary between regulatory and catalytic domains is analogous to the domain structure observed for the related enzyme tyrosine hydroxylase. PMID:9326303

  18. 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.

  19. 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

  20. Comprehensive analytical strategy for mutation screening in 21-hydroxylase deficiency.

    PubMed

    Krone, N; Roscher, A A; Schwarz, H P; Braun, A

    1998-10-01

    Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease with a wide range of clinical manifestations. It is most often caused by deficiency of steroid 21-hydroxylase, reflecting any of a wide range of mutations in the 21-hydroxylase (CYP21) gene. A major challenge in molecular diagnostics of CAH is the high homology between the CYP21 gene and the CYP21P pseudogene and the phenomenon of apparent gene conversion, which inactivates the functional gene. In this study we devised an improved stepwise diagnostic procedure involving nonradioactive Southern blotting and direct DNA sequencing. This strategy led to a successful elucidation of the molecular cause of the disease in 181 out of 182 unrelated alleles in a total of 91 clinically and biochemically characterized patients. We were able to identify all classical known disease-causing mutations of the 21-hydroxylase gene and a novel nonsense mutation (bp 670, A-->C, Y97X). Our method also allows the reliable, secure diagnosis of the heterozygous configuration and may therefore be used for pre-, peri-, and postnatal diagnosis of CAH, even when informative data of the index patient are lacking. Furthermore, it can be used to confirm the diagnosis of CAH in newborns detected in 17-hydroxyprogesterone screening programs. PMID:9761237

  1. Direct Binding of GTP Cyclohydrolase and Tyrosine Hydroxylase

    PubMed Central

    Bowling, Kevin M.; Huang, Zhinong; Xu, Dong; Ferdousy, Faiza; Funderburk, Christopher D.; Karnik, Nirmala; Neckameyer, Wendi; O'Donnell, Janis M.

    2008-01-01

    The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from end-product feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. Vmax is elevated, with no change in Km. These results demonstrate that these enzymes engage in mutual positive regulation. PMID:18801743

  2. Hypoxia-inducible factor asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the beta-carbon of asparagine-803.

    PubMed Central

    McNeill, Luke A; Hewitson, Kirsty S; Claridge, Timothy D; Seibel, Jürgen F; Horsfall, Louise E; Schofield, Christopher J

    2002-01-01

    Asparagine-803 in the C-terminal transactivation domain of human hypoxia-inducible factor (HIF)-1 alpha-subunit is hydroxylated by factor inhibiting HIF-1 (FIH-1) under normoxic conditions causing abrogation of the HIF-1alpha/p300 interaction. NMR and other analyses of a hydroxylated HIF fragment produced in vitro demonstrate that hydroxylation occurs at the beta-carbon of Asn-803 and imply production of the threo -isomer, in contrast with other known aspartic acid/asparagine hydroxylases that produce the erythro -isomer. PMID:12215170

  3. 20-HETE and CYP4A2 ω-hydroxylase contribute to the elevated blood pressure in hyperandrogenemic female rats.

    PubMed

    Dalmasso, Carolina; Maranon, Rodrigo; Patil, Chetan; Moulana, Mohadetheh; Romero, Damian G; Reckelhoff, Jane F

    2016-07-01

    In male rats, androgen supplements increase 20-hydroxyeicosatetraenoic acid (20-HETE) via cytochrome P-450 (CYP)4A ω-hydroxylase and cause an increase in blood pressure (BP). In the present study, we determined the roles of 20-HETE and CYP4A2 on the elevated BP in hyperandrogenemic female rats. Chronic dihydrotestosterone (DHT) increased mean arterial pressure (MAP) in female Sprague-Dawley rats (96 ± 2 vs. 108 ± 2 mmHg, P < 0.05) and was associated with increased renal microvascular CYP4A2 mRNA expression (15-fold), endogenous renal 20-HETE (5-fold), and ω-hydroxylase activity (3-fold). Chronic DHT also increased MAP in low salt-fed Dahl salt-resistant female rats (81 ± 4 vs. 95 ± 1 mmHg, P < 0.05) but had no effect on MAP in Dahl salt-sensitive female rats (154 ± 3 vs. 153 ± 3 mmHg), which are known to be 20-HETE deficient. To test the role of CYP4A2, female CYP4A2(-/-) and SS.5(Bn) (wild type) rats were treated with DHT. DHT increased MAP in SS.5(Bn) female rats (104 ± 1 vs. 128 ± 1 mmHg, P < 0.05) but had no effect in CYP4A2(-/-) female rats (118 ± 1 vs. 120 ± 1 mmHg). Renal microvascular 20-HETE was reduced in control CYP4A2(-/-) female rats and was increased with DHT in SS.5(Bn) female rats (6-fold) but not CYP4A2(-/-) female rats. ω-Hydroxylase activity was 40% lower in control CYP4A2(-/-) female rats than in SS.5(Bn) female rats, and DHT decreased ω-hydroxylase activity in SS.5(Bn) female rats (by 50%) but significantly increased ω-hydroxylase activity in CYP4A2(-/-) female rats (3-fold). These data suggest that 20-HETE via CYP4A2 contributes to the elevation in BP in hyperandrogenemic female rats. The data also suggest that 20-HETE synthesis inhibition may be effective in treating the elevated BP in women with hyperandrogenemia, such as women with polycystic ovary syndrome. PMID:27194719

  4. 25-Hydroxyvitamin D3-23-hydroxylase, a renal enzyme in several animal species.

    PubMed

    Engstrom, G W; Reinhardt, T A; Horst, R L

    1986-10-01

    The presence of 23,25-dihydroxyvitamin D3 has been demonstrated in vivo and in vitro by a number of laboratories. In order to evaluate the significance of 23-hydroxylation, renal 23-hydroxylase activity was compared to renal 24-hydroxylase activity in several species before and after treatment with 1,25-dihydroxyvitamin D3. The maximum activity of 23-hydroxylase varied widely among species. Treatment of animals with 1,25-dihydroxyvitamin D3 24 h and again 2 h prior to assay of renal tissue resulted in a 1.7- to 5.2-fold increase in 23-hydroxylase activity and a 3.8- to 20.6-fold increase in 24-hydroxylase activity compared to untreated controls. Maximum activity for both 23- and 24-hydroxylase required the enzyme substrate, 25-hydroxyvitamin D3, and an optimum concentration (30 mM) of an oxidizable substrate such as L-malate to supply the reducing equivalents of NADPH needed. Addition of 10 mumol of magnesium chloride resulted in 19 and 24% increases in activity for 23- and 24-hydroxylase, respectively. L-Malate supported the hydroxylation reactions better than succinate, alpha-ketoglutarate, or pyruvate. The apparent Km of calf renal 23-hydroxylase was 5.7 +/- 1.0 microM and of 24-hydroxylase, 2.0 +/- 0.2 microM. Apparent Km's for 23-hydroxylase varied from a low of 2.7 +/- 0.3 microM in the sheep to a high of 19.1 +/- 0.5 microM in the chick, and for 24-hydroxylase from 0.5 +/- 0.1 microM for the chick to 2.0 +/- 0.2 microM for the calf. Maximum velocity values (Vmax) ranged from 40 +/- 9 pmol/min/g for 23-hydroxylase in the chick to 396 +/- 92 in the calf, and for 24-hydroxylase from 108 +/- 89 pmol/min/g in the chick to 851 +/- 88 in the pig. These results help explain the in vivo metabolite concentrations and the predominance of the C(24)- over C(23)-oxidation pathways. Renal 23-hydroxylase was similar to 24-hydroxylase in that it was inhibited by carbon monoxide (63%), cyanide (51%), and antimycin (67%), required molecular oxygen, and functioned best at

  5. Human cholesterol 7alpha-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype.

    PubMed

    Pullinger, Clive R; Eng, Celeste; Salen, Gerald; Shefer, Sarah; Batta, Ashok K; Erickson, Sandra K; Verhagen, Andrea; Rivera, Christopher R; Mulvihill, Sean J; Malloy, Mary J; Kane, John P

    2002-07-01

    Bile acid synthesis plays a critical role in the maintenance of mammalian cholesterol homeostasis. The CYP7A1 gene encodes the enzyme cholesterol 7alpha-hydroxylase, which catalyzes the initial step in cholesterol catabolism and bile acid synthesis. We report here a new metabolic disorder presenting with hyperlipidemia caused by a homozygous deletion mutation in CYP7A1. The mutation leads to a frameshift (L413fsX414) that results in loss of the active site and enzyme function. High levels of LDL cholesterol were seen in three homozygous subjects. Analysis of a liver biopsy and stool from one of these subjects revealed double the normal hepatic cholesterol content, a markedly deficient rate of bile acid excretion, and evidence for upregulation of the alternative bile acid pathway. Two male subjects studied had hypertriglyceridemia and premature gallstone disease, and their LDL cholesterol levels were noticeably resistant to 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. One subject also had premature coronary and peripheral vascular disease. Study of the kindred, which is of English and Celtic background, revealed that individuals heterozygous for the mutation are also hyperlipidemic, indicating that this is a codominant disorder. PMID:12093894

  6. Decrease in the reactivity of locus coeruleus neurons to hypotension after an increase in their tyrosine hydroxylase content: a subregional in vivo voltammetry study in the rat.

    PubMed

    Vachette, C; Bourde, O; Gillon, J Y; Pujol, J F; Renaud, B

    1993-03-01

    The aim of the present work was to determine if noradrenergic neurons of the anterior and the posterior subregions of the locus coeruleus exhibit a difference in reactivity in response to sodium nitroprusside-induced arterial hypotension, and if the pharmacological induction of tyrosine hydroxylase by RU24722 modifies the reactivity of locus coeruleus neurons to this hypotensive stimulus. Previous findings have demonstrated that administration of RU24722 increases the concentration of tyrosine hydroxylase in the rat locus coeruleus by two different mechanisms in the anterior and in the posterior locus coeruleus subregions. The goal of the present study was to measure in vivo the changes in catecholaminergic metabolism in the locus coeruleus after treatment with RU24722 using differential normal pulse voltammetry (DNPV). In vehicle-treated rats, arterial hypotension increased catecholaminergic metabolism with the same pattern in the two locus coeruleus subregions. However, the changes in the magnitude of the catechol oxidation current throughout the recording period were significantly smaller in the posterior subregion (P < 0.001). In the RU24722-pretreated rats, there was a 39% increase in tyrosine hydroxylase and dihydroxyphenylacetic acid in the locus coeruleus. The functional reactivity to hypotension measured by DNPV was significantly decreased (P < 0.001) in both the anterior and posterior locus coeruleus subregions with RU24722 treatment. Therefore, this study suggests that the response of locus coeruleus cells to a hypotensive stimulus depends upon the intracellular tyrosine hydroxylase concentration both in the basal condition and during pharmacological induction of tyrosine hydroxylase gene expression. PMID:7903186

  7. Cholesterol 7α-hydroxylase-deficient mice are protected from high-fat/high-cholesterol diet-induced metabolic disorders.

    PubMed

    Ferrell, Jessica M; Boehme, Shannon; Li, Feng; Chiang, John Y L

    2016-07-01

    Cholesterol 7α-hydroxylase (CYP7A1) is the first and rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver. In addition to absorption and digestion of nutrients, bile acids play a critical role in the regulation of lipid, glucose, and energy homeostasis. We have backcrossed Cyp7a1(-/-) mice in a mixed B6/129Sv genetic background to C57BL/6J mice to generate Cyp7a1(-/-) mice in a near-pure C57BL/6J background. These mice survive well and have normal growth and a bile acid pool size ∼60% of WT mice. The expression of the genes in the alternative bile acid synthesis pathway are upregulated, resulting in a more hydrophilic bile acid composition with reduced cholic acid (CA). Surprisingly, Cyp7a1(-/-) mice have improved glucose sensitivity with reduced liver triglycerides and fecal bile acid excretion, but increased fecal fatty acid excretion and respiratory exchange ratio (RER) when fed a high-fat/high-cholesterol diet. Supplementing chow and Western diets with CA restored bile acid composition, reversed the glucose tolerant phenotype, and reduced the RER. Our current study points to a critical role of bile acid composition, rather than bile acid pool size, in regulation of glucose, lipid, and energy metabolism to improve glucose and insulin tolerance, maintain metabolic homeostasis, and prevent high-fat diet-induced metabolic disorders. PMID:27146480

  8. A heme peroxidase with a functional role as an L-tyrosine hydroxylase in the biosynthesis of anthramycin.

    PubMed

    Connor, Katherine L; Colabroy, Keri L; Gerratana, Barbara

    2011-10-18

    We report the first characterization and classification of Orf13 (S. refuineus) as a heme-dependent peroxidase catalyzing the ortho-hydroxylation of L-tyrosine to L-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis, and maximal L-tyrosine conversion to L-DOPA is observed in the presence of hydrogen peroxide. Preincubation of L-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for L-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady-state kinetic analysis of L-tyrosine hydroxylation revealed similar catalytic efficiency for both L-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO(g) UV-vis spectrum of Orf13 and electron paramagnetic resonance of ferric heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for L-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of L-tyrosine to L-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases. PMID:21919439

  9. 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.

  10. Bovine renal mitochondrial vitamin D3 hydroxylases: regulation of in vitro activities by inhibitors and antioxidants.

    PubMed

    Crivello, J F

    1985-08-01

    The regulation of bovine renal 1 alpha- and 24-hydroxylase activities was examined in primary bovine proximal tubule cell cultures. Maximal 1 alpha- and 24-hydroxylase activities in primary bovine proximal tubule cultures ranged from 1.5-1.8 and 2.0-2.7 pmol/min X 10(6) cells, respectively. The apparent Km was 795 nM for 1 alpha-hydroxylase activity and 1130 nM for 24-hydroxylase activity. 1 alpha- and 24-hydroxylase activities decreased in primary culture after cell plating. Activities decreased both as a function of cell number and as a function of the culture dish. 1 alpha-Hydroxylase activity decayed with a t1/2 of 37 h, while 24-hydroxylase activity decayed with a t1/2 of 45 h. Decreasing cell densities, at which cells were plated, increased the t1/2 for the decay of both activities [t1/2 = 21 h at 5,000 cells/cm vs. t1/2 = 37 h at 25,000 cells/cm for 1 alpha-hydroxylase (P greater than 0.001); t1/2 = 33 h at 5,000 cells/cm vs. t1/2 = 45 h at 25,000 cells/cm for 24-hydroxylase, (P greater than 0.0001)]. Direct addition of 0.25 mM metyrapone inhibited 1 alpha-hydroxylase activity by 33% and 24-hydroxylase activity by 51%. Long term incubation of cell cultures with 0.25 mM metyrapone resulted in a slowing in the loss of both hydroxylase activities, but did not stop the decay. 1 alpha-Hydroxylase activity in 4-day metyrapone-treated cultures was 35% higher than in 4-day untreated cultures. 24-Hydroxylase activity was increased 42% in treated cultures vs. that in untreated cell cultures. Both 1 alpha- and 24-hydroxylase activities were inhibited by direct addition of antioxidants. 1 alpha-Hydroxylase activity was directly inhibited 74% by the addition of 0.1 mM butylated hydroxyanisole (BHA), 69% by the addition of 0.1 mM butylated hydroxytoluene (BHT), and 56% by the addition of 0.05 mM benzyl sulfoxide (BS). 24-Hydroxylase activity was also directly inhibited 72% by 0.1 mM BHA, 55% by 0.1 mM BHT, and 73% 0.05 mM BS. There was no significant difference between

  11. 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

  12. Effect of squalene synthase inhibition on the expression of hepatic cholesterol biosynthetic enzymes, LDL receptor, and cholesterol 7 alpha hydroxylase.

    PubMed

    Ness, G C; Zhao, Z; Keller, R K

    1994-06-01

    Squalene synthase catalyzes the committed step in the biosynthesis of sterols. Treating rats with zaragozic acid A, a potent inhibitor of squalene synthase, caused marked increases in hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, squalene synthase, and LDL receptor mRNA levels. The increase in HMG-CoA reductase mRNA fully accounted for the increases seen in enzyme protein and activity. Farnesyl pyrophosphate synthase mRNA and activity were only slightly increased by zaragozic acid A, while cholesterol 7 alpha hydroxylase mRNA levels were decreased substantially. When rats were pretreated with zaragozic acid A, there was no change in mRNA levels for the cholesterol biosynthetic enzymes or cholesterol 7 alpha hydroxylase upon subsequent treatment with mevalonolactone. Under these same conditions, the enzymatic activity of HMG-CoA reductase was also unaffected. Mevalonolactone treatment reduced the zaragozic acid A-mediated increase in hepatic LDL receptor mRNA levels. Feeding cholesterol eliminated the zaragozic acid A-induced increase in HMG-CoA reductase mRNA levels. These results suggest that inhibition of squalene synthase decreases the level of a squalene-derived regulatory product, resulting in altered amounts of several mRNAs and coordinate increases in HMG-CoA reductase mRNA, protein, and activity. The increase in HMG-CoA reductase gene expression was closely related to the degree of inhibition of cholesterol synthesis caused by zaragozic acid A. PMID:7911291

  13. Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent N-Hydroxylase

    PubMed Central

    2015-01-01

    The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes. PMID:25184411

  14. Crystallographic evidence of drastic conformational changes in the active site of a flavin-dependent N-hydroxylase.

    PubMed

    Setser, Jeremy W; Heemstra, John R; Walsh, Christopher T; Drennan, Catherine L

    2014-09-30

    The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N(5)-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes. PMID:25184411

  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. The cytochrome P450 CYP86A22 is a fatty acyl-CoA omega-hydroxylase essential for Estolide synthesis in the stigma of Petunia hybrida.

    PubMed

    Han, Jixiang; Clement, Joel M; Li, Jia; King, Andrew; Ng, Shirley; Jaworski, Jan G

    2010-02-01

    The stigmatic estolide is a lipid-based polyester constituting the major component of exudate in solanaceous plants. Although the exudate is believed to play important roles in the pollination process, the biosynthetic pathway of stigmatic estolide, including genes encoding the key enzymes, remains unknown. Here we report the cloning and characterization of the cytochrome P450 gene CYP86A22, which encodes a fatty acyl-CoA omega-hydroxylase involved in estolide biosynthesis in the stigma of Petunia hybrida. A CYP86A22 cDNA was isolated from a developing stigma cDNA library, and the corresponding gene was shown to express predominantly in the developing stigma. Among six P450 genes isolated from this library, only CYP86A22 was implicated in omega-hydroxylation following RNA interference (RNAi)-mediated suppression. Unlike wild-type plants in which omega-hydroxy fatty acids (mainly in the form of 18-hydroxy oleic acid and 18-hydroxy linoleic acid) compose 96% of total stigma fatty acids, the omega-hydroxy fatty acids were essentially absent in the stigmas from 18 of 46 CYP86A22-RNAi transgenic plants and had varying levels of suppression in the remaining 28 plants. Furthermore, lipids in the 18 CYP86A22-RNAi stigmas were predominantly triacylglycerols and diacylglycerols instead of the estolides, which characterize the wild-type stigma. Analyses of recombinant CYP86A22 conclusively demonstrated that this P450 is a omega-hydroxylase with a substrate preference for both saturated and unsaturated acyl-CoAs rather than free fatty acids. We conclude that the cytochrome P450 enzyme CYP86A22 is the key fatty acyl-CoA omega-hydroxylase essential for the production of omega-hydroxy fatty acids and the biosynthesis of triacylglycerol-/diacylglycerol-based estolide polyesters in the petunia stigma. PMID:19940120

  17. 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.

  18. Significance of Plasma Dopamine β-Hydroxylase Activity as an Index of Sympathetic Neuronal Function

    PubMed Central

    Reid, John L.; Kopin, Irwin J.

    1974-01-01

    Plasma norepinephrine and dopamine β-hydroxylase (EC 1.14.17.1) activity were measured in rats. Adrenergic neuron blockade with bretylium for 4 hr and ganglion blockade with chlorisondamine for 72 hr lowered plasma norepinephrine. Neither treatment altered plasma dopamine β-hydroxylase activity. Phenoxybenzamine for up to 48 hr markedly raised plasma norepinephrine and transiently lowered plasma dopamine β-hydroxylase at 24 hr. Prolonged pharmacological modification of sympathetic nervous activity and plasma norepinephrine were not attended by parallel changes in circulating dopamine β-hydroxylase activity. Plasma dopamine β-hydroxylase activity does not appear to be a sensitive index of prolonged alterations in sympathetic neural activity. Norepinephrine in plasma, however, appears to reflect sensitively and accurately the rate of release of the neurotransmitter. PMID:4530990

  19. Crystal structures of the methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): implications for substrate gating and component interactions.

    PubMed

    Rosenzweig, A C; Brandstetter, H; Whittington, D A; Nordlund, P; Lippard, S J; Frederick, C A

    1997-10-01

    The crystal structure of the nonheme iron-containing hydroxylase component of methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) has been solved in two crystal forms, one of which was refined to 1.7 A resolution. The enzyme is composed of two copies each of three subunits (alpha 2 beta 2 gamma 2), and all three subunits are almost completely alpha-helical, with the exception of two beta hairpin structures in the alpha subunit. The active site of each alpha subunit contains one dinuclear iron center, housed in a four-helix bundle. The two iron atoms are octahedrally coordinated by 2 histidine and 4 glutamic acid residues as well as by a bridging hydroxide ion, a terminal water molecule, and at 4 degrees C, a bridging acetate ion, which is replaced at -160 degrees C with a bridging water molecule. Comparison of the results for two crystal forms demonstrates overall conservation and relative orientation of the domain structures. The most prominent structural differences identified between the two crystal forms is in an altered side chain conformation for Leu 110 at the active site cavity. We suggest that this residue serves as one component of a hydrophobic gate controlling access of substrates to and products from the active site. The leucine gate may be responsible for the effect of the B protein component on the reactivity of the reduced hydroxylase with dioxygen. A potential reductase binding site has been assigned based on an analysis of crystal packing in the two forms and corroborated by inhibition studies with a synthetic peptide corresponding to the proposed docking position. PMID:9329079

  20. Prolyl Hydroxylase PHD3 Activates Oxygen-dependent Protein Aggregation

    PubMed Central

    Rantanen, Krista; Pursiheimo, Juha; Högel, Heidi; Himanen, Virpi; Metzen, Eric

    2008-01-01

    The HIF prolyl hydroxylases (PHDs/EGLNs) are central regulators of the molecular responses to oxygen availability. One isoform, PHD3, is expressed in response to hypoxia and causes apoptosis in oxygenated conditions in neural cells. Here we show that PHD3 forms subcellular aggregates in an oxygen-dependent manner. The aggregation of PHD3 was seen under normoxia and was strongly reduced under hypoxia or by the inactivation of the PHD3 hydroxylase activity. The PHD3 aggregates were dependent on microtubular integrity and contained components of the 26S proteasome, chaperones, and ubiquitin, thus demonstrating features that are characteristic for aggresome-like structures. Forced expression of the active PHD3 induced the aggregation of proteasomal components and activated apoptosis under normoxia in HeLa cells. The apoptosis was seen in cells prone to PHD3 aggregation and the PHD3 aggregation preceded apoptosis. The data demonstrates the cellular oxygen sensor PHD3 as a regulator of protein aggregation in response to varying oxygen availability. PMID:18337469

  1. Regulation of the Extrarenal CYP27B1-Hydroxylase

    PubMed Central

    Adams, John S.; Rafison, Brandon; Witzel, Sten; Reyes, Rachel E.; Shieh, Albert; Chun, Rene; Zavala, Kathryn; Hewison, Martin; Liu, Philip T.

    2014-01-01

    Provided here is a collective review of research on the extrarenal CYP27B1-hydroxylase that shapes our current and expanding vision of the role this enzyme plays in the intracrinonology and paracrinology, as opposed to the traditional endocrinology, of vitamin D to regulate the innate and adaptive immune response, particularly in human granuloma-forming diseases like tuberculosis. Special emphasis is placed on soluble factors (i.e., cytokines) in the local microenvironment of these human diseases that coordinate amplification and feedback inhibition of the macrophage CYP27B1-hydroxylase. Principal among these factors are Type I and Type II interferons (IFNs); the Type II IFN, IFN-γ, stimulates the production of 1,25-dihydroxyvitamin D (1,25(OH)2D) from 25-hydroxyvitamin D (25OHD) by the granuloma-forming disease-activated macrophage, while the Type I IFNs, IFN-α and IFN-ß, block the hydroxylation reaction. The type I IFN response is associated with more aggressive disease, while the Type II IFN response, the one that promotes 1,25(OH)2D production by the macrophage, is associated with more confined disease. Tilting the balance in the human immune response toward a type II IFN, confined disease phenotype in enabled by the presence of extracellular 25OHD levels that are sufficient to enable the type II IFNγ-promoted, substrate 25OHD-driven intracellular synthesis of 1,25(OH)2D. PMID:24388948

  2. NF-Y activates mouse tryptophan hydroxylase transcription.

    PubMed

    Reed, G E; Kirchner, J E; Carr, L G

    1995-06-01

    Tryptophan hydroxylase catalyses the rate-limiting step in the biosynthesis of serotonin, a neurotransmitter which has been implicated in the etiologies of clinically important psychiatric illnesses. Tryptophan hydroxylase is expressed in a tissue-specific manner, but little is known about its transcriptional regulation. By analysing transcriptional activities of a set 5'-deletion constructs of promoter-reporter plasmids in P815-HTR mastocytoma cells, we found that transcription was activated by sequences between nucleotides -343 and -21. DNase I footprint analysis, using nuclear protein extracts from P815-HTR cells, revealed a protein-DNA interaction between nucleotides -77 and -46. A double stranded oligonucleotide, representing this binding site, specifically bound nuclear protein in a gel shift assay. Methylation interference analysis of this complex revealed that nuclear protein interacted with an inverted GGCCAAT element, which is a high-affinity binding motif for the transcription factor NF-Y (also known as CP1 or CBF). An NF-Y specific antibody abolished protein binding in a gel shift assay. Mutagenesis of specific base pairs abolished protein binding in vitro, and mutagenesis of the same base pairs in a reporter gene construct resulted in a 65% decrease in transcriptional activity. Our results suggest that the transcription factor NF-Y binds to a GGCCAAT motif in the tph proximal promoter and activates transcription. PMID:7552299

  3. The Solution Structure of the Regulatory Domain of Tyrosine Hydroxylase

    PubMed Central

    Zhang, Shengnan; Huang, Tao; Ilangovan, Udayar; Hinck, Andrew P.; Fitzpatrick, Paul F.

    2014-01-01

    Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine in the biosynthesis of the catecholamine neurotransmitters. The activity of the enzyme is regulated by phosphorylation of serine residues in a regulatory domain and by binding of catecholamines to the active site. Available structures of TyrH lack the regulatory domain, limiting the understanding of the effect of regulation on structure. We report the use of NMR spectroscopy to analyze the solution structure of the isolated regulatory domain of rat TyrH. The protein is composed of a largely unstructured N-terminal region (residues 1-71) and a well-folded C-terminal portion (residues 72-159). The structure of a truncated version of the regulatory domain containing residues 65-159 has been determined and establishes that it is an ACT domain. The isolated domain is a homodimer in solution, with the structure of each monomer very similar to that of the core of the regulatory domain of phenylalanine hydroxylase. Two TyrH regulatory domain monomers form an ACT domain dimer composed of a sheet of eight strands with four α-helices on one side of the sheet. Backbone dynamic analyses were carried out to characterize the conformational flexibility of TyrH65-159. The results provide molecular details critical for understanding the regulatory mechanism of TyrH. PMID:24361276

  4. The isolation and properties of phenylalanine hydroxylase from rat liver

    PubMed Central

    Gillam, Shirley Su; Woo, Savio L. C.; Woolf, Louis I.

    1974-01-01

    Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The Km and Vmax. values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide. PMID:4854920

  5. Characterisation of the Drosophila procollagen lysyl hydroxylase, dPlod

    PubMed Central

    Bunt, Stephanie; Denholm, Barry; Skaer, Helen

    2011-01-01

    The lysyl hydroxylase (LH) family of enzymes has important roles in the biosynthesis of collagen. In this paper we present the first description of Drosophila LH3 (dPlod), the only lysyl hydroxylase encoded in the fly genome. We have characterised in detail the developmental expression patterns of dPlod RNA and protein during embryogenesis. Consistent with its predicted function as a collagen-modifying enzyme, we find that dPlod is highly expressed in type-IV collagen-producing cells, particularly the haemocytes and fat body. Examination of dPlod subcellular localisation reveals that it is an endoplasmic reticulum resident protein, that partially overlaps with intracellular type-IV collagen. Furthermore, we show that dPlod is required for type-IV collagen secretion from haemocytes and fat body, and thus establish that LH3 enzyme function is conserved across widely separated animal phyla. Our findings, and the new tools we describe, establish the fly as an attractive model in which to study this important collagen biosynthesis enzyme. PMID:20888931

  6. Electrical stimulation increases phosphorylation of tyrosine hydroxylase in superior cervical ganglion of rat.

    PubMed Central

    Cahill, A L; Perlman, R L

    1984-01-01

    Electrical stimulation of the superior cervical ganglion of the rat increased the phosphorylation of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) in this tissue. Ganglia were incubated with [32P]Pi for 90 min and were then electrically stimulated via the preganglionic nerve. Tyrosine hydroxylase was isolated from homogenates of the ganglia by immunoprecipitation followed by polyacrylamide gel electrophoresis. 32P-labeled tyrosine hydroxylase was visualized by radioautography, and the incorporation of 32P into the enzyme was quantitated by densitometry of the radioautograms. Stimulation of ganglia at 20 Hz for 5 min increased the incorporation of 32P into tyrosine hydroxylase to a level 5-fold that found in unstimulated control ganglia. The increase in phosphorylation of tyrosine hydroxylase was dependent on the duration and frequency of stimulation. Preganglionic stimulation did not increase the phosphorylation of tyrosine hydroxylase in a medium that contained low Ca2+ and high Mg2+. Increases in phosphorylation were reversible; within 30 min after the cessation of stimulation, the incorporation of 32P into tyrosine hydroxylase decreased to the level found in unstimulated ganglia. The nicotinic antagonist hexamethonium reduced the increase in 32P incorporation into tyrosine hydroxylase by about 50%, while the muscarinic antagonist atropine had no effect. Thus, preganglionic stimulation appeared to increase the phosphorylation of tyrosine hydroxylase in part by a nicotinic mechanism and in part by a noncholinergic mechanism. Antidromic stimulation of ganglia also increased the phosphorylation of tyrosine hydroxylase. Two-dimensional gel electrophoresis revealed that electrical stimulation also increased the incorporation of 32P into at least six other phosphoproteins in the ganglion. Images PMID:6150485

  7. Kdo hydroxylase is an inner core assembly enzyme in the Ko-containing lipopolysaccharide biosynthesis.

    PubMed

    Chung, Hak Suk; Yang, Eun Gyeong; Hwang, Dohyeon; Lee, Ji Eun; Guan, Ziqiang; Raetz, Christian R H

    2014-09-26

    The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains d-glycero-d-talo-oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe(2+)/α-KG/O2 dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo2-lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo2-lipid IVA or Kdo2-lipid A as a substrate, but not Kdo-lipid IVAin vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro. These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis. PMID:25204504

  8. The frequency and the effects of 21-hydroxylase gene defects in congenital adrenal hyperplasia patients.

    PubMed

    Kirac, Deniz; Guney, Ahmet Ilter; Akcay, Teoman; Guran, Tulay; Ulucan, Korkut; Turan, Serap; Ergec, Deniz; Koc, Gulsah; Eren, Fatih; Kaspar, Elif Cigdem; Bereket, Abdullah

    2014-11-01

    Congenital adrenal hyperplasia (CAH) is a group of genetic endocrine disorders, caused by enzyme deficiencies in the conversion of cholesterol to cortisol. More than 90% of the cases have 21-hydroxylase deficiency (21-OHD). The clinical phenotype of the disease is classified as classic, the severe form, and nonclassic, the mild form. In this study, it was planned to characterize the mutations that cause 21-OHD in Turkish CAH patients by direct sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis and to investigate the type of CAH (classic or nonclassic type) that these mutations cause. A total of 124 CAH patients with 21-OHD and 100 healthy volunteers were recruited to the study. Most of the mutations were detected by direct sequencing. Large gene deletions/duplications/conversions were investigated with MLPA analysis. Results were evaluated statistically. At the end of our study, 66 different variations were detected including SNPs and deletions/duplications/conversions. Of these variations, 18 are novel, of which three cause amino acid substitutions. In addition, 15 SNPs which cause amino acid changes were identified among these variations. If similar results are obtained in different populations, these mutations, in particular the novel mutation 711 G>A, may be used as markers for prenatal diagnosis. PMID:25227725

  9. 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.

  10. 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. PMID:26672451

  11. Influence of the thyroid hormone status on tyrosine hydroxylase in central and peripheral catecholaminergic structures.

    PubMed

    Claustre, J; Balende, C; Pujol, J F

    1996-03-01

    We investigated the effect of hyper- and hypothyroidism on tyrosine hydroxylase protein concentration in the locus coeruleus (divided into anterior and posterior parts), the substantia nigra and the adrenals of adult rats. Rats were made hypothyroid with propylthiouracile (PTU, 0.02% in drinking water for 21 days) or hyperthyroid by thyroxine injection (100 or 250 micrograms/kg/day), for 3 or 17 days. PTU treatment resulted in statistically significant decrease of tyrosine hydroxylase in the anterior locus coeruleus (-13%) and the adrenals (-14%). After thyroxine treatment, in the anterior locus coeruleus, tyrosine hydroxylase was significantly higher (2 way ANOVA) after the 3 day treatment than after the 17 day treatment: tyrosine hydroxylase showed a trend to increase the 3 day treatment (+20% with the 250 micrograms/kg dose) and to decrease after the 17 day treatment (-15% with the 250 micrograms/kg dose). In the adrenals, tyrosine hydroxylase was increased by the 3 day treatment (+42% after the 250 micrograms/kg dose), but this increase was not observed after 17 days of treatment. Tyrosine hydroxylase was not altered in the posterior locus coeruleus and the substantia nigra, whatever the treatment. Together, our results support the hypothesis that in the anterior locus coeruleus and in the adrenals tyrosine hydroxylase level is positively modulated by thyroid hormones. After long-term treatment (17 days) this effect is not observed. PMID:8813245

  12. 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.

  13. 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. PMID:26808442

  14. Discovery of acyl guanidine tryptophan hydroxylase-1 inhibitors.

    PubMed

    Goldberg, Daniel R; De Lombaert, Stéphane; Aiello, Robert; Bourassa, Patricia; Barucci, Nicole; Zhang, Qing; Paralkar, Vishwas; Stein, Adam J; Valentine, Jim; Zavadoski, William

    2016-06-15

    An increasing number of diseases have been linked to a dysfunctional peripheral serotonin system. Given that tryptophan hydroxylase 1 (TPH1) is the rate limiting enzyme in the biosynthesis off serotonin, it represents an attractive target to regulate peripheral serotonin. Following up to our first disclosure, we report a new chemotype of TPH1 inhibitors where-by the more common central planar heterocycle has been replaced with an open-chain, acyl guanidine surrogate. Through our work, we found that compounds of this nature provide highly potent TPH1 inhibitors with favorable physicochemical properties that were effective in reducing murine intestinal 5-HT in vivo. Furthermore, we obtained a high resolution (1.90Å) X-ray structure crystal structure of one of these inhibitors (compound 51) that elucidated the active conformation along with revealing a dimeric form of TPH1 for the first time. PMID:27146606

  15. Lysyl Hydroxylase 3-mediated Glucosylation in Type I Collagen

    PubMed Central

    Sricholpech, Marnisa; Perdivara, Irina; Yokoyama, Megumi; Nagaoka, Hideaki; Terajima, Masahiko; Tomer, Kenneth B.; Yamauchi, Mitsuo

    2012-01-01

    Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing lysyl hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846–8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization. PMID:22573318

  16. Characterization of monoclonal antibodies specific to bovine renal vitamin D hydroxylases.

    PubMed

    Bort, R E; Crivello, J F

    1988-11-01

    Monoclonal antibodies (MAbs) have been produced which recognize specific epitopes on bovine renal mitochondrial vitamin D3 1 alpha- and 24-hydroxylases. Renal mitochondria cytochrome P-450s were partially purified to 0.5-2 nmol/mg by Emulgen 911 and cholate solubilization, followed by chromatography on a 2-(4,6-dichloro-O-biphenyloxy)ethylamine HBR affinity column. Reduced carbon monoxide difference spectra determined that this preparation contained 0.5-2 nmol P-450/mg protein. This preparation contained both 1 alpha- and 24-hydroxylase activities, and Eadie-Hofstee plots of product formation as a function of substrate concentrations have maximum velocities of 1.4 and 4 pmol product/30 min.mg protein and Km values of 690 and 1300 nM, respectively. Bovine renal hydroxylases were isolated by immunoprecipitation from this partially purified P-450 preparation with a polyclonal antibody specific for rat liver microsomal cytochrome P-450 RLM5. This polyclonal antibody immunoprecipitated both 1 alpha- and 24-hydroxylase activities as well as renal mitochondrial cytochrome P-450, as determined by reduced CO spectra. Bovine renal mitochondrial components were immunoisolated and used to immunize BALB/c mouse spleen cells in vitro. MAbs then produced were screened for 1) immunoisolation of renal mitochondrial hydroxylase activity from a partially purified preparation, 2) immunohistochemical detection of antigen in renal proximal tubule cells, and 3) immunoquantitation of renal hydroxylases in a solid phase sandwich (enzyme-linked immunosorbent assay) and by 4) Western blot analysis. MAbs were isolated with specifically immunoprecipitated 1 alpha-hydroxylase activity, 24-hydroxylase activity, or both. In 10 micron sections of bovine kidney, antibodies detected antigen only in proximal tubule cells on the basal surface, which is rich in mitochondria. No antigen was detected in sections of pancreas or liver. In the solid phase sandwich enzyme-linked immunosorbent assay, MAbs

  17. A Novel Mutation in the CYP11B1 Gene Causes Steroid 11β-Hydroxylase Deficient Congenital Adrenal Hyperplasia with Reversible Cardiomyopathy.

    PubMed

    Alqahtani, Mohammad A; Shati, Ayed A; Zou, Minjing; Alsuheel, Ali M; Alhayani, Abdullah A; Al-Qahtani, Saleh M; Gilban, Hessa M; Meyer, Brain F; Shi, Yufei

    2015-01-01

    Congenital adrenal hyperplasia (CAH) due to steroid 11β-hydroxylase deficiency is the second most common form of CAH, resulting from a mutation in the CYP11B1 gene. Steroid 11β-hydroxylase deficiency results in excessive mineralcorticoids and androgen production leading to hypertension, precocious puberty with acne, enlarged penis, and hyperpigmentation of scrotum of genetically male infants. In the present study, we reported 3 male cases from a Saudi family who presented with penile enlargement, progressive darkness of skin, hypertension, and cardiomyopathy. The elder patient died due to heart failure and his younger brothers were treated with hydrocortisone and antihypertensive medications. Six months following treatment, cardiomyopathy disappeared with normal blood pressure and improvement in the skin pigmentation. The underlying molecular defect was investigated by PCR-sequencing analysis of all coding exons and intron-exon boundary of the CYP11B1 gene. A novel biallelic mutation c.780 G>A in exon 4 of the CYP11B1 gene was found in the patients. The mutation created a premature stop codon at amino acid 260 (p.W260 (∗) ), resulting in a truncated protein devoid of 11β-hydroxylase activity. Interestingly, a somatic mutation at the same codon (c.779 G>A, p.W260 (∗) ) was reported in a patient with papillary thyroid cancer (COSMIC database). In conclusion, we have identified a novel nonsense mutation in the CYP11B1 gene that causes classic steroid 11β-hydroxylase deficient CAH. Cardiomyopathy and cardiac failure can be reversed by early diagnosis and treatment. PMID:26265915

  18. 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.

  19. Effects of omega-hydroxylase product on distal human pulmonary arteries.

    PubMed

    Morin, Caroline; Guibert, Christelle; Sirois, Marco; Echave, Vincent; Gomes, Marcio M; Rousseau, Eric

    2008-03-01

    The aim of the present study was to provide a mechanistic insight into how 20-hydroxyeicosatetraenoic acid (20-HETE) relaxes distal human pulmonary arteries (HPAs). This compound is produced by omega-hydroxylase from free arachidonic acid. Tension measurements, performed on either fresh or 1 day-cultured pulmonary arteries, revealed that the contractile responses to 1 microM 5-hydroxytryptamine were largely relaxed by 20-HETE in a concentration-dependent manner (0.01-10 microM). Iberiotoxin pretreatments (10 nM) partially decreased 20-HETE-induced relaxations. However, 10 microM indomethacin and 3 microM SC-560 pretreatments significantly reduced the relaxations to 20-HETE in these tissues. The relaxing responses induced by the eicosanoid were likely related to a reduced Ca2+ sensitivity of the myofilaments since free Ca2+ concentration ([Ca2+])-response curves performed on beta-escin-permeabilized cultured explants were shifted toward higher [Ca2+]. 20-HETE also abolished the tonic responses induced by phorbol-ester-dibutyrate (a PKC-sensitizing agent). Western blot analyses, using two specific primary antibodies against the PKC-potentiated inhibitory protein CPI-17 and its PKC-dependent phosphorylated isoform pCPI-17, confirmed that 20-HETE interferes with this intracellular process. We also investigated the effect of 20-HETE on the activation of Rho-kinase pathway-induced Ca2+ sensitivity. The data demonstrated that 20-HETE decreased U-46619-induced Ca2+ sensitivity on arteries. Hence, this observation was correlated with an increased staining of p116(Rip), a RhoA-binding protein. Together, these results strongly suggest that the 20-hydroxyarachidonic acid derivative is a potent modulator of tone in HPAs in vitro. PMID:18203846

  20. 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

  1. Evaluation of aspirin metabolites as inhibitors of hypoxia-inducible factor hydroxylases.

    PubMed

    Lienard, Benoit M; Conejo-García, Ana; Stolze, Ineke; Loenarz, Christoph; Oldham, Neil J; Ratcliffe, Peter J; Schofield, Christopher J

    2008-12-21

    Known and potential aspirin metabolites were evaluated as inhibitors of oxygen-sensing hypoxia-inducible transcription factor (HIF) hydroxylases; some of the metabolites were found to stabilise HIF-alpha in cells. PMID:19048166

  2. 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.

  3. PARGYLINE AND Y-BUTYROLACTONE ENHANCE TYROSINE HYDROXYLASE IMMUNOSTAINING OF NIGROSTRIATAL AXONS

    EPA Science Inventory

    Administration of pargyline or q-butyrolactone enhanced immunostaining of tyrosine hydroxylase immunoreactive axons within the striatum. he qualitative characteristics of this enhancement were compound dependent and the enhancement was not associated with an increase in the amoun...

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

    MedlinePlus

    ... Intersex Society of North America MalaCards: adrenal hyperplasia, congenital, due to 11-beta-hydroxylase deficiency March of Dimes: Genital and Urinary Tract Defects Merck Manual Consumer Version: The Body's Control ...

  5. 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

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

    PubMed

    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 5(th) 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

  7. Incorporation of a Prolyl Hydroxylase Inhibitor into Scaffolds: A Strategy for Stimulating Vascularization

    PubMed Central

    Sham, Adeline; Martinez, Eliana C.; Beyer, Sebastian; Trau, Dieter W.

    2015-01-01

    Clinical applications of tissue engineering are constrained by the ability of the implanted construct to invoke vascularization in adequate extent and velocity. To overcome the current limitations presented by local delivery of single angiogenic factors, we explored the incorporation of prolyl hydroxylase inhibitors (PHIs) into scaffolds as an alternative vascularization strategy. PHIs are small molecule drugs that can stabilize the alpha subunit of hypoxia-inducible factor-1 (HIF-1), a key transcription factor that regulates a variety of angiogenic mechanisms. In this study, we conjugated the PHI pyridine-2,4-dicarboxylic acid (PDCA) through amide bonds to a gelatin sponge (Gelfoam®). Fibroblasts cultured on PDCA-Gelfoam were able to infiltrate and proliferate in these scaffolds while secreting significantly more vascular endothelial growth factor than cells grown on Gelfoam without PDCA. Reporter cells expressing green fluorescent protein-tagged HIF-1α exhibited dose-dependent stabilization of this angiogenic transcription factor when growing within PDCA-Gelfoam constructs. Subsequently, we implanted PDCA-Gelfoam scaffolds into the perirenal fat tissue of Sprague Dawley rats for 8 days. Immunostaining of explants revealed that the PDCA-Gelfoam scaffolds were amply infiltrated by cells and promoted vascular ingrowth in a dose-dependent manner. Thus, the incorporation of PHIs into scaffolds appears to be a feasible strategy for improving vascularization in regenerative medicine applications. PMID:25370818

  8. Regulation of ferulate-5-hydroxylase expression in Arabidopsis in the context of sinapate ester biosynthesis

    SciTech Connect

    Ruegger, M.; Meyer, K.; Cusumano, J.C.; Chapple, C.

    1999-01-01

    Sinapic acid is an intermediate in syringyl lignin biosynthesis in angiosperms, and in some taxa serves as a precursor for soluble secondary metabolites. The biosynthesis and accumulation of the sinapate esters sinapoylglucose, sinapolymalate, and sinapolycholine are developmentally regulated in Arabidopsis and other members of the Brassicaceae. The FAH1 locus of Arabidopsis encodes the enzyme ferulate-5-hydroxylase (F5H), which catalyzes the rate-limiting step in syringyl lignin biosynthesis and is required for the production of sinapate esters. Here the authors show that F5H expression parallels sinapate ester accumulation in developing siliques and seedlings, but is not rate limiting for their biosynthesis. RNA gel-blot analysis indicated that the tissue-specific and developmentally regulated expression of F5H mRNA is distinct from that of other phenylpropanoid genes. Efforts to identify constructs capable of complementing the sinapate ester-deficient phenotype of fah1 mutants demonstrated that F5H expression in leaves is dependent on sequences 3{prime} of the F5H coding region. In contrast, the positive regulatory function of the downstream region is not required for F5H transcript or sinapolycholine accumulation in embryos.

  9. 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.

  10. 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

  11. Alu-alu recombination results in a duplication of seven exons in the lysyl hydroxylase gene in a patient with the type VI variant of Ethlers-Danlos syndrome

    SciTech Connect

    Pousi, B.; Hautala, T.; Heikkinen, J.; Pajunen, L.; Kivirikko, K.I.; Myllylae, R.

    1994-11-01

    The type VI variant of the Ethlers-Danlos syndrome (EDS) is a recessively inherited connective-tissue disorder. The characteristic features of the variant are muscular hyptonia, kyphoscoliosis, ocular manifestations, joint hypermobility, skin fragility and hyperextensibility, and other signs of connective-tissue involvement. The biochemical defect in most but not all patients is a deficiency in lysyl hydroxylase activity. Lysyl hydroxylase is an enzyme that catalyzes the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. We have recently reported an apparently homozygous large-duplication rearrangement in the gene for lysyl hydroxylase, leading to the type VI variant of EDS in two siblings. We now report an identical, apparently homozygous large duplication in an unrelated 49-year-old female originally analyzed by Sussman et al. Our simple-sequence-repeat-polymorphism analysis does not support uniparental isodisomy inheritance for either of the two duplications. Furthermore, we indicate in this study that the duplication in the lysyl hydroxylase gene is caused by an Alu-Alu recombination in both families. Cloning of the junction fragment of the duplication has allowed synthesis of appropriate primers for rapid screening for this rearrangement in other families with the type VI variant of EDS. 38 refs., 6 figs.

  12. Molecular screening for alkane hydroxylase genes in Gram-negative and Gram-positive strains.

    PubMed

    Smits, T H; Röthlisberger, M; Witholt, B; van Beilen, J B

    1999-08-01

    We have developed highly degenerate oligonucleotides for polymerase chain reaction (PCR) amplification of genes related to the Pseudomonas oleovorans GPo1 and Acinetobacter sp. ADP1 alkane hydroxylases, based on a number of highly conserved sequence motifs. In all Gram-negative and in two out of three Gram-positive strains able to grow on medium- (C6-C11) or long-chain n-alkanes (C12-C16), PCR products of the expected size were obtained. The PCR fragments were cloned and sequenced and found to encode peptides with 43.2-93.8% sequence identity to the corresponding fragment of the P. oleovorans GPo1 alkane hydroxylase. Strains that were unable to grow on n-alkanes did not yield PCR products with homology to alkane hydroxylase genes. The alkane hydroxylase genes of Acinetobacter calcoaceticus EB104 and Pseudomonas putida P1 were cloned using the PCR products as probes. The two genes allow an alkane hydroxylase-negative mutant of Acinetobacter sp. ADP1 and an Escherichia coli recombinant containing all P. oleovorans alk genes except alkB, respectively, to grow on n-alkanes, showing that the cloned genes do indeed encode alkane hydroxylases. PMID:11207749

  13. 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

  14. 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

    2015-01-01

    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. PMID:26125800

  15. Product bound structures of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): protein motion in the alpha-subunit.

    PubMed

    Sazinsky, Matthew H; Lippard, Stephen J

    2005-04-27

    The soluble methane monooxygenase hydroxylase (MMOH) alpha-subunit contains a series of cavities that delineate the route of substrate entrance to and product egress from the buried carboxylate-bridged diiron center. The presence of discrete cavities is a major structural difference between MMOH, which can hydroxylate methane, and toluene/o-xylene monooxygenase hydroxylase (ToMOH), which cannot. To understand better the functions of the cavities and to investigate how an enzyme designed for methane hydroxylation can also accommodate larger substrates such as octane, methylcubane, and trans-1-methyl-2-phenylcyclopropane, MMOH crystals were soaked with an assortment of different alcohols and their X-ray structures were solved to 1.8-2.4 A resolution. The product analogues localize to cavities 1-3 and delineate a path of product exit and/or substrate entrance from the active site to the surface of the protein. The binding of the alcohols to a position bridging the two iron atoms in cavity 1 extends and validates previous crystallographic, spectroscopic, and computational work indicating this site to be where substrates are hydroxylated and products form. The presence of these alcohols induces perturbations in the amino acid side-chain gates linking pairs of cavities, allowing for the formation of a channel similar to one observed in ToMOH. Upon binding of 6-bromohexan-1-ol, the pi helix formed by residues 202-211 in helix E of the alpha-subunit is extended through residue 216, changing the orientations of several amino acid residues in the active site cavity. This remarkable secondary structure rearrangement in the four-helix bundle has several mechanistic implications for substrate accommodation and the function of the effector protein, MMOB. PMID:15839679

  16. 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

  17. Phenol hydroxylase from Bacillus thermoglucosidasius A7, a two-protein component monooxygenase with a dual role for FAD.

    PubMed

    Kirchner, Ulrike; Westphal, Adrie H; Müller, Rudolf; van Berkel, Willem J H

    2003-11-28

    A novel phenol hydroxylase (PheA) that catalyzes the first step in the degradation of phenol in Bacillus thermoglucosidasius A7 is described. The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) and is optimally active at 55 degrees C. PheA1 and PheA2 were separately expressed in recombinant Escherichia coli BL21(DE3) pLysS cells and purified to apparent homogeneity. The pheA1 gene codes for a protein of 504 amino acids with a predicted mass of 57.2 kDa. PheA1 exists as a homodimer in solution and has no enzyme activity on its own. PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. The hydroxylase activity is strictly FAD-dependent, and neither FMN nor riboflavin can replace FAD in this reaction. The pheA2 gene codes for a protein of 161 amino acids with a predicted mass of 17.7 kDa. PheA2 is also a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group. PheA2 catalyzes the NADH-dependent reduction of free flavins according to a Ping Pong Bi Bi mechanism. PheA2 is structurally related to ferric reductase, an NAD(P)H-dependent reductase from the hyperthermophilic Archaea Archaeoglobus fulgidus that catalyzes the flavin-mediated reduction of iron complexes. However, PheA2 displays no ferric reductase activity and is the first member of a newly recognized family of short-chain flavin reductases that use FAD both as a substrate and as a prosthetic group. PMID:12968028

  18. Pitx2 Regulates Procollagen Lysyl Hydroxylase (Plod) Gene Expression

    PubMed Central

    Hjalt, Tord A.; Amendt, Brad A.; Murray, Jeffrey C.

    2001-01-01

    The Rieger syndrome is an autosomal dominant disease characterized by ocular, craniofacial, and umbilical defects. Patients have mutations in PITX2, a paired-bicoid homeobox gene, also involved in left/right polarity determination. In this study we have identified a family of genes for enzymes responsible for hydroxylizing lysines in collagens as one group of likely cognate targets of PITX2 transcriptional regulation. The mouse procollagen lysyl hydroxylase (Plod)-2 gene was enriched for by chromatin precipitation using a PITX2/Pitx2-specific antibody. Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements. We show these elements to bind PITX2 specifically in vitro. The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments. The Rieger syndrome causing PITX2 mutant T68P fails to induce PLOD-1–luciferase. Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]). Several of the same organ systems are involved in Rieger syndrome and EDVI. PMID:11157981

  19. 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

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

    PubMed Central

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

    2014-01-01

    Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes phenylalanine oxidation to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH features a regulatory domain where binding of the substrate leads to allosteric activation of the enzyme. However, existence of PAH regulation in evolutionarily distant organisms, such as certain bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum (cPAH), a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site, 15.7Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 µM for phenylalanine. Under the same conditions, no detectable binding was observed in ITC for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) lead to impaired binding, consistent with the presence of distal site binding in solution. Kinetic analysis reveals that the distal site mutants suffer a discernible loss in their catalytic activity. However, x-ray structures of Y155A and F258A, two of the mutants showing more noticeable defect in their activity, show no discernible change in their active site structure, suggesting that the effect of distal binding may transpire through protein dynamics in solution. PMID:23860686

  1. Myoclonus-dystonia syndrome due to tyrosine hydroxylase deficiency

    PubMed Central

    Mencacci, Niccolo E.; Cordivari, Carla; Batla, Amit; Wood, Nick W.; Houlden, Henry; Hardy, John; Bhatia, Kailash P.

    2012-01-01

    Objective: To present a new family with tyrosine hydroxylase deficiency (THD) that presented with a new phenotype of predominant, levodopa-responsive myoclonus with dystonia due to compound heterozygosity of one previously reported mutation in the promoter region and a novel nonsynonymous mutation in the other allele, thus expanding the clinical and genetic spectrum of this disorder. Methods: We performed detailed clinical examination of the family and electrophysiology to characterize the myoclonus. We performed analysis of the TH gene and in silico prediction of the possible effect of nonsynonymous substitutions on protein structure. Results: Electrophysiology suggested that the myoclonus was of subcortical origin. Genetic analysis of the TH gene revealed compound heterozygosity of a point mutation in the promoter region (c.1-71 C>T) and a novel nonsynonymous substitution in exon 12 (c.1282G>A, p.Gly428Arg). The latter is a novel variant, predicted to have a deleterious effect on the TH protein function and is the first pathogenic TH mutation in patients of African ancestry. Conclusion: We presented a THD family with predominant myoclonus-dystonia and a new genotype. It is important to consider THD in the differential diagnosis of myoclonus-dystonia, because early treatment with levodopa is crucial for these patients. PMID:22815559

  2. Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans.

    PubMed

    Calvo, Ana C; Pey, Angel L; Ying, Ming; Loer, Curtis M; Martinez, Aurora

    2008-08-01

    In humans, liver phenylalanine hydroxylase (PAH) has an established catabolic function, and mutations in PAH cause phenylketonuria, a genetic disease characterized by neurological damage, if not treated. To obtain novel evolutionary insights and information on molecular mechanisms operating in phenylketonuria, we investigated PAH in the nematode Caenorhabditis elegans (cePAH), where the enzyme is coded by the pah-1 gene, expressed in the hypodermis. CePAH presents similar molecular and kinetic properties to human PAH [S(0.5)(L-Phe) approximately 150 microM; K(m) for tetrahydrobiopterin (BH(4)) approximately 35 microM and comparable V(max)], but cePAH is devoid of positive cooperativity for L-Phe, an important regulatory mechanism of mammalian PAH that protects the nervous system from excess L-Phe. Pah-1 knockout worms show no obvious neurological defects, but in combination with a second cuticle synthesis mutation, they display serious cuticle abnormalities. We found that pah-1 knockouts lack a yellow-orange pigment in the cuticle, identified as melanin by spectroscopic techniques, and which is detected in C. elegans for the first time. Pah-1 mutants show stimulation of superoxide dismutase activity, suggesting that cuticle melanin functions as oxygen radical scavenger. Our results uncover both an important anabolic function of PAH and the change in regulation of the enzyme along evolution. PMID:18460651

  3. Tryptophan Hydroxylase 2 Gene and Alcohol Use among College Students

    PubMed Central

    Gacek, Paul; Conner, Tamlin S.; Tennen, Howard; Kranzler, Henry R.; Covault, Jonathan

    2009-01-01

    Objective Genes that regulate serotonin activity are regarded as promising predictors of heavy alcohol use. Tryptophan Hydroxylase (TPH2) plays an important role in serotonergic neurotransmission by serving as the rate-limiting enzyme for serotonin biosynthesis in the midbrain and serotonergic neurons. Despite the link between TPH2 and serotonergic function, TPH2’s role in the pathogenesis of alcohol use disorders remains unclear. The goal of this study was to examine whether variation in the TPH2 gene is associated with risky alcohol consumption. Specifically, this study examined whether the TPH2 G-703T polymorphism predicted alcohol consumption among college students. Methods In two successive years, 351 undergraduates were asked to record their alcohol use each day for 30 days using an internet-based electronic diary. Participants’ DNA was collected and polymerase chain reaction genotyping was performed. Results Alcohol consumption was not associated with the TPH2 G-703T polymorphism alone, or the interaction of TPH2 with two other candidate polymorphisms (TPH1 C218A, and the SLC6A4 tri-allelic 5-HTTLPR) or negative life events. Conclusions This study supports recent null findings relating TPH2 to drinking outcomes. It also extends these findings by showing null interactions with the TPH1 C218A polymorphism, the SLC6A4 tri-allelic 5-HTTLPR polymorphism, and environmental stressors in predicting sub-clinical alcohol use among Caucasian American young adults. PMID:18782386

  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. PMID:26975317

  5. Immunochemical characterization of brain and pineal tryptophan hydroxylase.

    PubMed Central

    Chung, Y. I.; Park, D. H.; Kim, M.; Baker, H.; Joh, T. H.

    2001-01-01

    Recombinant mouse tryptophan hydroxylase (TPH) was expressed in Escherichia coli, using a bacterial expression vector and has been purified to homogeneity by sonication followed by Sepharose 4B column chromatography and native slab gel electrophoresis. This purified enzymatically active TPH protein was used for production of a specific antiserum. This antiserum identified the predicted TPH band (molecular weight, 54 kDa) on Western blot of crude extracts from the rat and mouse dorsal raphe, and the rat pineal gland. However, this antiserum recognized an additional protein band of lower molecular weight (48 kDa) in pineal extract. It is not clear whether the 48 kDa TPH band represents an isozyme or a protease cleavage product of TPH. Since the pineal gland contains higher TPH mRNA and lower TPH activity when it is compared with dorsal raphe nucleus enzyme, this lower molecular weight TPH may participate in the reduced TPH specific activity. In addition, there are no specific TPH inhibitors in the pineal gland and this lower molecular weight TPH is inactive or has a very low specific activity. This antiserum specifically immunostained serotonergic cell bodies in the dorsal raphe nuclei, some large caliber serotonergic processes in the dorsal raphe area as well as terminals in the olfactory bulb. It also immunolabeled the pineal gland and immunoprecipitated equally well TPH protein from the dorsal raphe nucleus and the pineal gland in a concentration-dependent manner. PMID:11511796

  6. Colour variation in red grapevines (Vitis vinifera L.): genomic organisation, expression of flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin

    PubMed Central

    Castellarin, Simone D; Di Gaspero, Gabriele; Marconi, Raffaella; Nonis, Alberto; Peterlunger, Enrico; Paillard, Sophie; Adam-Blondon, Anne-Francoise; Testolin, Raffaele

    2006-01-01

    Background Structural genes of the phenyl-propanoid pathway which encode flavonoid 3'- and 3',5'-hydroxylases (F3'H and F3'5'H) have long been invoked to explain the biosynthesis of cyanidin- and delphinidin-based anthocyanin pigments in the so-called red cultivars of grapevine. The relative proportion of the two types of anthocyanins is largely under genetic control and determines the colour variation among red/purple/blue berry grape varieties and their corresponding wines. Results Gene fragments of VvF3'H and VvF3'5'H, that were isolated from Vitis vinifera 'Cabernet Sauvignon' using degenerate primers designed on plant homologous genes, translated into 313 and 239 amino acid protein fragments, respectively, with up to 76% and 82% identity to plant CYP75 cytochrome P450 monooxygenases. Putative function was assigned on the basis of sequence homology, expression profiling and its correlation with metabolite accumulation at ten different ripening stages. At the onset of colour transition, transcriptional induction of VvF3'H and VvF3'5'H was temporally coordinated with the beginning of anthocyanin biosynthesis, the expression being 2-fold and 50-fold higher, respectively, in red berries versus green berries. The peak of VvF3'5'H expression was observed two weeks later concomitantly with the increase of the ratio of delphinidin-/cyanidin-derivatives. The analysis of structural genomics revealed that two copies of VvF3'H are physically linked on linkage group no. 17 and several copies of VvF3'5'H are tightly clustered and embedded into a segmental duplication on linkage group no. 6, unveiling a high complexity when compared to other plant flavonoid hydroxylase genes known so far, mostly in ornamentals. Conclusion We have shown that genes encoding flavonoid 3'- and 3',5'-hydroxylases are expressed in any tissues of the grape plant that accumulate flavonoids and, particularly, in skin of ripening red berries that synthesise mostly anthocyanins. The correlation between

  7. Trans-activity of plasma membrane-associated ganglioside sialyltransferase in mammalian cells.

    PubMed

    Vilcaes, Aldo A; Demichelis, Vanina Torres; Daniotti, Jose L

    2011-09-01

    Gangliosides are acidic glycosphingolipids that contain sialic acid residues and are expressed in nearly all vertebrate cells. They are synthesized at the Golgi complex by a combination of glycosyltransferase activities followed by vesicular delivery to the plasma membrane, where they participate in a variety of physiological as well as pathological processes. Recently, a number of enzymes of ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane. In particular, it was observed that CMP-NeuAc:GM3 sialyltransferase (Sial-T2) is able to sialylate GM3 at the plasma membrane (cis-catalytic activity). In this work, we demonstrated that plasma membrane-integrated ecto-Sial-T2 also displays a trans-catalytic activity at the cell surface of epithelial and melanoma cells. By using a highly sensitive enzyme-linked immunosorbent assay combined with confocal fluorescence microscopy, we observed that ecto-Sial-T2 was able to sialylate hydrophobically or covalently immobilized GM3 onto a solid surface. More interestingly, we observed that ecto-Sial-T2 was able to sialylate GM3 exposed on the membrane of neighboring cells by using both the exogenous and endogenous donor substrate (CMP-N-acetylneuraminic acid) available at the extracellular milieu. In addition, the trans-activity of ecto-Sial-T2 was considerably reduced when the expression of the acceptor substrate was inhibited by using a specific inhibitor of biosynthesis of glycolipids, indicating the lipidic nature of the acceptor. Our findings provide the first direct evidence that an ecto-sialyltransferase is able to trans-sialylate substrates exposed in the plasma membrane from mammalian cells, which represents a novel insight into the molecular events that regulate the local glycosphingolipid composition. PMID:21768099

  8. Trans-activity of Plasma Membrane-associated Ganglioside Sialyltransferase in Mammalian Cells*

    PubMed Central

    Vilcaes, Aldo A.; Demichelis, Vanina Torres; Daniotti, Jose L.

    2011-01-01

    Gangliosides are acidic glycosphingolipids that contain sialic acid residues and are expressed in nearly all vertebrate cells. They are synthesized at the Golgi complex by a combination of glycosyltransferase activities followed by vesicular delivery to the plasma membrane, where they participate in a variety of physiological as well as pathological processes. Recently, a number of enzymes of ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane. In particular, it was observed that CMP-NeuAc:GM3 sialyltransferase (Sial-T2) is able to sialylate GM3 at the plasma membrane (cis-catalytic activity). In this work, we demonstrated that plasma membrane-integrated ecto-Sial-T2 also displays a trans-catalytic activity at the cell surface of epithelial and melanoma cells. By using a highly sensitive enzyme-linked immunosorbent assay combined with confocal fluorescence microscopy, we observed that ecto-Sial-T2 was able to sialylate hydrophobically or covalently immobilized GM3 onto a solid surface. More interestingly, we observed that ecto-Sial-T2 was able to sialylate GM3 exposed on the membrane of neighboring cells by using both the exogenous and endogenous donor substrate (CMP-N-acetylneuraminic acid) available at the extracellular milieu. In addition, the trans-activity of ecto-Sial-T2 was considerably reduced when the expression of the acceptor substrate was inhibited by using a specific inhibitor of biosynthesis of glycolipids, indicating the lipidic nature of the acceptor. Our findings provide the first direct evidence that an ecto-sialyltransferase is able to trans-sialylate substrates exposed in the plasma membrane from mammalian cells, which represents a novel insight into the molecular events that regulate the local glycosphingolipid composition. PMID:21768099

  9. 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

  10. The regulation of pulmonary inflammation by the hypoxia-inducible factor-hydroxylase oxygen-sensing pathway.

    PubMed

    Whyte, Moira K B; Walmsley, Sarah R

    2014-12-01

    Although the hypoxia-inducible factor (HIF)-hydroxylase oxygen-sensing pathway has been extensively reviewed in the context of cellular responses to hypoxia and cancer biology, its importance in regulating innate immune biology is less well described. In this review, we focus on the role of the HIF-hydroxylase pathway in regulating myeloid cell responses and its relevance to inflammatory lung disease. The more specific roles of individual HIF/ prolyl hydroxylase pathway members in vivo are discussed in the context of lineage-specific rodent models of inflammation, with final reference made to the therapeutic challenges of targeting the HIF/hydroxylase pathway in immune cells. PMID:25525731

  11. 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. PMID:19537927

  12. 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

  13. Neurocognitive Function in Dopamine-β-Hydroxylase Deficiency

    PubMed Central

    Jepma, Marieke; Deinum, Jaap; Asplund, Christopher L; Rombouts, Serge ARB; Tamsma, Jouke T; Tjeerdema, Nathanja; Spapé, Michiel M; Garland, Emily M; Robertson, David; Lenders, Jacques WM; Nieuwenhuis, Sander

    2011-01-01

    Dopamine-β-hydroxylase (DβH) deficiency is a rare genetic syndrome characterized by the complete absence of norepinephrine in the peripheral and the central nervous system. DβH-deficient patients suffer from several physical symptoms, which can be treated successfully with L-threo-3,4-dihydroxyphenylserine, a synthetic precursor of norepinephrine. Informal clinical observations suggest that DβH-deficient patients do not have obvious cognitive impairments, even when they are not medicated, which is remarkable given the important role of norepinephrine in normal neurocognitive function. This study provided the first systematic investigation of neurocognitive function in human DβH deficiency. We tested 5 DβH-deficient patients and 10 matched healthy control participants on a comprehensive cognitive task battery, and examined their pupil dynamics, brain structure, and the P3 component of the electroencephalogram. All participants were tested twice; the patients were tested once ON and once OFF medication. Magnetic resonance imaging scans of the brain revealed that the patients had a smaller total brain volume than the control group, which is in line with the recent hypothesis that norepinephrine has a neurotrophic effect. In addition, the patients showed an abnormally small or absent task-evoked pupil dilation. However, we found no substantial differences in cognitive performance or P3 amplitude between the patients and the control participants, with the exception of a temporal-attention deficit in the patients OFF medication. The largely spared neurocognitive function in DβH-deficient patients suggests that other neuromodulators have taken over the function of norepinephrine in the brains of these patients. PMID:21471955

  14. The sustained phase of tyrosine hydroxylase activation in vivo.

    PubMed

    Ong, Lin Kooi; Sominsky, Luba; Dickson, Phillip W; Hodgson, Deborah M; Dunkley, Peter R

    2012-09-01

    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthetic pathway for catecholamine synthesis. Stress triggers an increase in TH activity, resulting in increased release of catecholamines from both neurons and the adrenal medulla. In response to stress three phases of TH activation have been identified (acute, sustained and chronic) and each phase has a unique mechanism. The acute and chronic phases have been studied in vivo in a number of animal models, but to date the sustained phase has only been characterised in vitro. We aimed to investigate the effects of dual exposure to lipopolysaccharide (LPS) in neonatal rats on TH protein, TH phosphorylation at serine residues 19, 31 and 40 and TH activity in the adrenal gland over the sustained phase. Wistar rats were administered LPS (0.05 mg/kg, intraperitoneal injection) or an equivolume of non-pyrogenic saline on days 3 and 5 postpartum. Adrenal glands were collected at 4, 24 and 48 h after the drug exposure on day 5. Neonatal LPS treatment resulted in increases in TH phosphorylation of Ser40 at 4 and 24 h, TH phosphorylation of Ser31 at 24 h, TH activity at 4 and 24 h and TH protein at 48 h. We therefore have provided evidence for the first time that TH phosphorylation at Ser31 and Ser40 occurs for up to 24 h in vivo and leads to TH activation independent of TH protein synthesis, suggesting that the sustained phase of TH activation occurs in vivo. PMID:22684282

  15. Profiles of 21-Carbon Steroids in 21-hydroxylase Deficiency

    PubMed Central

    Turcu, Adina F.; Rege, Juilee; Chomic, Robert; Liu, Jiayan; Nishimoto, Hiromi K.; Else, Tobias; Moraitis, Andreas G.; Palapattu, Ganesh S.; Rainey, William E.

    2015-01-01

    Context: Marked elevations of 17-hydroxyprogesterone (17OHP) are characteristic of classic 21-hydroxylase deficiency (21OHD). Testing of 17OHP provides the basis for 21OHD diagnosis, although it suffers from several pitfalls. False-positive or false-negative results and poor discrimination of nonclassic 21OHD from carriers limit the utility of serum 17OHP and necessitate dynamic testing after cosyntropin stimulation when values are indeterminate. Objective: The objective was to provide a detailed characterization of 21-carbon (C21) steroids in classic 21OHD, which might identify other candidate steroids that could be employed for the diagnosis of 21OHD. Setting and Participants: Patients (11 women, 10 men) with classic 21OHD and 21 sex- and age-matched controls seen in a tertiary referral center were studied. Methods: C21 steroids in the peripheral sera from all subjects, as well as in media from cultured testicular adrenal rest tumor (TART) cells and normal adrenal (NA) cells, were analyzed using liquid chromatography/tandem mass spectrometry (10 steroids). Additionally, the dynamics of C21 steroid metabolism in TART and NA cells were assessed with radiotracer studies. Results: Five C21 steroids were significantly higher in 21OHD patients: 17OHP (67-fold; P < .01), 21-deoxycortisol (21dF; 35-fold; P < .01), 16α-hydroxyprogesterone (16OHP; 28-fold; P < .01), progesterone (2-fold; P < .01), and 11β-hydroxyprogesterone (11OHP; not detected in controls; P < .01). The same steroids were the highest in media from TART cells relative to the NA cells: 11OHP, 58- to 65-fold; 21dF, 30- to 41-fold; 17OHP, 9-fold; progesterone, 9- to 12-fold; and 16OHP, 7-fold. Conclusion: Measurement of 16OHP and 11OHP along with 17OHP and 21dF by liquid chromatography/tandem mass spectrometry might comprise a biomarker panel to accurately diagnose all forms of 21OHD. PMID:25850025

  16. Reduced striatal tyrosine hydroxylase in incidental Lewy body disease

    PubMed Central

    Adler, Charles H.; Sue, Lucia I.; Peirce, Jeffrey B.; Bachalakuri, Jyothi; Dalsing-Hernandez, Jessica E.; Lue, Lih Fen; Caviness, John N.; Connor, Donald J.; Sabbagh, Marwan N.; Walker, Douglas G.

    2009-01-01

    Incidental Lewy body disease (ILBD) is the term used when Lewy bodies are found in the nervous system of subjects without clinically documented parkinsonism or dementia. The prevalence of ILBD in the elderly population has been estimated at between 3.8 and 30%, depending on subject age and anatomical site of sampling. It has been speculated that ILBD represents the preclinical stage of Parkinson’s disease (PD) and/or dementia with Lewy bodies (DLB). Studies of ILBD could potentially identify early diagnostic signs of these disorders. At present, however, it is impossible to know whether ILBD is a precursor to PD or DLB or is just a benign finding of normal aging. We hypothesized that, if ILBD represents an early stage of PD or DLB, it should be associated with depletion of striatal dopaminergic markers. Eleven subjects with ILBD and 27 control subjects were studied. The ILBD subjects ranged in age from 74 to 96 years (mean 86.5) while the control subjects’ age ranged from 75 to 102 years (mean 86.7). Controls and subjects did not differ in terms of age, postmortem interval, gender distribution, medical history conditions, brain weight, neuritic plaque density or Braak neurofibrillary stage. Quantitative ELISA measurement of striatal tyrosine hydroxylase (TH), the principal enzyme for dopamine synthesis, showed a 49.8% (P = 0.01) reduction in ILBD cases, as compared with control cases. The finding suggests that ILBD is not a benign condition but is likely a precursor to PD and/or DLB. PMID:17985144

  17. Taxol biosynthesis: taxane 13 alpha-hydroxylase is a cytochrome P450-dependent monooxygenase.

    PubMed

    Jennewein, S; Rithner, C D; Williams, R M; Croteau, R B

    2001-11-20

    A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10 beta-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5 alpha-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10 beta-hydroxylase) as a taxane 13 alpha-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species. PMID:11707604

  18. Beta hydroxylation of glycolipids from Ustilago maydis and Pseudozyma flocculosa by an NADPH-dependent β-hydroxylase.

    PubMed

    Teichmann, Beate; Lefebvre, François; Labbé, Caroline; Bölker, Michael; Linne, Uwe; Bélanger, Richard R

    2011-11-01

    Flocculosin and ustilagic acid (UA), two highly similar antifungal cellobiose lipids, are respectively produced by Pseudozyma flocculosa, a biocontrol agent, and Ustilago maydis, a plant pathogen. Both glycolipids contain a short-chain fatty acid hydroxylated at the β position but differ in the long fatty acid, which is hydroxylated at the α position in UA and at the β position in flocculosin. In both organisms, the biosynthesis genes are arranged in large clusters. The functions of most genes have already been characterized, but those of the P. flocculosa fhd1 gene and its homolog from U. maydis, uhd1, have remained undefined. The deduced amino acid sequences of these genes show homology to those of short-chain dehydrogenases and reductases (SDR). We disrupted the uhd1 gene in U. maydis and analyzed the secreted UA. uhd1 deletion strains produced UA lacking the β-hydroxyl group of the short-chain fatty acid. To analyze the function of P. flocculosa Fhd1, the corresponding gene was used to complement U. maydis Δuhd1 mutants. Fhd1 was able to restore wild-type UA production, indicating that Fhd1 is responsible for β hydroxylation of the flocculosin short-chain fatty acid. We also investigated a P. flocculosa homolog of the U. maydis long-chain fatty-acid alpha hydroxylase Ahd1. The P. flocculosa ahd1 gene, which does not reside in the flocculosin gene cluster, was introduced into U. maydis Δahd1 mutant strains. P. flocculosa Ahd1 neither complemented the U. maydis Δahd1 phenotype nor resulted in the production of β-hydroxylated UA. This suggests that P. flocculosa Ahd1 is not involved in flocculosin hydroxylation. PMID:21926207

  19. 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. PMID:26884182

  20. Identification of four variants in the tryptophan hydroxylase promoter and association to behavior.

    PubMed

    Rotondo, A; Schuebel, K; Bergen, A; Aragon, R; Virkkunen, M; Linnoila, M; Goldman, D; Nielsen, D

    1999-07-01

    One of the most replicated findings in biological psychiatry is the observation of lower 5-hydroxyindoleacetic acid concentrations, the major metabolite of serotonin, in the brain and cerebrospinal fluid of subjects with impulsive aggression. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of serotonin, however functional variants have not been reported from the coding sequence of this gene. Therefore, we screened the human TPH promoter (TPH-P) for genetic variants which could modulate TPH gene transcription. The TPH-P (2093 nucleotides) was screened for sequence variation by SSCP analysis of 260 individuals from Finnish, Italian, American Caucasian, and American Indian populations. Four common polymorphisms were identified: -7180T>G, -7065C>T, -6526A>G, and -5806G>T (designated as nucleotides upstream of the translation start site). In the Finns, the four polymorphisms had a minor allele frequency of 0.40 and in this population linkage disequilibrium between the four loci was complete. In the other populations the minor allele frequencies ranged from 0.40 to 0.45. TPH -6526A>G genotype was determined in 167 unrelated Finnish offenders and 153 controls previously studied for the TPH IVS7+779C>A polymorphism. A significant association was observed between -6526A>G and suicidality in the offenders. TPH -6526A>G and the previously reported intron seven polymorphism, TPH IVS7+779C>A, exhibited a normalised linkage disequilibrium of 0.89 in Finns. Normalized linkage disequilibrium was reduced in other populations, being 0.49 and 0.21 in Italians and American Indians, respectively. In conclusion, four TPH-P variants were identified which can be used for haplotype-based analysis to localize functional TPH alleles influencing behavior. PMID:10483053

  1. Identification of amino-terminal sequences contributing to tryptophan hydroxylase tetramer formation.

    PubMed

    Yohrling, G J; Mockus, S M; Vrana, K E

    1999-02-01

    Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of serotonin. In the rabbit, TPH exists as a tetramer of four identical 51-kDa subunits comprised of 444 amino acids each. The enzyme consists of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Previous studies demonstrated that within the carboxyl-terminus of TPH, there resides an intersubunit binding domain (a leucine zipper) that is essential for tetramer formation. However, it is hypothesized that a 4,3-hydrophobic repeat identified within the regulatory domain of TPH (residues 21-41) may also be involved in macromolecular assembly. To test this hypothesis, a series of amino-terminal deletions (Ndelta15, 30, 41, and 90) were created and assessed for macromolecular structure using size-exclusion chromatography. The amino-terminal deletion Ndelta15, upstream from the 4,3-hydrophobic repeat, was capable of forming tetramers. However, when a portion of the 4,3-hydrophobic repeat was deleted (Ndelta30), a heterogeneous elution pattern of tetramers, dimers, and monomers was observed. Complete removal of the 4,3-hydrophobic repeat (Ndelta41) rendered the enzyme incapable of forming tetramers; a monomeric form predominated. In addition, a double-point mutation (V28R-L31R) was created in the hydrophobic region of the enzyme. The introduction of two arginines (R) at positions 28 and 31 respectively, in the helix disrupted the native tetrameric state of TPH. According to size-exclusion chromatography analysis, the double-point mutant (V28R-L31R) formed dimers of 127 kDa. Thus, it is concluded that there is information within the amino-terminus that is necessary for tetramer formation of TPH. This additional intersubunit binding domain in the amino-terminus is similar to that found in the carboxyl-terminus. PMID:10636468

  2. Light response and potential interacting proteins of a grape flavonoid 3'-hydroxylase gene promoter.

    PubMed

    Sun, Run-Ze; Pan, Qiu-Hong; Duan, Chang-Qing; Wang, Jun

    2015-12-01

    Flavonoid 3'-hydroxylase (F3'H), a member of cytochrome P450 protein family, introduces B-ring hydroxyl group in the 3' position of the flavonoid. In this study, the cDNA sequence of a F3'H gene (VviF3'H), which contains an open reading frame of 1530 bp encoding a polypeptide of 509 amino acids, was cloned and characterized from Vitis vinifera L. cv. Cabernet Sauvignon. VviF3'H showed high homology to known F3'H genes, especially F3'Hs from the V. vinifera reference genome (Pinot Noir) and lotus. Expression profiling analysis using real-time PCR revealed that VviF3'H was ubiquitously expressed in all tested tissues including berries, leaves, flowers, roots, stems and tendrils, suggesting its important physiological role in plant growth and development. Moreover, the transcript level of VviF3'H gene in grape berries was relatively higher at early developmental stages and gradually decreased during véraison, and then increased in the mature phase. In addition, the promoter of VviF3'H was isolated by using TAIL-PCR. Yeast one-hybrid screening of the Cabernet Sauvignon cDNA library and subsequent in vivo/vitro validations revealed the interaction between VviF3'H promoter and several transcription factors, including members of HD-Zip, NAC, MYB and EIN families. A transcriptional regulation mechanism of VviF3'H expression is proposed for the first time. PMID:26433636

  3. 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. PMID:27070290

  4. Cloning of cDNA encoding steroid 11. beta. -hydroxylase (P450c11)

    SciTech Connect

    Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

    1987-10-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11..beta..-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage lambda vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11..beta..-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia.

  5. The Tryptophan Hydroxylase Inhibitor LX1031 Shows Clinical Benefit in Patients With Nonconstipating Irritable Bowel Syndrome

    PubMed Central

    Brown, Philip M.; Drossman, Douglas A.; Wood, Alastair J. J.; Cline, Gary A.; Frazier, Kenny S.; Jackson, Jessica I.; Bronner, Johanna; Freiman, Joel; Zambrowicz, Brian; Sands, Arthur; Gershon, Michael D.

    2016-01-01

    BACKGROUND & AIMS Serotonin (5-hydroxytryptamine [5-HT]) has an important role in gastrointestinal function. LX1031 is an oral, locally acting, small molecule inhibitor of tryptophan hydroxylase (TPH). Local inhibition of TPH in the gastrointestinal tract might reduce mucosal production of serotonin (5-HT) and be used to treat patients with nonconstipating irritable bowel syndrome (IBS). METHODS We evaluated 2 dose levels of LX1031 (250 mg or 1000 mg, given 4 times/day) in a 28-day, multicenter, randomized, double-blind, placebo-controlled study of 155 patients with nonconstipating IBS. 5-hydroxyindoleacetic acid (5-HIAA), a biomarker of pharmacodynamic activity, was measured in urine samples at baseline (24 hours after LX1031 administration), and at weeks 4 and 6 (n = 76). RESULTS Each dose of LX1031 was safe and well-tolerated. The primary efficacy end point, relief of IBS pain and discomfort, improved significantly in patients given 1000 mg LX1031 (25.5%), compared with those given placebo, at week 1 (P = .018); with nonsignificant improvements at weeks 2, 3, and 4 (17.9%, 16.3%, and 11.6%, respectively). Symptom improvement correlated with a dose-dependent reduction in 5-HIAA, a marker for TPH inhibition, from baseline until week 4. This suggests the efficacy of LX1031 is related to the extent of inhibition of 5-HT biosynthesis. Stool consistency significantly improved, compared with the group given placebo, at weeks 1 and 4 (P < .01) and at week 2 (P < .001). CONCLUSIONS In a phase 2 study, LX1031 was well tolerated, relieving symptoms and increasing stool consistency in patients with nonconstipating IBS. Symptom relief was associated with reduced levels of 5-HIAA in urine samples. This marker might be used to identify patients with nonconstipating IBS who respond to inhibitors of 5-HT synthesis. PMID:21684281

  6. 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. PMID:22664582

  7. Multiplicity of 3-Ketosteroid-9α-Hydroxylase Enzymes in Rhodococcus rhodochrous DSM43269 for Specific Degradation of Different Classes of Steroids ▿ †

    PubMed Central

    Petrusma, Mirjan; Hessels, Gerda; Dijkhuizen, Lubbert; van der Geize, Robert

    2011-01-01

    The well-known large catabolic potential of rhodococci is greatly facilitated by an impressive gene multiplicity. This study reports on the multiplicity of kshA, encoding the oxygenase component of 3-ketosteroid 9α-hydroxylase, a key enzyme in steroid catabolism. Five kshA homologues (kshA1 to kshA5) were previously identified in Rhodococcus rhodochrous DSM43269. These KshADSM43269 homologues are distributed over several phylogenetic groups. The involvement of these KshA homologues in the catabolism of different classes of steroids, i.e., sterols, pregnanes, androstenes, and bile acids, was investigated. Enzyme activity assays showed that all KSH enzymes with KshADSM43269 homologues are C-9 α-hydroxylases acting on a wide range of 3-ketosteroids, but not on 3-hydroxysteroids. KshA5 appeared to be the most versatile enzyme, with the broadest substrate range but without a clear substrate preference. In contrast, KshA1 was found to be dedicated to cholic acid catabolism. Transcriptional analysis and functional complementation studies revealed that kshA5 supported growth on any of the different classes of steroids tested, consistent with its broad expression induction pattern. The presence of multiple kshA genes in the R. rhodochrous DSM43269 genome, each displaying unique steroid induction patterns and substrate ranges, appears to facilitate a dynamic and fine-tuned steroid catabolism, with C-9 α-hydroxylation occurring at different levels during microbial steroid degradation. PMID:21642460

  8. 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

  9. Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-beta-hydroxylase.

    PubMed

    Laduron, P M

    1984-01-01

    Presynaptic muscarinic receptors labeled with [3H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [3H]quinuclidinyl benzilate or [3H]dexetimide binding revealed a distribution profile similar to that of dopamine-beta-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-beta-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-beta-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle. PMID:6198205

  10. Functional analysis of the effect of monoclonal antibodies on monkey liver phenylalanine hydroxylase.

    PubMed Central

    Jennings, I G; Russell, R G; Armarego, W L; Cotton, R G

    1986-01-01

    An analysis of the effect of eleven monoclonal antibodies on the functional characteristics of monkey liver phenylalanine hydroxylase is presented. These eleven antibodies have been found to react with eight distinct regions on the phenylalanine hydroxylase protein. PH1 antibody inhibits enzyme activity, is dependent on phenylalanine for its binding, and appears to be related to structural changes occurring during phenylalanine activation of the enzyme activity. PH2 and PH3 antibodies stimulate enzyme activity, their binding is inhibited by lysolecithin and this group apparently is recognizing structures involved in lysolecithin activation of the enzyme activity. PH5, PH10, PH12 and PH6 recognise sites on phenylalanine hydroxylase affected by lysolecithin activation. PMID:2427069