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Sample records for s-adenosylmethionine decarboxylase adometdc

  1. Conformational stabilization of rat s-adenosylmethionine decarboxylase by putrescine.

    PubMed

    Wada, Makiko; Shirahata, Akira

    2010-01-01

    The activity and processing of mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is stimulated by putrescine. To obtain new insights into the mechanism through which putrescine stimulates AdoMetDC, we investigated conformational changes in rat prostate AdoMetDC in the presence or absence of putrescine. We examined the reactivity of purified rat prostate AdoMetDC to the SH-reagent iodoacetic acid (IAA) and its susceptibility to proteolysis in the presence or absence of putrescine using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The activity of AdoMetDC treated with IAA in the absence of putrescine was reduced, but about 80% of its activity remained after treatment with IAA in the presence of putrescine. In the presence of putrescine, IAA incorporation was 1.9 mol IAA/mol of AdoMetDC α-subunit, while there was no incorporation of IAA in the β-subunit of AdoMetDC. In the absence of putrescine, 5.0 mol of IAA/mol of α-subunit and 0.9 mol of IAA/mol of β-subunit were incorporated. Only Cys292 and Cys310 were carboxymethylated by IAA in the presence of putrescine. In contrast, in the absence of putrescine all cysteines were carboxymethylated by IAA. In addition, putrescine slowed the rate of AdoMetDC degradation by trypsin. These results demonstrate that the conformation of AdoMetDC purified from rat prostate is stabilized by putrescine.

  2. Complexes of Thermotoga maritima S-adenosylmethionine decarboxylase provide insights into substrate specificity

    SciTech Connect

    Bale, Shridhar; Baba, Kavita; McCloskey, Diane E.; Pegg, Anthony E.; Ealick, Steven E.

    2010-06-25

    The polyamines putrescine, spermidine and spermine are ubiquitous aliphatic cations and are essential for cellular growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical pyruvoyl-dependent enzyme in the polyamine-biosynthetic pathway. The crystal structures of AdoMetDC from humans and plants and of the AdoMetDC proenzyme from Thermotoga maritima have been obtained previously. Here, the crystal structures of activated T. maritima AdoMetDC (TmAdoMetDC) and of its complexes with S-adenosylmethionine methyl ester and 5{prime}-deoxy-5{prime}-dimethylthioadenosine are reported. The results demonstrate for the first time that TmAdoMetDC autoprocesses without the need for additional factors and that the enzyme contains two complete active sites, both of which use residues from both chains of the homodimer. The complexes provide insights into the substrate specificity and ligand binding of AdoMetDC in prokaryotes. The conservation of the ligand-binding mode and the active-site residues between human and T. maritima AdoMetDC provides insight into the evolution of AdoMetDC.

  3. Novel protein–protein interaction between spermidine synthase and S-adenosylmethionine decarboxylase from Leishmania donovani

    SciTech Connect

    Mishra, Arjun K.; Agnihotri, Pragati; Srivastava, Vijay Kumar; Pratap, J. Venkatesh

    2015-01-09

    Highlights: • L. donovani spermidine synthase and S-adenosylmethionine decarboxylase have been cloned and purified. • S-adenosylmethionine decarboxylase has autocatalytic property. • GST pull down assay shows the two proteins to form a metabolon. • Isothermal titration calorimetry shows that binding was exothermic having K{sub d} value of 0.4 μM. • Interaction confirmed by fluorescence spectroscopy and size exclusion chromatography. - Abstract: Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein–protein interaction between SpdSyn and AdoMetDc. The protein–protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes.

  4. Trypanosoma cruzi has not lost its S-adenosylmethionine decarboxylase: characterization of the gene and the encoded enzyme.

    PubMed Central

    Persson, K; Aslund, L; Grahn, B; Hanke, J; Heby, O

    1998-01-01

    All attempts to identify ornithine decarboxylase in the human pathogen Trypanosoma cruzi have failed. The parasites have instead been assumed to depend on putrescine uptake and S-adenosylmethionine decarboxylase (AdoMetDC) for their synthesis of the polyamines spermidine and spermine. We have now identified the gene encoding AdoMetDC in T. cruzi by PCR cloning, with degenerate primers corresponding to conserved amino acid sequences in AdoMetDC proteins of other trypanosomatids. The amplified DNA fragment was used as a probe to isolate the complete AdoMetDC gene from a T. cruzi genomic library. The AdoMetDC gene was located on chromosomes with a size of approx. 1.4 Mbp, and contained a coding region of 1110 bp, specifying a sequence of 370 amino acid residues. The protein showed a sequence identity of only 25% with human AdoMetDC, the major differences being additional amino acids present in the terminal regions of the T. cruzi enzyme. As expected, a higher sequence identity (68-72%) was found in comparison with trypanosomatid AdoMetDCs. When the coding region was expressed in Escherichia coli, the recombinant protein underwent autocatalytic cleavage, generating a 33-34 kDa alpha subunit and a 9 kDa beta subunit. The encoded protein catalysed the decarboxylation of AdoMet (Km 0.21 mM) and was stimulated by putrescine but inhibited by the polyamines, weakly by spermidine and strongly by spermine. Methylglyoxal-bis(guanylhydrazone) (MGBG), a potent inhibitor of human AdoMetDC, was a poor inhibitor of the T. cruzi enzyme. This differential sensitivity to MGBG suggests that the two enzymes are sufficiently different to warrant the search for compounds that might interfere with the progression of Chagas' disease by selectively inhibiting T. cruzi AdoMetDC. PMID:9677309

  5. Product feedback regulation implicated in translational control of the Trypanosoma brucei S-adenosylmethionine decarboxylase regulatory subunit prozyme

    PubMed Central

    Xiao, Yanjing; Nguyen, Suong; Kim, Sok Ho; Volkov, Oleg A.; Tu, Benjamin P.; Phillips, Margaret A.

    2013-01-01

    Summary Human African sleeping sickness (HAT) is caused by the parasitic protozoan Trypanosoma brucei. Polyamine biosynthesis is an important drug target in the treatment of HAT. Previously we showed that trypanosomatid S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme for biosynthesis of the polyamine spermidine, is activated by heterodimer formation with an inactive paralog termed prozyme. Furthermore, prozyme protein levels were regulated in response reduced AdoMetDC activity. Herein we show that T. brucei encodes three prozyme transcripts. The 3’UTRs of these transcripts were mapped and chloramphenicol acetyltransferase (CAT) reporter constructs were used to identify a 1.2 kb region that contained a 3’UTR prozyme regulatory element sufficient to up regulate CAT protein levels (but not RNA) upon AdoMetDC inhibition, supporting the hypothesis that prozyme expression is regulated translationally. To gain insight into trans-acting factors, genetic rescue of AdoMetDC RNAi knockdown lines with human AdoMetDC was performed leading to rescue of the cell growth block, and restoration of prozyme protein to wild-type levels. Polyamine and AdoMet metabolite analysis showed that prozyme protein levels were inversely proportional to intracellular levels of decarboxylated AdoMet (dcAdoMet). These data suggest that prozyme translation may be regulated by dcAdoMet, a metabolite not previously identified to play a regulatory role. PMID:23634831

  6. New Insights into the Design of Inhibitors of Human S-Adenosylmethionine Decarboxylase: Studies of Adenine C[superscript 8] Substitution in Structural Analogues of S-Adenosylmethionine

    SciTech Connect

    McCloskey, Diane E.; Bale, Shridhar; Secrist, III, John A.; Tiwari, Anita; Moss, III, Thomas H.; Valiyaveettil, Jacob; Brooks, Wesley H.; Guida, Wayne C.; Pegg, Anthony E.; Ealick, Steven E.

    2009-04-02

    S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical enzyme in the polyamine biosynthetic pathway and depends on a pyruvoyl group for the decarboxylation process. The crystal structures of the enzyme with various inhibitors at the active site have shown that the adenine base of the ligands adopts an unusual syn conformation when bound to the enzyme. To determine whether compounds that favor the syn conformation in solution would be more potent AdoMetDC inhibitors, several series of AdoMet substrate analogues with a variety of substituents at the 8-position of adenine were synthesized and analyzed for their ability to inhibit hAdoMetDC. The biochemical analysis indicated that an 8-methyl substituent resulted in more potent inhibitors, yet most other 8-substitutions provided no benefit over the parent compound. To understand these results, we used computational modeling and X-ray crystallography to study C{sup 8}-substituted adenine analogues bound in the active site.

  7. Monomeric S-adenosylmethionine decarboxylase from plants provides an alternative to putrescine stimulation.

    PubMed

    Bennett, Eric M; Ekstrom, Jennifer L; Pegg, Anthony E; Ealick, Steven E

    2002-12-10

    S-Adenosylmethionine decarboxylase has been implicated in cell growth and differentiation and is synthesized as a proenzyme, which undergoes autocatalytic cleavage to generate an active site pyruvoyl group. In mammals, S-adenosylmethionine decarboxylase is active as a dimer in which each protomer contains one alpha subunit and one beta subunit. In many higher organisms, autocatalysis and decarboxylation are stimulated by putrescine, which binds in a buried site containing numerous negatively charged residues. In contrast, plant S-adenosylmethionine decarboxylases are fully active in the absence of putrescine, with rapid autocatalysis that is not stimulated by putrescine. We have determined the structure of the S-adenosylmethionine decarboxylase from potato, Solanum tuberosum, to 2.3 A resolution. Unlike the previously determined human enzyme structure, the potato enzyme is a monomer in the crystal structure. Ultracentrifugation studies show that the potato enzyme is also a monomer under physiological conditions, with a weak self-association constant of 6.5 x 10(4) M(-)(1) for the monomer-dimer association. Although the potato enzyme contains most of the buried charged residues that make up the putrescine binding site in the human enzyme, there is no evidence for a putrescine binding site in the potato enzyme. Instead, several amino acid substitutions, including Leu13/Arg18, Phe111/Arg114, Asp174/Val181, and Phe285/His294 (human/potato), provide side chains that mimic the role of putrescine in the human enzyme. In the potato enzyme, the positively charged residues form an extensive network of hydrogen bonds bridging a cluster of highly conserved negatively charged residues and the active site, including interactions with the catalytic residues Glu16 and His249. The results explain the constitutively high activity of plant S-adenosylmethionine decarboxylases in the absence of putrescine and are consistent with previously proposed models for how putrescine together

  8. Discovery of novel inhibitors of human S-adenosylmethionine decarboxylase based on in silico high-throughput screening and a non-radioactive enzymatic assay.

    PubMed

    Liao, Chenzeng; Wang, Yanlin; Tan, Xiao; Sun, Lidan; Liu, Sen

    2015-06-01

    Natural polyamines are small polycationic molecules essential for cell growth and development, and elevated level of polyamines is positively correlated with various cancers. As a rate-limiting enzyme of the polyamine biosynthetic pathway, S-adenosylmethionine decarboxylase (AdoMetDC) has been an attractive drug target. In this report, we present the discovery of novel human AdoMetDC (hAdoMetDC) inhibitors by coupling computational and experimental tools. We constructed a reasonable computational structure model of hAdoMetDC that is compatible with general protocols for high-throughput drug screening, and used this model in in silico screening of hAdoMetDC inhibitors against a large compound library using a battery of computational tools. We also established and validated a simple, economic, and non-radioactive enzymatic assay, which can be adapted for experimental high-throughput screening of hAdoMetDC inhibitors. Finally, we obtained an hAdoMetDC inhibitor lead with a novel scaffold. This study provides both new tools and a new lead for the developing of novel hAdoMetDC inhibitors.

  9. In vitro translation of the upstream open reading frame in the mammalian mRNA encoding S-adenosylmethionine decarboxylase.

    PubMed

    Raney, A; Baron, A C; Mize, G J; Law, G L; Morris, D R

    2000-08-11

    The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive element that suppresses translation of the associated downstream cistron in vivo. In this paper, we provide the first direct evidence of peptide synthesis from the S-adenosylmethionine decarboxylase uORF using an in vitro translation system. We examine both the influence of cation concentration on peptide synthesis and the effect of altering the uORF sequence on peptide synthesis. Synthesis of wild type and altered peptides was similar at all concentrations of magnesium tested. In contrast, synthesis of the wild type peptide was more sensitive than that of altered peptides to elevated concentrations of the naturally occurring polyamines, spermidine and spermine, as well as several polyamine analogs. The sensitivity of in vitro synthesis to spermidine was influenced by both the amino acid sequence and the length of the peptide product of the uORF. Findings from the present study correlate with the effects of the uORF and polyamines on translation of a downstream cistron in vivo and support the hypothesis that polyamines and the structure of the nascent peptide create a rate-limiting step in uORF translation, perhaps through a ribosome stalling mechanism.

  10. Spermine facilitates recovery from drought but does not confer drought tolerance in transgenic rice plants expressing Datura stramonium S-adenosylmethionine decarboxylase.

    PubMed

    Peremarti, Ariadna; Bassie, Ludovic; Christou, Paul; Capell, Teresa

    2009-06-01

    Polyamines are known to play important roles in plant stress tolerance but it has been difficult to determine precise functions for each type of polyamine and their interrelationships. To dissect the roles of putrescine from the higher polyamines spermidine and spermine, we generated transgenic rice plants constitutively expressing a heterologous S-adenosylmethionine decarboxylase (SAMDC) gene from Datura stramonium so that spermidine and spermine levels could be investigated while maintaining a constant putrescine pool. Whereas transgenic plants expressing arginine decarboxylase (ADC) produced higher levels of putrescine, spermidine and spermine, and were protected from drought stress, transgenic plants expressing SAMDC produced normal levels of putrescine and showed drought symptoms typical of wild type plants under stress, but the transgenic plants showed a much more robust recovery on return to normal conditions (90% full recovery compared to 25% partial recovery for wild type plants). At the molecular level, both wild type and transgenic plants showed transient reductions in the levels of endogenous ADC1 and SAMDC mRNA, but only wild type plants showed a spike in putrescine levels under stress. In transgenic plants, there was no spike in putrescine but a smooth increase in spermine levels at the expense of spermidine. These results confirm and extend the threshold model for polyamine activity in drought stress, and attribute individual roles to putrescine, spermidine and spermine.

  11. Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus embryos activates maternal program of apoptosis as a "fail-safe" mechanism of early embryogenesis.

    PubMed

    Kai, Masatake; Kaito, Chikara; Fukamachi, Hiroshi; Higo, Takayasu; Takayama, Eiji; Hara, Hiroshi; Ohya, Yoshikazu; Igarashi, Kazuei; Shiokawa, Koichiro

    2003-06-01

    In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or 2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due to activation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animal side blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of the apoptotic reaction. In the injection at 4- and 8-cell stages, a considerable number of embryos developed into tadpoles and in the injection at 16- and 32-cell stages, all the embryos became tadpoles, although tadpoles obtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cells of the blastomere injected with SAMDC mRNA at 8- to 32-cell stages are confined within the blastocoel at the early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continued development of the injected embryos. These results indicate that cells overexpressed with SAMDC undergo apoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection. We assume that apoptosis is executed in Xenopus early gastrulae as a "fail-safe" mechanism to eliminate physiologically-severely damaged cells to save the rest of the embryo.

  12. Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos.

    PubMed

    Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Takai, Jun-Ichi; Yoshida, Junki; Mishina, Takamichi; Fuchimukai, Kota; Ogasawara, Tsukasa; Kariya, Taro; Tashiro, Kosuke; Igarashi, Kazuei

    2010-02-01

    We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.

  13. Overexpression of carnation S-adenosylmethionine decarboxylase gene generates a broad-spectrum tolerance to abiotic stresses in transgenic tobacco plants.

    PubMed

    Wi, Soo Jin; Kim, Woo Taek; Park, Ky Young

    2006-10-01

    Polyamines (PAs), such as putrescine, spermidine, and spermine, are present in all living organism and implicate in a wide range of cellular physiological processes. We have used transgenic technology in an attempt to evaluate their potential for mitigating the adverse effects of several abiotic stresses in plants. Sense construct of full-length cDNA for S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in PA biosynthesis, from carnation (Dianthus caryophyllus L.) flower was introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium tumefaciens-mediated transformation. Several transgenic lines overexpressing SAMDC gene under the control of cauliflower mosaic virus 35S promoter accumulated soluble total PAs by 2.2 (S16-S-4) to 3.1 (S16-S-1) times than wild-type plants. The transgenic tobacco did not show any difference in organ phenotype compared to the wild-type. The number and weight of seeds increased, and net photosynthetic rate also increased in transgenic plants. Stress-induced damage was attenuated in these transgenic plants, in the symptom of visible yellowing and chlorophyll degradation after all experienced stresses such as salt stress, cold stress, acidic stress, and abscisic acid treatment. H2O2-induced damage was attenuated by spermidine treatment. Transcripts for antioxidant enzymes (ascorbate peroxidase, manganase superoxide dismutase, and glutathione S-transferase) in transgenic plants and GUS activity transformed with SAMDC promoter::GUS fusion were induced more significantly by stress treatment, compared to control. These results that the transgenic plants with sense SAMDC cDNA are more tolerant to abiotic stresses than wild-type plants suggest that PAs may play an important role in contributing stress tolerance in plants.

  14. Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli.

    PubMed

    Makhoba, Xolani Henry; Burger, Adélle; Coertzen, Dina; Zininga, Tawanda; Birkholtz, Lyn-Marie; Shonhai, Addmore

    2016-01-01

    S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial

  15. Enhanced susceptibility of photosynthesis to low-temperature photoinhibition due to interruption of chill-induced increase of S-adenosylmethionine decarboxylase activity in leaves of spinach (Spinacia oleracea L.).

    PubMed

    He, Lixiong; Nada, Kazuyoshi; Kasukabe, Yoshihisa; Tachibana, Shoji

    2002-02-01

    The possible involvement of polyamines in the chilling tolerance of spinach (Spinacia oleracea L.) was investigated focusing on photosynthesis. During chilling at 8/5C (day/night) for 6 d, S-adenosylmethionine decarboxylase (SAMDC) activity increased significantly in leaves in parallel with the increase in putrescine and spermidine (Spd) content in leaves and chloroplasts. Treatment of leaves with methylglyoxal-bis(guanylhydrazone) (MGBG), an SAMDC inhibitor, resulted in the deterioration of plant growth and photosynthesis under chilling conditions, which was reversed by the concomitant treatment with Spd through the roots. Plants treated with MGBG showed lower photochemical efficiency of PSII than either the control or plants treated with MGBG plus Spd during chilling and even after transfer to warm conditions, suggesting an increase of photoinhibition due to low Spd in chloroplasts. Indeed, MGBG-treated plants had much lower activities of thylakoid electron transport and enzymes in carbon metabolism as well as higher degrees of lipid peroxidation of thylakoid membranes compared to the control. These results indicate that the enhanced activity of SAMDC with a consequential rise of Spd in chloroplasts is crucial for the cold acclimation of the photosynthetic apparatus in spinach leaves.

  16. Expression of an antisense Datura stramonium S-adenosylmethionine decarboxylase cDNA in tobacco: changes in enzyme activity, putrescine-spermidine ratio, rhizogenic potential, and response to methyl jasmonate.

    PubMed

    Torrigiani, Patrizia; Scaramagli, Sonia; Ziosi, Vanina; Mayer, Melinda; Biondi, Stefania

    2005-05-01

    S-adenosylmethionine decarboxylase activity (SAMDC; EC 4.1.1.21) leads to spermidine and spermine synthesis through specific synthases which use putrescine, spermidine and decarboxylated S-adenosylmethionine as substrates. In order to better understand the regulation of polyamine (PA), namely spermidine and spermine, biosynthesis, a SAMDC cDNA of Datura stramonium was introduced in tobacco (Nicotiana tabacum L. cv. Xanthi) in antisense orientation under the CaMV 35S promoter, by means of Agrobacterium tumefaciens and leaf disc transformation. The effect of the genetic manipulation on PA metabolism, ethylene production and plant morphology was analysed in primary transformants (R0), and in the transgenic progeny (second generation, R1) of self-fertilised primary transformants, relative to empty vector-transformed (pBin19) and wild-type (WT) controls. All were maintained in vitro by micropropagation. Primary transformants, which were confirmed by Southern and northern analyses, efficiently transcribed the antisense SAMDC gene, but SAMDC activity and PA titres did not change. By contrast, in most transgenic R1 shoots, SAMDC activity was remarkably lower than in controls, and the putrescine-to-spermidine ratio was altered, mainly due to increased putrescine, even though putrescine oxidising activity (diamine oxidase, EC 1.4.3.6) did not change relative to controls. Despite the reduction in SAMDC activity, the production of ethylene, which shares with PAs the common precursor SAM, was not influenced by the foreign gene. Some plants were transferred to pots and acclimatised in a growth chamber. In these in vivo-grown second generation transgenic plants, at the vegetative stage, SAMDC activity was scarcely reduced, and PA titres did not change. Finally, the rhizogenic potential of in vitro-cultured leaf explants excised from antisense plants was significantly diminished as compared with WT ones, and the response to methyl jasmonate, a stress-mimicking compound, in terms

  17. Identification of Trypanosoma brucei AdoMetDC Inhibitors Using a High-Throughput Mass Spectrometry-Based Assay.

    PubMed

    Volkov, Oleg A; Cosner, Casey C; Brockway, Anthony J; Kramer, Martin; Booker, Michael; Zhong, Shihua; Ketcherside, Ariel; Wei, Shuguang; Longgood, Jamie; McCoy, Melissa; Richardson, Thomas E; Wring, Stephen A; Peel, Michael; Klinger, Jeffrey D; Posner, Bruce A; De Brabander, Jef K; Phillips, Margaret A

    2017-04-07

    Human African trypanosomiasis (HAT) is a fatal infectious disease caused by the eukaryotic pathogen Trypanosoma brucei (Tb). Available treatments are difficult to administer and have significant safety issues. S-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the parasite polyamine biosynthetic pathway. Previous attempts to develop TbAdoMetDC inhibitors into anti-HAT therapies failed due to poor brain exposure. Here, we describe a large screening campaign of two small-molecule libraries (∼400,000 compounds) employing a new high-throughput (∼7 s per sample) mass spectrometry-based assay for AdoMetDC activity. As a result of primary screening, followed by hit confirmation and validation, we identified 13 new classes of reversible TbAdoMetDC inhibitors with low-micromolar potency (IC50) against both TbAdoMetDC and T. brucei parasite cells. The majority of these compounds were >10-fold selective against the human enzyme. Importantly, compounds from four classes demonstrated high propensity to cross the blood-brain barrier in a cell monolayer assay. Biochemical analysis demonstrated that compounds from eight classes inhibited intracellular TbAdoMetDC in the parasite, although evidence for a secondary off-target component was also present. The discovery of several new TbAdoMetDC inhibitor chemotypes provides new hits for lead optimization programs aimed to deliver a novel treatment for HAT.

  18. S-adenosylmethionine and Pneumocystis.

    PubMed

    Merali, Salim; Clarkson, Allen Boykin

    2004-08-15

    Pneumocystis is a parasitic fungus causing pneumonia in immunosuppressed mammals and S-adenosylmethionine a key intermediary metabolite for all cells. Other than a species of Rickettsia bacteria and an aberrant strain of the protozoan Amoeba proteus, Pneumocystis is the only cell known unable to synthesize AdoMet; it must extract this key compound from its host. This was discovered using a culture system and confirmed by observing depletion of AdoMet in the plasma of infected animals. Depletion also occurs in patients with Pneumocystis pneumonia (PcP), a phenomenon suggested as a basis for a method for diagnosis and evaluation of response to therapy. Preliminary data indicate that deliberate reduction of host lung AdoMet by nicotine treatment is therapeutic in the rat model of Pneumocystis pneumonia.

  19. S-Adenosylmethionine — EDRN Public Portal

    Cancer.gov

    S-adenosylmethionine, also known as AdoMet or SAMe, is a biochemical intermediate involved in methyl group transfers. S-adenosylmethionine is formed from from adenosine triphosphate (ATP) and methionine, catalyzed by the enzyme methionine adenosyltransferase. S-adeosylmethionine is sold as a supplement under the common names SAM, SAMe, and SAM-e. A synthesized form of SAM-e is considered a supplement in the U.S., but SAM-e has been sold as a prescription drug in parts of Europe for decades. As a supplement, SAMe has been used to treat osteoarthritis, depression, fibromyalgia, and other conditions. Scientific studies are conflicted on the benefits of SAMe as a supplement.

  20. Inhibition of angiogenesis by S-adenosylmethionine

    SciTech Connect

    Sahin, Mehmet; Sahin, Emel; Guemueslue, Saadet; Erdogan, Abdullah; Gueltekin, Meral

    2011-04-29

    Highlights: {yields} Effects of S-adenosylmethionine (SAM) were investigated in endothelial cells. {yields} Our results showed that SAM decreased proliferation of endothelial cells. {yields} SAM influentially inhibited the percentage of cell migration. {yields} SAM probably stopped migration as independent from its effects on proliferation. {yields} SAM was shown to suppress in vitro angiogenesis. -- Abstract: Metastasis is a leading cause of mortality and morbidity in cancer. One of the steps in metastasis process is the formation of new blood vessels. Aberrant DNA methylation patterns are common in cancer cells. In recent studies, S-adenosylmethionine (SAM), which is a DNA methylating agent, has been found to have inhibitory effects on some carcinoma cells in vivo and in vitro. In the present study, we have used SAM to investigate whether it is effective against angiogenesis in vitro. Our results have shown that SAM can reduce the formation and organization of capillary-like structures of endothelial cells in tumoral environment. Besides, we have found SAM can block endothelial cell proliferation and the migration of cells towards growth factors-rich media. In conclusion, our study suggests that SAM may be used against angiogenesis as a natural bio-product.

  1. Natural history of S-adenosylmethionine-binding proteins

    PubMed Central

    Kozbial, Piotr Z; Mushegian, Arcady R

    2005-01-01

    Background S-adenosylmethionine is a source of diverse chemical groups used in biosynthesis and modification of virtually every class of biomolecules. The most notable reaction requiring S-adenosylmethionine, transfer of methyl group, is performed by a large class of enzymes, S-adenosylmethionine-dependent methyltransferases, which have been the focus of considerable structure-function studies. Evolutionary trajectories of these enzymes, and especially of other classes of S-adenosylmethionine-binding proteins, nevertheless, remain poorly understood. We addressed this issue by computational comparison of sequences and structures of various S-adenosylmethionine-binding proteins. Results Two widespread folds, Rossmann fold and TIM barrel, have been repeatedly used in evolution for diverse types of S-adenosylmethionine conversion. There were also cases of recruitment of other relatively common folds for S-adenosylmethionine binding. Several classes of proteins have unique unrelated folds, specialized for just one type of chemistry and unified by the theme of internal domain duplications. In several cases, functional divergence is evident, when evolutionarily related enzymes have changed the mode of binding and the type of chemical transformation of S-adenosylmethionine. There are also instances of functional convergence, when biochemically similar processes are performed by drastically different classes of S-adenosylmethionine-binding proteins. Comparison of remote sequence similarities and analysis of phyletic patterns suggests that the last universal common ancestor of cellular life had between 10 and 20 S-adenosylmethionine-binding proteins from at least 5 fold classes, providing for S-adenosylmethionine formation, polyamine biosynthesis, and methylation of several substrates, including nucleic acids and peptide chain release factor. Conclusion We have observed several novel relationships between families that were not known to be related before, and defined 15

  2. Metabolism of S-adenosylmethionine in rat hepatocytes: transfer of methyl group from S-adenosylmethionine by methyltransferase reactions

    SciTech Connect

    Tsukada, K.; Abe, T.; Kuwahata, T.; Mitsui, K.

    1985-08-19

    Treatment of rats with a methionine diet leads not only to a marked increase of S-adenosylmethionine synthetase in liver, but also to the increase of glycine, guanidoacetate and betaine-homocysteine methyltransferases. The activity of tRNA methyltransferase decreased with the increased amounts of methionine in the diets. However, the activities of phospholipids and S-adenosylmethionine-homocysteine methyltransferases did not show any significant change. When hepatocarcinogenesis induced by 2-fluorenylacetamide progresses, the activities of glycine and guanidoacetate methyltransferases in rat liver decreased, and could not be detected in tumorous areas 8 months after treatment. The levels of S-adenosylmethionine in the liver also decreased to levels of one-fifth of control animals at 8 months. The uptake and metabolism of (methyl-/sup 3/H)-methionine and -S-adenosylmethionine have been investigated by in vivo and isolated hepatocytes. The uptake of methionine and transfer of methyl group to phospholipid in the cells by methionine were remarkably higher than those by S-adenosylmethionine. These results indicate that phospholipids in hepatocytes accept methyl group from S-adenosylmethionine immediately, when it is synthesized from methionine, before mixing its pool in the cells. 39 references, 1 figure, 2 tables.

  3. S-adenosylmethionine levels regulate the Schwann cell DNA methylome

    PubMed Central

    Varela-Rey, Marta; Iruarrizaga-Lejarreta, Marta; Lozano, Juan José; Aransay, Ana María; Fernandez, Agustín F.; Lavin, José Luis; Mósen-Ansorena, David; Berdasco, María; Turmaine, Marc; Luka, Zigmund; Wagner, Conrad; Lu, Shelly C.; Esteller, Manel; Mirsky, Rhona; Jessen, Kristján R.; Fraga, Mario F.; Martínez-Chantar, María L.; Mato, José M.; Woodhoo, Ashwin

    2014-01-01

    SUMMARY Axonal myelination is essential for rapid saltatory impulse conduction in the nervous system, and malformation or destruction of myelin sheaths leads to motor and sensory disabilities. DNA methylation is an essential epigenetic modification during mammalian development, yet its role in myelination remains obscure. Here, using high-resolution methylome maps, we show that DNA methylation could play a key gene regulatory role in peripheral nerve myelination and that S-adenosylmethionine (SAMe), the principal methyl donor in cytosine methylation, regulates the methylome dynamics during this process. Our studies also point to a possible role of SAMe in establishing the aberrant DNA methylation patterns in a mouse model of diabetic neuropathy, implicating SAMe in the pathogenesis of this disease. These critical observations establish a link between SAMe and DNA methylation status in a defined biological system, and provides a novel mechanism that could direct methylation changes during cellular differentiation and in diverse pathological situations. PMID:24607226

  4. Folates and S-adenosylmethionine for major depressive disorder.

    PubMed

    Papakostas, George I; Cassiello, Clair F; Iovieno, Nadia

    2012-07-01

    Interest in nonpharmaceutical supplements for treating major depressive disorder (MDD) has increased significantly, both among patients and among clinicians during the past decades. Despite the large array of antidepressants (ADs) available, many patients continue to experience relatively modest response and remission rates, in addition to a burden of side effects that can hinder treatment compliance and acceptability. In this article, we review the literature on folates and S-adenosylmethionine (SAMe), 2 natural compounds linked in the 1-carbon cycle metabolic pathway, for which substantial evidence supports their involvement in mood disorders. Background information, efficacy data, proposed mechanisms of action, and side effects are reviewed. Based on existing data, supplementation with SAMe, as well as with various formulations of folates, appears to be efficacious and well tolerated in reducing depressive symptoms. Compared with other forms of folates, 5-methyltetrahydrofolate (L-methylfolate or 5-MTHF) may represent a preferable treatment option for MDD given its greater bioavailability in patients with a genetic polymorphism, and the lower risk of specific side effects associated with folic acid. Although further randomized controlled trials in this area appear warranted, SAMe and L-methylfolate may represent a useful addition to the AD armamentarium.

  5. Paramagnetic Intermediates Generated by Radical S-Adenosylmethionine (SAM) Enzymes

    PubMed Central

    2015-01-01

    Conspectus A [4Fe–4S]+ cluster reduces a bound S-adenosylmethionine (SAM) molecule, cleaving it into methionine and a 5′-deoxyadenosyl radical (5′-dA•). This step initiates the varied chemistry catalyzed by each of the so-called radical SAM enzymes. The strongly oxidizing 5′-dA• is quenched by abstracting a H-atom from a target species. In some cases, this species is an exogenous molecule of substrate, for example, l-tyrosine in the [FeFe] hydrogenase maturase, HydG. In other cases, the target is a proteinaceous residue as in all the glycyl radical forming enzymes. The generation of this initial radical species and the subsequent chemistry involving downstream radical intermediates is meticulously controlled by the enzyme so as to prevent unwanted reactions. But the manner in which this control is exerted is unknown. Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool used to gain insight into these mechanisms. In this Account, we summarize efforts to trap such radical intermediates in radical SAM enzymes and highlight four examples in which EPR spectroscopic results have shed significant light on the corresponding mechanism. For lysine 2,3-aminomutase, nearly each possible intermediate, from an analogue of the initial 5′-dA• to the product radical l-β-lysine, has been explored. A paramagnetic intermediate observed in biotin synthase is shown to involve an auxiliary [FeS] cluster whose bridging sulfide is a co-substrate for the final step in the biosynthesis of vitamin B7. In HydG, the l-tyrosine substrate is converted in unprecedented fashion to a 4-oxidobenzyl radical on the way to generating CO and CN– ligands for the [FeFe] cluster of hydrogenase. And finally, EPR has confirmed a mechanistic proposal for the antibiotic resistance protein Cfr, which methylates the unactivated sp2-hybridized C8-carbon of an adenosine base of 23S ribosomal RNA. These four systems provide just a brief survey of the ever-growing set

  6. S-adenosylmethionine prevents total parenteral nutrition-induced cholestasis in the rat.

    PubMed

    Belli, D C; Fournier, L A; Lepage, G; Yousef, I; Roy, C C

    1994-07-01

    Both an excess and an imbalance of amino acids have been associated with total parenteral nutrition-induced cholestasis. The present study was undertaken to further our understanding of this condition in light of observations that methyl donor amino acids may be protective. Rats were maintained on Travasol (3.4 g amino acids/24 h) and dextrose (10.2 g/24 h) with and without the "active methyl" S-adenosylmethionine at a dose of 75 mg/kg/24 h for 5 days, and compared to control rats on dextrose alone (10.2 g/24 h) with free access to rat chow. Bile flow (microliters/min) was lower (p < 0.025) in the Travasol (8.65 +/- 0.78) than in the control group (12.30 +/- 0.52) and was restored in the Travasol+S-adenosylmethionine animals (11.42 +/- 10). Furthermore, the bile acid secretory rate (mumol/h) was higher (p < 0.05) with S-adenosylmethionine (23.34 +/- 3.71) than without S-adenosylmethionine (14.16 +/- 2.19). As expected, the molar ratio of biliary cholesterol was lower (p < 0.005) in both total parenteral nutrition groups. However, in the total parenteral nutrition group without S-adenosylmethionine, there was also a decrease in the molar ratio of phospholipids which correlated well with the bile acid secretory rate. Analysis of liver plasma membranes showed that a lower activity of Na+K(+)-ATPase (mumol Pi/mg protein/h) (p < 0.005) in the Travasol animals (6.26 +/- 0.53) was restored to control values (15.20 +/- 1.43) by the addition of S-adenosylmethionine (17.07 +/- 2.87). In the three groups, a close correlation was observed between Na+K(+)-ATPase activity and bile flow.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. S-adenosylmethionine lowers the inflammatory response in macrophages associated with changes in DNA methylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    S-adenosylmethionine (SAM), the unique methyl donor in DNA methylation, has been shown to lower inflammation. We assessed whether epigenetic mechanisms mediate this effect. Human THP-1 cells were differentiated into macrophages and treated with 0 micromole/L, 500 micromole/L or 1000 micromole/L SAM ...

  8. S-Adenosylmethionine Synthetase 3 Is Important for Pollen Tube Growth1[OPEN

    PubMed Central

    Zou, Ting

    2016-01-01

    S-Adenosylmethionine is widely used in a variety of biological reactions and participates in the methionine (Met) metabolic pathway. In Arabidopsis (Arabidopsis thaliana), one of the four S-adenosylmethionine synthetase genes, METHIONINE ADENOSYLTRANSFERASE3 (MAT3), is highly expressed in pollen. Here, we show that mat3 mutants have impaired pollen tube growth and reduced seed set. Metabolomics analyses confirmed that mat3 pollen and pollen tubes overaccumulate Met and that mat3 pollen has several metabolite profiles, such as those of polyamine biosynthesis, which are different from those of the wild type. Additionally, we show that disruption of Met metabolism in mat3 pollen affected transfer RNA and histone methylation levels. Thus, our results suggest a connection between metabolism and epigenetics. PMID:27482079

  9. Effects of selenomethionine on cell growth and on S-adenosylmethionine metabolism in cultured malignant cells.

    PubMed Central

    Kajander, E O; Harvima, R J; Kauppinen, L; Akerman, K K; Martikainen, H; Pajula, R L; Kärenlampi, S O

    1990-01-01

    The effects of selenomethionine (SeMet) on the growth of 17 cultured cell lines were studied. SeMet in the culture medium of three hepatoma cell lines promoted cell growth at subcytotoxic levels (1-20 microM), but the growth of malignant lymphoid and myeloid cells was not stimulated. L-SeMet was cytotoxic to all 17 cell lines when assayed after culture for 3-10 days. A 50% growth inhibition was observed by 30-160 microM-SeMet in a culture medium containing 100 microM-methionine. SeMet cytotoxicity to normal (fibroblasts) and malignant cells was rather similar, excluding specific antineoplastic cytotoxicity. Cytotoxicity was increased by decreasing concentrations of methionine. The DL form of SeMet was less cytotoxic than the L form. L-SeMet was metabolized to a selenium analogue of S-adenosylmethionine approximately as effectively as the natural sulphur analogue methionine in malignant R1.1 lymphoblasts. Concomitantly, S-adenosylmethionine pools were decreased. This occurred early and at cytotoxic SeMet levels. Methionine adenosyltransferase activity was not altered by SeMet treatment. ATP pools were not affected early, and decreases in the synthesis of DNA and protein took place late and were apparently related to cell death. RNA synthesis was slightly stimulated at low cytotoxic SeMet levels by 24 h, but was markedly inhibited after 48 h. The SeMet analogue of S-adenosylmethionine could be effectively utilized in a specific enzymic transmethylation. Neither S-adenosylhomocysteine nor its selenium analogue accumulated in the treated cells. These findings together suggest a direct or indirect involvement of S-adenosylmethionine metabolism in SeMet cytotoxicity, but exclude a gross blockage of transmethylations. PMID:2339986

  10. Inclusion of thiamine diphosphate and S-adenosylmethionine at their chemically active sites.

    PubMed

    Schrader, Thomas; Fokkens, Michael; Klärner, Frank-Gerrit; Polkowska, Jolanta; Bastkowski, Frank

    2005-12-09

    [structure: see text] Molecular clips functionalized by phosphonate or phosphate groups bind thiamine diphosphate (TPP) and S-adenosylmethionine (SAM) with high affinity in water; both sulfur-based cofactors transfer organic groups to biomolecules. For TPP, various analytical tools point toward a simultaneous insertion of both heterocyclic rings into the electron-rich clip cavity. Similarly, SAM is also embedded with its sulfonium moiety inside the receptor cavity. This paves the way for enzyme models and direct interference with enzymatic processes.

  11. Marine-Derived Metabolites of S-Adenosylmethionine as Templates for New Anti-Infectives

    PubMed Central

    Sufrin, Janice R.; Finckbeiner, Steven; Oliver, Colin M.

    2009-01-01

    S-Adenosylmethionine (AdoMet) is a key biochemical co-factor whose proximate metabolites include methylated macromolecules (e.g., nucleic acids, proteins, phospholipids), methylated small molecules (e.g., sterols, biogenic amines), polyamines (e.g., spermidine, spermine), ethylene, and N-acyl-homoserine lactones. Marine organisms produce numerous AdoMet metabolites whose novel structures can be regarded as lead compounds for anti-infective drug design. PMID:19841722

  12. Mutations in the Drosophila melanogaster gene encoding S-adenosylmethionine suppress position-effect variegation

    SciTech Connect

    Larsson, J.; Rasmuson-Lestander, A.; Zhang, Jingpu

    1996-06-01

    In Drosophila melanogaster, the study of trans-acting modifier mutations of position-effect variegation and Polycomb group (Pc-G) genes have been useful tools to investigate genes involved in chromatin structure. We have cloned a modifier gene, Suppressor of zeste 5 (Su(z)5), which encodes S-adenosylmethionine synthetase, and we present here molecular results and data concerning its expression in mutants and genetic interactions. The mutant alleles Su(z)5, l(2)R23 and l(2)M6 show suppression of w{sup m4} and also of two white mutants induced by roo element insertions in the regulatory region i.e., w{sup is} (in combination with z{sup 1}) and w{sup sp1}. Two of the Su(z)5 alleles, as well as a deletion of the gene, also act as enhancers of Polycomb by increasing the size of sex combes on midleg. The results suggest that Su(z)5 is connected with regulation of chromatin structure. The enzyme S-adenosylmethionine synthetase is involved in the synthesis of S-adenosylmethionine, a methyl group donor and also, after decarboxylation, a propylamino group donor in the biosynthesis of polyamines. Our results from HPLC analysis show that in ovaries from heterozygous Su(z)5 mutants the content of spermine is significantly reduced. Results presented here suggest that polyamines are an important molecule class in the regulation of chromatin structure. 50 refs., 5 figs., 3 tabs.

  13. Lysine 2,3-aminomutase. Support for a mechanism of hydrogen transfer involving S-adenosylmethionine.

    PubMed

    Baraniak, J; Moss, M L; Frey, P A

    1989-01-25

    The conversion of L-lysine to L-beta-lysine is catalyzed by lysine 2,3-aminomutase. The reaction involves the interchange of the 2-amino group of lysine with a hydrogen at carbon 3. As such the reaction is formally analogous to adenosylcobalamin-dependent rearrangements. However, the enzyme does not contain and is not activated by this coenzyme. Instead it contains iron and pyridoxal phosphate and is activated by S-adenosylmethionine. Earlier experiments implicated adenosyl-C-5' of S-adenosylmethionine in the hydrogen transfer mechanism, apparently in a role similar or analogous to that of adenosyl moiety of adenosylcobalamin in the B12-dependent rearrangements. The question of whether both hydrogens or only one hydrogen at adenosyl-C-5' participate in the hydrogen-transfer process has been addressed by carrying out the lysine 2,3-aminomutase reaction with S-[5'-3H] adenosylmethionine in the presence of 10 times its molar concentration of enzyme. Under these conditions all of the tritium appeared in lysine and beta-lysine, showing that C-5'-hydrogens participate. To determine whether hydrogen transfer is compulsorily intermolecular and intramolecular, various molar ratios of [3,3-2H2]lysine and unlabeled lysine were submitted to the action of lysine 2,3-aminomutase under conditions in which 10-15% conversion to beta-lysine occurred. Mass spectral analysis of the beta-lysine for monodeutero and dideutero species showed conclusively that hydrogen transfer is both intramolecular and intermolecular. The results quantitatively support our postulate that activation of the enzyme involves a transformation of S-adenosylmethionine into a form that promotes the generation of an adenosyl-5' free radical, which abstracts hydrogen from lysine to form 5'-deoxyadenosine as an intermediate.

  14. Dietary betaine promotes generation of hepatic S-adenosylmethionine and protects the liver from ethanol-induced fatty infiltration.

    PubMed

    Barak, A J; Beckenhauer, H C; Junnila, M; Tuma, D J

    1993-06-01

    Previous studies have shown that ethanol feeding to rats alters methionine metabolism by decreasing the activity of methionine synthetase. This is the enzyme that converts homocysteine in the presence of vitamin B12 and N5-methyltetrahydrofolate to methionine. The action of the ethanol results in an increase in the hepatic level of the substrate N5-methyltetrahydrofolate but as an adaptive mechanism, betaine homocysteine methyltransferase, is induced in order to maintain hepatic S-adenosylmethionine at normal levels. Continued ethanol feeding, beyond 2 months, however, produces depressed levels of hepatic S-adenosylmethionine. Because betaine homocysteine methyltransferase is induced in the livers of ethanol-fed rats, this study was conducted to determine what effect the feeding of betaine, a substrate of betaine homocysteine methyltransferase, has on methionine metabolism in control and ethanol-fed animals. Control and ethanol-fed rats were given both betaine-lacking and betaine-containing liquid diets for 4 weeks, and parameters of methionine metabolism were measured. These measurements demonstrated that betaine administration doubled the hepatic levels of S-adenosylmethionine in control animals and increased by 4-fold the levels of hepatic S-adenosylmethionine in the ethanol-fed rats. The ethanol-induced infiltration of triglycerides in the liver was also reduced by the feeding of betaine to the ethanol-fed animals. These results indicate that betaine administration has the capacity to elevate hepatic S-adenosylmethionine and to prevent the ethanol-induced fatty liver.

  15. Defective cystathionine beta-synthase regulation by S-adenosylmethionine in a partially pyridoxine responsive homocystinuria patient.

    PubMed Central

    Kluijtmans, L A; Boers, G H; Stevens, E M; Renier, W O; Kraus, J P; Trijbels, F J; van den Heuvel, L P; Blom, H J

    1996-01-01

    We determined the molecular basis of cystathionine beta-synthase (CBS) deficiency in a partially pyridoxine-responsive homocystinuria patient. Direct sequencing of the entire CBS cDNA revealed the presence of a homozygous G1330A transition. This mutation causes an amino acid change from aspartic acid to asparagine (D444N) in the regulatory domain of the protein and abolishes a TaqI restriction site at DNA level. Despite the homozygous mutation, CBS activities in extracts of cultured fibroblasts of this patient were not in the homozygous but in the heterozygous range. Furthermore, we observed no stimulation of CBS activity by S-adenosylmethionine, contrary to a threefold stimulation in control fibroblast extract. The mutation was introduced in an E. coli expression system and CBS activities were measured after addition of different S-adenosylmethionine concentrations (0-200 microM). Again, we observed a defective stimulation of CBS activity by S-adenosylmethionine in the mutated construct, whereas the normal construct showed a threefold stimulation in activity. These data suggest that this D444N mutation interferes in S-adenosylmethionine regulation of CBS. Furthermore, it indicates the importance of S-adenosylmethionine regulation of the transsulfuration pathway in homocysteine homeostasis in humans. PMID:8755636

  16. The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor.

    PubMed Central

    Eliasson, R; Fontecave, M; Jörnvall, H; Krook, M; Pontis, E; Reichard, P

    1990-01-01

    Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2+ ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E. coli. After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions. One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several defined protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein B1) of the aerobic reductase but not by monoclonal anti-B1 antibodies. Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component. Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established. The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction. One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal. We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP. Images PMID:2185465

  17. Comparative Effects of Triflusal, S-Adenosylmethionine, and Dextromethorphan over Intestinal Ischemia/Reperfusion Injury

    PubMed Central

    Cámara-Lemarroy, Carlos R.; Guzmán-de la Garza, Francisco J.; Cordero-Pérez, Paula; Alarcón-Galván, Gabriela; Torres-Gonzalez, Liliana; Muñoz-Espinosa, Linda E.; Fernández-Garza, Nancy E.

    2011-01-01

    Ischemia/reperfusion (I/R) is a condition that stimulates an intense inflammatory response. No ideal treatment exists. Triflusal is an antiplatelet salicylate derivative with anti-inflammatory effects. S-adenosylmethionine is a metabolic precursor for glutathione, an endogenous antioxidant. Dextromethorphan is a low-affinity N-methyl-D-aspartate receptor inhibitor. There is evidence that these agents modulate some of the pathways involved in I/R physiopathology. Intestinal I/R was induced in rats by clamping the superior mesenteric artery for 60 minutes, followed by 60 minutes of reperfusion. Rats either received saline or the drugs studied. At the end of the procedure, serum concentrations of tumor necrosis factor-alpha (TNF-alpha), malonaldehyde (MDA), and total antioxidant capacity (TAC) were determined and intestinal morphology analyzed. I/R resulted in tissue damage, serum TNF-alpha and MDA elevations, and depletion of TAC. All drugs showed tissue protection. Only triflusal reduced TNF-alpha levels. All drugs lowered MDA levels, but only triflusal and S-adenosylmethionine maintained the serum TAC. PMID:22125445

  18. SPASM and Twitch Domains in S-Adenosylmethionine (SAM) Radical Enzymes*

    PubMed Central

    Grell, Tsehai A. J.; Goldman, Peter J.; Drennan, Catherine L.

    2015-01-01

    S-Adenosylmethionine (SAM, also known as AdoMet) radical enzymes use SAM and a [4Fe-4S] cluster to catalyze a diverse array of reactions. They adopt a partial triose-phosphate isomerase (TIM) barrel fold with N- and C-terminal extensions that tailor the structure of the enzyme to its specific function. One extension, termed a SPASM domain, binds two auxiliary [4Fe-4S] clusters and is present within peptide-modifying enzymes. The first structure of a SPASM-containing enzyme, anaerobic sulfatase-maturating enzyme (anSME), revealed unexpected similarities to two non-SPASM proteins, butirosin biosynthetic enzyme 2-deoxy-scyllo-inosamine dehydrogenase (BtrN) and molybdenum cofactor biosynthetic enzyme (MoaA). The latter two enzymes bind one auxiliary cluster and exhibit a partial SPASM motif, coined a Twitch domain. Here we review the structure and function of auxiliary cluster domains within the SAM radical enzyme superfamily. PMID:25477505

  19. [Double-blind polycentric study of the action of S-adenosylmethionine in hepatic cirrhosis].

    PubMed

    Labo, G; Miglio, F; D'Ambro, A; Bellobuono, A; Ideo, G; Dioguardi, N; Bernardi, M; Corazza, G R; Gasbarrini, G; Avogaro, P; Pasquino, M

    1975-05-02

    Two comparable groups of patients with hepatic cirrhosis of different genesis in a compensation phase have been treated for 30 days with S-adenosylmethionine and vitamine B-12 (28 cases) or with vitamine B-12 alone (25 cases). The drugs were given by slow intravenous route at the daily dose of 150 mg of SAMe and 2000 gamma of vit. B-12 or of 2000 gamma of vit. B-12 alone, in two adminstrations. An evaluation of the results was carried out mostly on the laboratory data testing the liver function. Only the group of patients who had received SAMe showed significant modifications of all the parameters considered. This is confirming SAMe ability to restore hepatocyte activity bringing also to normal the protein synthesis.

  20. As(III) S-adenosylmethionine methyltransferases and other arsenic binding proteins

    PubMed Central

    Ajees, A. Abdul; Rosen, Barry P.

    2014-01-01

    Efflux is by far the most common means of arsenic detoxification is by methylation catalyzed by a family of As(III) S-adenosylmethionine (SAM) methyltransferases (MTs) enzymes designated ArsM in microbes or AS3MT in higher eukaryotes. The protein sequence of more than 5000 AS3MT/ArsM orthologues have been deposited in the NCBI database, mostly in prokaryotic and eukaryotic microbes. As(III) SAM MTs are members of a large superfamily of MTs involved in numerous physiological functions. ArsMs detoxify arsenic by conversion of inorganic trivalent arsenic (As(III)) into mono-, di- and trimethylated species that may be more toxic and carcinogenic than inorganic arsenic. The pathway of methylation remains controversial. Several hypotheses will be examined in this review. PMID:26366023

  1. Boron Deprivation Decreases Liver S-Adenosylmethionine and Spermidine and Increases Plasma Homocysteine and Cysteine in Rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two experiments were conducted with weanling Sprague-Dawley rats to determine whether changes in S-adenosylmethionine utilization or metabolism contribute to the diverse responses to boron deprivation. In both experiments, four treatment groups of 15 male rats were fed ground corn-casein based diets...

  2. Boron deprivation decreases liver S-adenosylmethionine and spermidine and increases plasma homocysteine and cysteine in rats.

    PubMed

    Nielsen, Forrest Harold

    2009-01-01

    Two experiments were conducted with weanling Sprague-Dawley rats to determine whether changes in S-adenosylmethionine utilization or metabolism contribute to the diverse responses to boron deprivation. In both experiments, four treatment groups of 15 male rats were fed ground corn-casein based diets that contained an average of 0.05 mg (experiment 1) or 0.15 mg (experiment 2) boron/kg. In experiment 2, some ground corn was replaced by sucrose and fructose to increase oxidative stress. The dietary variables were supplemental 0 (boron-deprived) or 3 (boron-adequate) mg boron/kg and different fat sources (can affect the response to boron) of 75 g corn oil/kg or 65 g fish (menhaden) oil/kg plus 10 linoleic acid/kg. When euthanized at age 20 (experiment 1) and 18 (experiment 2) weeks, rats fed the low-boron diet were considered boron-deprived because they had decreased boron concentrations in femur and kidney. Boron deprivation regardless of dietary oil increased plasma cysteine and homocysteine and decreased liver S-adenosylmethionine, S-adenosylhomocysteine, and spermidine. Plasma concentration of 8-iso-prostaglandin F2alpha (indicator of oxidative stress) was not affected by boron, but was decreased by feeding fish oil instead of corn oil. Fish oil instead of corn oil decreased S-adenosylmethionine, increased spermidine, and did not affect S-adenosylhomocysteine concentrations in liver. Additionally, fish oil versus corn oil did not affect plasma homocysteine in experiment 1, and slightly increased it in experiment 2. The findings suggest that boron is bioactive through affecting the formation or utilization of S-adenosylmethionine. Dietary fatty acid composition also affects S-adenosylmethionine formation or utilization, but apparently through a mechanism different from that of boron.

  3. Identification of Catalytic Residues in the As(III) S-Adenosylmethionine Methyltransferase

    PubMed Central

    Marapakala, Kavitha; Qin, Jie; Rosen, Barry P.

    2012-01-01

    The enzyme As(III) S-adenosylmethionine methyltransferase (EC 2.1.1.137) (ArsM or AS3MT) is found in members of every kingdom, from bacteria to humans. In these enzymes, there are three conserved cysteine residues at positions 72, 174, and 224 in the CmArsM orthologue from the thermophilic eukaryotic alga Cyanidioschyzon sp. 5508. Substitution of any of the three led to loss of As(III) methylation. In contrast, a C72A mutant still methylated trivalent methylarsenite [MAs(III)]. Protein fluorescence of a single-tryptophan mutant reported binding of As(III) or MAs(III). As(GS)3 and MAs(GS)2 bound significantly faster than As(III), suggesting that the glutathionylated arsenicals are preferred substrates for the enzyme. Protein fluorescence also reported binding of Sb(III), and the purified enzyme methylated and volatilized Sb(III). The results suggest that all three cysteine residues are necessary for the first step in the reaction, As(III) methylation, but that only Cys174 and Cys224 are required for the second step, methylation of MAs(III) to dimethylarsenite [DMAs(III)]. The rate-limiting step was identified as the conversion of DMAs(III) to trimethylarsine, and DMAs(III) accumulates as the principal product. PMID:22257120

  4. S-Adenosylmethionine-Binding Properties of a Bacterial Phospholipid N-Methyltransferase▿†

    PubMed Central

    Aktas, Meriyem; Gleichenhagen, Jan; Stoll, Raphael; Narberhaus, Franz

    2011-01-01

    The presence of the membrane lipid phosphatidylcholine (PC) in the bacterial membrane is critically important for many host-microbe interactions. The phospholipid N-methyltransferase PmtA from the plant pathogen Agrobacterium tumefaciens catalyzes the formation of PC by a three-step methylation of phosphatidylethanolamine via monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine. The methyl group is provided by S-adenosylmethionine (SAM), which is converted to S-adenosylhomocysteine (SAH) during transmethylation. Despite the biological importance of bacterial phospholipid N-methyltransferases, little is known about amino acids critical for binding to SAM or phospholipids and catalysis. Alanine substitutions in the predicted SAM-binding residues E58, G60, G62, and E84 in A. tumefaciens PmtA dramatically reduced SAM-binding and enzyme activity. Homology modeling of PmtA satisfactorily explained the mutational results. The enzyme is predicted to exhibit a consensus topology of the SAM-binding fold consistent with cofactor interaction as seen with most structurally characterized SAM-methyltransferases. Nuclear magnetic resonance (NMR) titration experiments and 14C-SAM-binding studies revealed binding constants for SAM and SAH in the low micromolar range. Our study provides first insights into structural features and SAM binding of a bacterial phospholipid N-methyltransferase. PMID:21602340

  5. Arsenic methylation and volatilization by arsenite S-adenosylmethionine methyltransferase in Pseudomonas alcaligenes NBRC14159.

    PubMed

    Zhang, Jun; Cao, Tingting; Tang, Zhu; Shen, Qirong; Rosen, Barry P; Zhao, Fang-Jie

    2015-04-01

    Inorganic arsenic (As) is highly toxic and ubiquitous in the environment. Inorganic As can be transformed by microbial methylation, which constitutes an important part of the As biogeochemical cycle. In this study, we investigated As biotransformation by Pseudomonas alcaligenes NBRC14159. P. alcaligenes was able to methylate arsenite [As(III)] rapidly to dimethylarsenate and small amounts of trimethylarsenic oxide. An arsenite S-adenosylmethionine methyltransferase, PaArsM, was identified and functionally characterized. PaArsM shares low similarities with other reported ArsM enzymes (<55%). When P. alcaligenes arsM gene (PaarsM) was disrupted, the mutant lost As methylation ability and became more sensitive to As(III). PaarsM was expressed in the absence of As(III) and the expression was further enhanced by As(III) exposure. Heterologous expression of PaarsM in an As-hypersensitive strain of Escherichia coli conferred As(III) resistance. Purified PaArsM protein methylated As(III) to dimethylarsenate as the main product in the medium and also produced dimethylarsine and trimethylarsine gases. We propose that PaArsM plays a role in As methylation and detoxification of As(III) and could be exploited in bioremediation of As-contaminated environments.

  6. Elements involved in S-adenosylmethionine-mediated regulation of the Saccharomyces cerevisiae MET25 gene.

    PubMed Central

    Thomas, D; Cherest, H; Surdin-Kerjan, Y

    1989-01-01

    In Saccharomyces cerevisiae, the MET25 gene encodes O-acetylhomoserine sulfhydrylase. Synthesis of this enzyme is repressed by the presence of S-adenosylmethionine (AdoMet) in the growth medium. We identified cis elements required for MET25 expression by analyzing small deletions in the MET25 promoter region. The results revealed a regulatory region, acting as an upstream activation site, that activated transcription of MET25 in the absence of methionine or AdoMet. We found that, for the most part, repression of MET25 expression was due to a lack of activation at this site, reinforced by an independent repression mechanism. The activation region contained a repeated dyad sequence that is also found in the promoter regions of other unlinked but coordinately regulated genes (MET3, MET2, and SAM2). We show that the presence of the two dyads is necessary for maximal gene expression. Moreover, we demonstrate that in addition to this transcriptional regulation, a posttranscriptional regulation, probably targeted at the 5' region of mRNA, is involved in MET25 expression. Images PMID:2552290

  7. S-adenosylmethionine stimulates fatty acid metabolism-linked gene expression in porcine muscle satellite cells.

    PubMed

    Yue, Tao; Fang, Qian; Yin, JingDong; Li, DeFa; Li, Wei

    2010-10-01

    Evidence indicates that both S-adenosylmethionine (SAMe) metabolism and intramuscular fat are associated with insulin resistance and type II diabetes. However, it is still unknown whether SAMe have effects on intramuscular adipogenesis. The present study investigated the roles of SAMe in the adipogenic differentiation of porcine muscle satellite cells. Cells isolated from neonatal pig muscle were treated with different concentrations of SAMe (0, 0.5 and 1.0 mM) for 24 h, induced for a 9-day adipogenic differentiation and were finally stained by oil red O staining. The adipocyte determination and differentiation factor-1 (ADD1) and peroxisome proliferator-activated receptor gamma (PPARγ) mRNA and protein were stimulated by SAMe treatment in a dose-dependent manner. Lipoprotein lipase (LPL) mRNA and protein were enhanced in 1.0 mM treatment group, compared with the control. No significant difference was observed in the intracellular lipid content among treatments. These results provide evidence that SAMe may be associated with intramuscular adipogenesis and indicate a novel action of SAMe in fat metabolism.

  8. Functional proteomics of nonalcoholic steatohepatitis: Mitochondrial proteins as targets of S-adenosylmethionine

    PubMed Central

    Santamaría, Enrique; Avila, Matías A.; Latasa, M. Ujue; Rubio, Angel; Martín-Duce, Antonio; Lu, Shelly C.; Mato, José M.; Corrales, Fernando J.

    2003-01-01

    Recent work shows that S-adenosylmethionine (AdoMet) helps maintain normal liver function as chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. The mechanisms by which these nontraditional functions of AdoMet occur are unknown. Here, we use knockout mice deficient in hepatic AdoMet synthesis (MAT1A−/−) to study the proteome of the liver during the development of steatohepatitis. One hundred and seventeen protein spots, differentially expressed during the development of steatohepatitis, were selected and identified by peptide mass fingerprinting. Among them, 12 proteins were found to be affected from birth, when MAT1A−/− expression is switched on in WT mouse liver, to the rise of histological lesions, which occurs at ≈8 months. Of the 12 proteins, 4 [prohibitin 1 (PHB1), cytochrome c oxidase I and II, and ATPase β-subunit] have known roles in mitochondrial function. We show that the alteration in expression of PHB1 correlates with a loss of mitochondrial function. Experiments in isolated rat hepatocytes indicate that AdoMet regulates PHB1 content, thus suggesting ways by which steatohepatitis may be induced. Importantly, we found the expression of these mitochondrial proteins was abnormal in ob/ob mice and obese patients who are at risk for nonalcoholic steatohepatitis. PMID:12631701

  9. Associations between S-adenosylmethionine, S-adenosylhomocysteine, and colorectal adenoma risk are modified by sex

    PubMed Central

    Shrubsole, Martha J; Wagner, Conrad; Zhu, Xiangzhu; Hou, Lifang; Loukachevitch, Lioudmila V; Ness, Reid M; Zheng, Wei

    2015-01-01

    Methionine metabolism is an important component of one-carbon metabolism. S-adenosylmethionine (SAM), the methyl donor for nearly all methylation reactions, is irreversibly converted to S-adenosylhomocysteine (SAH), an inhibitor of methyltransferases, some of which are key enzymes for methylation. Changes in DNA methylation are common in colorectal cancers. We evaluated plasma SAM and SAH with colorectal adenoma risk in a matched case-control study conducted among individuals undergoing routine colonoscopy. 216 cases were individually matched to polyp-free controls in a 1:1 ratio on age (± 5 years), sex, race (white/non-white), study site (academic medical center/VA hospital) and date of sample collection (± 60 days). Sex-specific quantiles were evaluated based on the control distribution due to vastly different metabolite levels by sex. Conditional logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Among males, both higher SAM (OR = 0.38, 95% CI: 0.18-0.77, p for trend = 0.007) and higher SAH (OR = 0.45, 95% CI: 0.22-0.91, p for trend = 0.02) were associated with statistically significantly decreased risks of colorectal adenoma in comparison to lowest plasma SAM or SAH tertile. Conversely, among females, both higher SAM and higher SAH were associated with increased risk of colorectal adenoma, which was statistically significant for SAH (OR = 5.18, 95% CI: 1.09-24.62, p for trend = 0.04). The difference in these associations between men and women was statistically significant (p < 0.05). The ratio of SAM/SAH was not associated with colorectal adenoma risk among males or females. These findings suggest SAM and SAH may be involved in the development of colorectal adenoma and the association may be modified by sex. PMID:25628954

  10. Arsenic methylation by an arsenite S-adenosylmethionine methyltransferase from Spirulina platensis.

    PubMed

    Guo, Yuqing; Xue, Ximei; Yan, Yu; Zhu, Yongguan; Yang, Guidi; Ye, Jun

    2016-11-01

    Arsenic-contaminated water is a serious hazard for human health. Plankton plays a critical role in the fate and toxicity of arsenic in water by accumulation and biotransformation. Spirulina platensis (S. platensis), a typical plankton, is often used as a supplement or feed for pharmacy and aquiculture, and may introduce arsenic into the food chain, resulting in a risk to human health. However, there are few studies about how S. platensis biotransforms arsenic. In this study, we investigated arsenic biotransformation by S. platensis. When exposed to arsenite (As(III)), S. platensis accumulated arsenic up to 4.1mg/kg dry weight. After exposure to As(III), arsenate (As(V)) was the predominant species making up 64% to 86% of the total arsenic. Monomethylarsenate (MMA(V)) and dimethylarsenate (DMA(V)) were also detected. An arsenite S-adenosylmethionine methyltransferase from S. platensis (SpArsM) was identified and characterized. SpArsM showed low identity with other reported ArsM enzymes. The Escherichia coli AW3110 bearing SparsM gene resulted in As(III) methylation and conferring resistance to As(III). The in vitro assay showed that SpArsM exhibited As(III) methylation activity. DMA(V) and a small amount of MMA(V) were detected in the reaction system within 0.5hr. A truncated SpArsM derivative lacking the last 34 residues still had the ability to methylate As(III). The three single mutants of SpArsM (C59S, C186S, and C238S) abolished the capability of As(III) methylation, suggesting the three cysteine residues are involved in catalysis. We propose that SpArsM is responsible for As methylation and detoxification of As(III) and may contribute to As biogeochemistry.

  11. Identification and Characterization of the Chlamydia trachomatis L2 S-Adenosylmethionine Transporter

    PubMed Central

    Binet, Rachel; Fernandez, Reinaldo E.; Fisher, Derek J.; Maurelli, Anthony T.

    2011-01-01

    ABSTRACT Methylation is essential to the physiology of all cells, including the obligate intracellular bacterium Chlamydia. Nevertheless, the methylation cycle is under strong reductive evolutionary pressure in Chlamydia. Only Parachlamydia acanthamoebae and Waddlia chondrophila genome sequences harbor homologs to metK, encoding the S-adenosylmethionine (SAM) synthetase required for synthesis of SAM, and to sahH, which encodes the S-adenosylhomocysteine (SAH) hydrolase required for detoxification of SAH formed after the transfer of the methyl group from SAM to the methylation substrate. Transformation of a conditional-lethal ΔmetK mutant of Escherichia coli with a genomic library of Chlamydia trachomatis L2 identified CTL843 as a putative SAM transporter based on its ability to allow the mutant to survive metK deficiency only in the presence of extracellular SAM. CTL843 belongs to the drug/metabolite superfamily of transporters and allowed E. coli to transport S-adenosyl-l-[methyl-14C]methionine with an apparent Km of 5.9 µM and a Vmax of 32 pmol min−1 mg−1. Moreover, CTL843 conferred a growth advantage to a Δpfs E. coli mutant that lost the ability to detoxify SAH, while competition and back-transport experiments further implied that SAH was an additional substrate for CTL843. We propose that CTL843 acts as a SAM/SAH transporter (SAMHT) serving a dual function by allowing Chlamydia to acquire SAM from the host cell and excrete the toxic by-product SAH. The demonstration of a functional SAMHT provides further insight into the reductive evolution associated with the obligate intracellular lifestyle of Chlamydia and identifies an excellent chemotherapeutic target. PMID:21558433

  12. LINE-1 hypomethylation induced by reactive oxygen species is mediated via depletion of S-adenosylmethionine.

    PubMed

    Kloypan, Chiraphat; Srisa-art, Monpicha; Mutirangura, Apiwat; Boonla, Chanchai

    2015-08-01

    Whether long interspersed nuclear element-1 (LINE-1) hypomethylation induced by reactive oxygen species (ROS) was mediated through the depletion of S-adenosylmethionine (SAM) was investigated. Bladder cancer (UM-UC-3 and TCCSUP) and human kidney (HK-2) cell lines were exposed to 20 μM H2O2 for 72 h to induce oxidative stress. Level of LINE-1 methylation, SAM and homocysteine (Hcy) was measured in the H2O2 -exposed cells. Effects of α-tocopheryl acetate (TA), N-acetylcysteine (NAC), methionine, SAM and folic acid on oxidative stress and LINE-1 methylation in the H2O2 -treated cells were explored. Viabilities of cells treated with H2O2 were not significantly changed. Intracellular ROS production and protein carbonyl content were significantly increased, but LINE-1 methylation was significantly decreased in the H2O2 -treated cells. LINE-1 methylation was restored by TA, NAC, methionine, SAM and folic acid. SAM level in H2O2 -treated cells was significantly decreased, while total glutathione was significantly increased. SAM level in H2O2 -treated cells was restored by NAC, methionine, SAM and folic acid; while, total glutathione level was normalized by TA and NAC. Hcy was significantly decreased in the H2O2 -treated cells and subsequently restored by NAC. In conclusion, in bladder cancer and normal kidney cells exposed to H2O2 , SAM and Hcy were decreased, but total glutathione was increased. Treatments with antioxidants (TA and NAC) and one-carbon metabolites (SAM, methionine and folic acid) restored these changes. This pioneer finding suggests that exposure of cells to ROS activates glutathione synthesis via the transsulfuration pathway leading to deficiency of Hcy, which consequently causes SAM depletion and eventual hypomethylation of LINE-1.

  13. Evidence for the existence of independent chloromethane- and S-adenosylmethionine-utilizing systems for methylation in Phanerochaete chrysosporium.

    PubMed Central

    Coulter, C; Hamilton, J T; Harper, D B

    1993-01-01

    O methylation of acetovanillone at 4 position by C2H3Cl and S-adenosyl[methyl-2H3]methionine was monitored in whole mycelia of Phanerochaete chrysosporium in the presence and absence of S-adenosylhomocysteine. Both the amount of the methylation product, 3,4-dimethoxyacetophenone, and the percent C2H3 incorporation into the 4-methoxyl group of the compound were determined. The results strongly suggest the presence of biochemically distinct systems for O methylation of acetovanillone utilizing S-adenosylmethionine and chloromethane, respectively, as the methyl donor. The S-adenosylmethionine-dependent enzyme is induced early in the growth cycle, with activity attaining an initial maximum after 55 h of incubation. Methylation by this enzyme is totally suppressed by 1 mM S-adenosylhomocysteine over almost the entire growth cycle. S-Adenosylmethionine-dependent O-methyltransferase activity is detectable in cell extracts, and the purification and characterization of the enzyme are described elsewhere (C. Coulter, J. T. Kennedy, W. C. McRoberts, and D. B. Harper, Appl. Environ. Microbiol. 59:706-711, 1993). The chloromethane-utilizing methylation system is absent in early growth but attains peak activity in the mid-growth phase after 72 h of incubation. The system is not significantly inhibited by S-adenosylhomocysteine at any stage of growth. No chloromethane-dependent O-methyltransferase activity is detectable in cell extract, suggesting that the enzyme is membrane bound and/or part of a multienzyme complex. Although the biochemical role of the chloromethane-dependent methylation system in metabolism is not known, one possible function could be the regeneration of veratryl alcohol degraded by the attack of lignin peroxidase. PMID:8517739

  14. Analysis of S-methylmethionine and S-adenosylmethionine in plant tissue by a dansylation, Dual-isotope method

    SciTech Connect

    Macnicol, P.K.

    1986-10-01

    A method is presented for determining the levels of S-methylmethionine (MeMet) and S-adenosylmethionine (AdoMet) in the same plant tissue sample, utilizing readily available equipment. The bottom limit of sensitivity, ca. 100 pmol, can be lowered if required. A trichloracetic acid homogenate of the tissue is supplemented with (carboxyl-/sup 14/C)MeMet and (carboxyl-/sup 14/C)AdoMet. After separation of MeMet and AdoMet from each other and from endogenous homoserine on a phosphocellulose column, the two fractions are heat treated at appropriate pH values to liberate (/sup 14/C)homoserine. Quantitation is via the /sup 3/H//sup 14/C ratio of (/sup 3/H)dansyl-(/sup 14/C)homoserine isolated by thin-layer chromatography. The method is validated with pea cotyledon, corn root, and cauliflower leaf.

  15. A functional tomato ACC synthase expressed in Escherichia coli demonstrates suicidal inactivation by its substrate S-adenosylmethionine.

    PubMed

    Li, N; Wiesman, Z; Liu, D; Mattoo, A K

    1992-07-20

    1-Aminocyclopropane-1-carboxylate (ACC) synthase is a key enzyme in the biosynthesis of the plant hormone, ethylene. We have isolated, sequenced and expressed a functional tomato (cv Pik-Red) ACC synthase gene in Escherichia coli. ACC synthase expressed in E. coli was inactivated by incubation with S-adenosylmethionine (SAM), the half-time of which was concentration dependent. Mixing the tomato fruit protein extract with the cell-free extract from transformed E. coli did not affect SAM-dependent inactivation of ACC synthase activity. Thus, single isoforms of the ACC synthase enzyme, which demonstrate the biochemical features expected of the tomato fruit enzyme, can be expressed in E. coli and their structure-function relationships investigated.

  16. Purification and Properties of an S-Adenosylmethionine: 2,4-Disubstituted Phenol O-Methyltransferase from Phanerochaete chrysosporium

    PubMed Central

    Coulter, Catherine; Kennedy, James T.; McRoberts, W. Colin; Harper, David B.

    1993-01-01

    An enzyme catalyzing the O-methylation of acetovanillone (3-methoxy-4-hydroxyacetophenone) by S-adeno-sylmethionine was isolated from Phanerochaete chrysosporium and purified 270-fold by ultrafiltration, anion-exchange chromatography, and gel filtration. The enzyme exhibited a pH optimum between 7 and 9 and was rapidly denatured at temperatures above 55°C. The Km values for acetovanillone and S-adenosylmethionine were 34 and 99 μM, respectively. S-Adenosylhomocysteine acted as a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 41 μM. The enzyme was also susceptible to inhibition by thiol reagents and low concentrations of heavy metal ions. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was monomeric and had a molecular weight of approximately 53,000. Substrate specificity studies showed that 3-methoxy- and 3,5-dimethoxy-substituted 4-hydroxy-benzaldehydes, -benzoic acids, and -acetophenones were the preferred substrates for the enzyme. The corresponding 3,4-dihydroxy compounds were methylated relatively slowly, while the 3-hydroxy-4-methoxy compounds were almost inactive as substrates. Substituents in both the 2 and 4 positions relative to the hydroxyl group appeared to be essential for significant enzyme attack of a substrate. Provided that certain steric criteria were satisfied, the nature of the substituent was not critical. Hence, xenobiotic compounds such as 2,4-dichlorophenol and 2,4-dibromophenol were methylated almost as readily as acetovanillone. However, an extended side chain in the 4 position was not compatible with activity as a substrate, and neither homovanillic, caffeic, nor ferulic acid was methylated. The substrate range of the O-methyltransferase tends to imply a role in the catabolism or detoxification of lignin degradation products such as vanillic and syringic acids. PMID:16348886

  17. A consistent S-Adenosylmethionine force field improved by dynamic Hirshfeld-I atomic charges for biomolecular simulation.

    PubMed

    Saez, David Adrian; Vöhringer-Martinez, Esteban

    2015-10-01

    S-Adenosylmethionine (AdoMet) is involved in many biological processes as cofactor in enzymes transferring its sulfonium methyl group to various substrates. Additionally, it is used as drug and nutritional supplement to reduce the pain in osteoarthritis and against depression. Due to the biological relevance of AdoMet it has been part of various computational simulation studies and will also be in the future. However, to our knowledge no rigorous force field parameter development for its simulation in biological systems has been reported. Here, we use electronic structure calculations combined with molecular dynamics simulations in explicit solvent to develop force field parameters compatible with the AMBER99 force field. Additionally, we propose new dynamic Hirshfeld-I atomic charges which are derived from the polarized electron density of AdoMet in aqueous solution to describe its electrostatic interactions in biological systems. The validation of the force field parameters and the atomic charges is performed against experimental interproton NOE distances of AdoMet in aqueous solution and crystal structures of AdoMet in the cavity of three representative proteins.

  18. High Affinity S-Adenosylmethionine Plasma Membrane Transporter of Leishmania Is a Member of the Folate Biopterin Transporter (FBT) Family*

    PubMed Central

    Dridi, Larbi; Ahmed Ouameur, Amin; Ouellette, Marc

    2010-01-01

    S-Adenosylmethionine (AdoMet) is an important methyl group donor that plays a central role in many essential biochemical processes. The parasite Leishmania can both synthesize and transport AdoMet. Leishmania cells resistant to the antifolate methotrexate due to a rearrangement in folate biopterin transporter (FBT) genes were cross-resistant to sinefungin, an AdoMet analogue. FBT gene rearrangements were also observed in Leishmania major cells selected for sinefungin resistance. One of the rearranged FBT genes corresponded to the main AdoMet transporter (AdoMetT1) of Leishmania as determined by gene transfection and gene inactivation experiments. AdoMetT1 was determined to be a high affinity plasma membrane transporter expressed constitutively throughout the growth phases of the parasite. Leishmania cells selected for resistance or naturally insensitive to sinefungin had lower expression of AdoMetT1. A new function in one carbon metabolism, also a pathway of interest for chemotherapeutic interventions, is described for a novel class of membrane proteins found in diverse organisms. PMID:20406813

  19. The Endosymbiont Amoebophilus asiaticus Encodes an S-Adenosylmethionine Carrier That Compensates for Its Missing Methylation Cycle

    PubMed Central

    Haferkamp, Ilka; Penz, Thomas; Geier, Melanie; Ast, Michelle; Mushak, Tanja; Horn, Matthias

    2013-01-01

    All organisms require S-adenosylmethionine (SAM) as a methyl group donor and cofactor for various biologically important processes. However, certain obligate intracellular parasitic bacteria and also the amoeba symbiont Amoebophilus asiaticus have lost the capacity to synthesize this cofactor and hence rely on its uptake from host cells. Genome analyses revealed that A. asiaticus encodes a putative SAM transporter. The corresponding protein was functionally characterized in Escherichia coli: import studies demonstrated that it is specific for SAM and S-adenosylhomocysteine (SAH), the end product of methylation. SAM transport activity was shown to be highly dependent on the presence of a membrane potential, and by targeted analyses, we obtained direct evidence for a proton-driven SAM/SAH antiport mechanism. Sequence analyses suggest that SAM carriers from Rickettsiales might operate in a similar way, in contrast to chlamydial SAM transporters. SAM/SAH antiport is of high physiological importance, as it allows for compensation for the missing methylation cycle. The identification of a SAM transporter in A. asiaticus belonging to the Bacteroidetes phylum demonstrates that SAM transport is more widely spread than previously assumed and occurs in bacteria belonging to three different phyla (Proteobacteria, Chlamydiae, and Bacteroidetes). PMID:23667233

  20. A consistent S-Adenosylmethionine force field improved by dynamic Hirshfeld-I atomic charges for biomolecular simulation

    NASA Astrophysics Data System (ADS)

    Saez, David Adrian; Vöhringer-Martinez, Esteban

    2015-10-01

    S-Adenosylmethionine (AdoMet) is involved in many biological processes as cofactor in enzymes transferring its sulfonium methyl group to various substrates. Additionally, it is used as drug and nutritional supplement to reduce the pain in osteoarthritis and against depression. Due to the biological relevance of AdoMet it has been part of various computational simulation studies and will also be in the future. However, to our knowledge no rigorous force field parameter development for its simulation in biological systems has been reported. Here, we use electronic structure calculations combined with molecular dynamics simulations in explicit solvent to develop force field parameters compatible with the AMBER99 force field. Additionally, we propose new dynamic Hirshfeld-I atomic charges which are derived from the polarized electron density of AdoMet in aqueous solution to describe its electrostatic interactions in biological systems. The validation of the force field parameters and the atomic charges is performed against experimental interproton NOE distances of AdoMet in aqueous solution and crystal structures of AdoMet in the cavity of three representative proteins.

  1. Identification of Small Molecule Inhibitors of Human As(III) S-Adenosylmethionine Methyltransferase (AS3MT)

    PubMed Central

    2015-01-01

    Arsenic is the most ubiquitous environmental toxin and carcinogen. Long-term exposure to arsenic is associated with human diseases including cancer, cardiovascular disease, and diabetes. Human As(III) S-adenosylmethionine (SAM) methyltransferases (hAS3MT) methylates As(III) to trivalent mono- and dimethyl species that are more toxic and potentially more carcinogenic than inorganic arsenic. Modulators of hAS3MT activity may be useful for the prevention or treatment of arsenic-related diseases. Using a newly developed high-throughput assay for hAS3MT activity, we identified 10 novel noncompetitive small molecule inhibitors. In silico docking analysis with the crystal structure of an AS3MT orthologue suggests that the inhibitors bind in a cleft between domains that is distant from either the As(III) or SAM binding sites. This suggests the presence of a possible allosteric and regulatory site in the enzyme. These inhibitors may be useful tools for future research in arsenic metabolism and are the starting-point for the development of drugs against hAS3MT. PMID:26577531

  2. Identification of the human mitochondrial S-adenosylmethionine transporter: bacterial expression, reconstitution, functional characterization and tissue distribution.

    PubMed Central

    Agrimi, G; Di Noia, M A; Marobbio, C M T; Fiermonte, G; Lasorsa, F M; Palmieri, F

    2004-01-01

    The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix. SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins. The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity. By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed. This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC). Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple. SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria. The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine. This is the first report describing the identification and characterization of the human SAMC and its gene. PMID:14674884

  3. Significantly enhanced production of acarbose in fed-batch fermentation with the addition of S-adenosylmethionine.

    PubMed

    Sun, Li-Hui; Li, Ming-Gang; Wang, Yuan-Shan; Zheng, Yu-Guo

    2012-06-01

    Acarbose, a pseudo-oligosaccharide, is widely used clinically in therapies for non-insulin-dependent diabetes. In the present study, S-adenosylmethionine (SAM) was added to selected media in order to investigate its effect on acarbose fermentation by Actinoplanes utahensis ZJB- 08196. Acarbose titer was seen to increase markedly when concentrations of SAM were added over a period of time. The effects of glucose and maltose on the production of acarbose were investigated in both batch and fed-batch fermentation. Optimal acarbose production was observed at relatively low glucose levels and high maltose levels. Based on these results, a further fed-batch experiment was designed so as to enhance the production of acarbose. Fed-batch fermentation was carried out at an initial glucose level of 10 g/l and an initial maltose level of 60 g/l. Then, 12 h post inoculation, 100 micromol/l SAM was added. In addition, 8 g/l of glucose was added every 24 h, and 20 g/l of maltose was added at 96 h. By way of this novel feeding strategy, the maximum titer of acarbose achieved was 6,113 mg/l at 192 h. To our knowledge, the production level of acarbose achieved in this study is the highest ever reported.

  4. Effect of Nifedipine and S-Adenosylmethionine in the liver of rats treated with CCl[sub 4] and ethanol for one month

    SciTech Connect

    Cutrin, C.; Menino, J.M.; Otero, X.; Miguez, J.; Perez-becerra, E.; Barrio, E. )

    1992-01-01

    An experimental model of toxic liver injury in rats was employed to assay the effect of Nifedipine (a calcium antagonist blocker) and S-Adenosylmethionine. An important decrease in both perivenular fibrosis and cirrhosis was found. Furthermore, a significant decrease in lactic acid levels was found in the group of animals treated with pharmacologic therapy, although no correlation was seen between lactic acid levels and the different degrees of perivenular fibrosis. No significant variations in ALT and AST enzymes were observed between both groups, as opposed to a significant decrease in LDH enzyme in the Nifedipine+S-Adenosylmethionine group. The results indicate an improvement in the histologic picture of the liver in rats treated by means of pharmacological association, without any change in inflammatory infiltrate and with a slight decrease in necrosis, indicating an action mechanism via creeping fibrosis.

  5. Structure and Function of 4-Hydroxyphenylacetate Decarboxylase and Its Cognate Activating Enzyme.

    PubMed

    Selvaraj, Brinda; Buckel, Wolfgang; Golding, Bernard T; Ullmann, G Matthias; Martins, Berta M

    2016-01-01

    4-Hydroxyphenylacetate decarboxylase (4Hpad) is the prototype of a new class of Fe-S cluster-dependent glycyl radical enzymes (Fe-S GREs) acting on aromatic compounds. The two-enzyme component system comprises a decarboxylase responsible for substrate conversion and a dedicated activating enzyme (4Hpad-AE). The decarboxylase uses a glycyl/thiyl radical dyad to convert 4-hydroxyphenylacetate into p-cresol (4-methylphenol) by a biologically unprecedented Kolbe-type decarboxylation. In addition to the radical dyad prosthetic group, the decarboxylase unit contains two [4Fe-4S] clusters coordinated by an extra small subunit of unknown function. 4Hpad-AE reductively cleaves S-adenosylmethionine (SAM or AdoMet) at a site-differentiated [4Fe-4S]2+/+ cluster (RS cluster) generating a transient 5'-deoxyadenosyl radical that produces a stable glycyl radical in the decarboxylase by the abstraction of a hydrogen atom. 4Hpad-AE binds up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert that is C-terminal to the RS cluster-binding motif. The ferredoxin-like domain with its two auxiliary clusters is not vital for SAM-dependent glycyl radical formation in the decarboxylase, but facilitates a longer lifetime for the radical. This review describes the 4Hpad and cognate AE families and focuses on the recent advances and open questions concerning the structure, function and mechanism of this novel Fe-S-dependent class of GREs.

  6. In Vivo Evidence that S-Adenosylmethionine and Fatty Acid Synthesis Intermediates Are the Substrates for the LuxI Family of Autoinducer Synthases

    PubMed Central

    Val, Dale L.; Cronan, John E.

    1998-01-01

    Many gram-negative bacteria synthesize N-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In Escherichia coli, synthesis of N-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a fadD mutant blocked in β-oxidative fatty acid degradation. Also, conditions known to induce the fad regulon did not increase autoinducer synthesis. In contrast, cerulenin and diazoborine, specific inhibitors of fatty acid synthesis, both blocked autoinducer synthesis even in a strain dependent on β-oxidative fatty acid degradation for growth. These data provide the first in vivo evidence that the acyl chains in autoinducers synthesized by LuxI-family synthases are derived from acyl-acyl carrier protein substrates rather than acyl coenzyme A substrates. Also, we show that decreased levels of intracellular S-adenosylmethionine caused by expression of bacteriophage T3 S-adenosylmethionine hydrolase result in a marked reduction in autoinducer synthesis, thus providing direct in vivo evidence that the homoserine lactone ring of LuxI-family autoinducers is derived from S-adenosylmethionine. PMID:9573148

  7. Methionine and S-Adenosylmethionine levels are critical regulators of PP2A activity modulating lipophagy during steatosis

    PubMed Central

    Zubiete-Franco, Imanol; García-Rodríguez, Juan Luis; Martínez-Uña, Maite; Martínez-Lopez, Nuria; Woodhoo, Ashwin; Juan, Virginia Gutiérrez-De; Beraza, Naiara; Lage-Medina, Sergio; Andrade, Fernando; Fernandez, Marta Llarena; Aldámiz-Echevarría, Luis; Fernández-Ramos, David; Falcon-Perez, Juan Manuel; Lopitz-Otsoa, Fernando; Fernandez-Tussy, Pablo; Barbier-Torres, Lucía; Luka, Zigmund; Wagner, Conrad; García-Monzón, Carmelo; Lu, Shelly C.; Aspichueta, Patricia; Mato, José María; Martínez-Chantar, María Luz; Varela-Rey, Marta

    2015-01-01

    Background & Aims Glycine N-methyltransferase (GNMT) expression is decreased in some patients with severe NAFLD. Gnmt deficiency in mice (Gnmt-KO) results in abnormally elevated serum levels of methionine and its metabolite S-adenosylmethionine (SAMe), and this leads to rapid liver steatosis development. Autophagy plays a critical role in lipid catabolism (lipophagy), and defects in autophagy have been related to liver steatosis development. Since methionine and its metabolite SAMe are well known inactivators of autophagy, we aimed to examine whether high levels of both metabolites could block autophagy-mediated lipid catabolism. Methods We examined methionine levels in a cohort of 358 serum samples from steatotic patients. We used hepatocytes cultured with methionine and SAMe, and hepatocytes and livers from Gnmt-KO mice. Results We detected a significant increase in serum methionine levels in steatotic patients. We observed that autophagy and lipophagy were impaired in hepatocytes cultured with high methionine and SAMe, and that Gnmt-KO livers were characterized by an impairment in autophagy functionality, likely caused by defects at the lysosomal level. Elevated levels of methionine and SAMe activated PP2A by methylation, while blocking PP2A activity restored autophagy flux in Gnmt-KO hepatocytes, and in hepatocytes treated with SAMe and Methionine. Finally, normalization of methionine and SAMe levels in Gnmt-KO mice using a methionine deficient diet normalized the methylation capacity, PP2A methylation, autophagy, and ameloriated liver steatosis. Conclusions These data suggest that elevated levels of methionine and SAMe can inhibit autophagic catabolism of lipids contributing to liver steatosis. PMID:26394163

  8. The Effect of S-Adenosylmethionine on Cognitive Performance in Mice: An Animal Model Meta-Analysis

    PubMed Central

    Montgomery, Sarah E.; Sepehry, Amir A.; Wangsgaard, John D.; Koenig, Jeremy E.

    2014-01-01

    Background Alzheimer's disease (AD) is the most frequently diagnosed form of dementia resulting in cognitive impairment. Many AD mouse studies, using the methyl donor S-adenosylmethionine (SAM), report improved cognitive ability, but conflicting results between and within studies currently exist. To address this, we conducted a meta-analysis to evaluate the effect of SAM on cognitive ability as measured by Y maze performance. As supporting evidence, we include further discussion of improvements in cognitive ability, by SAM, as measured by the Morris water maze (MWM). Methods We conducted a comprehensive literature review up to April 2014 based on searches querying MEDLINE, EMBASE, Web of Science, the Cochrane Library and Proquest Theses and Dissertation databases. We identified three studies containing a total of 12 experiments that met our inclusion criteria and one study for qualitative review. The data from these studies were used to evaluate the effect of SAM on cognitive performance according to two scenarios: 1. SAM supplemented folate deficient (SFD) diet compared to a folate deficient (FD) diet and 2. SFD diet compared to a nutrient complete (NC) diet. Hedge's g was used to calculate effect sizes and mixed effects model meta-regression was used to evaluate moderating factors. Results Our findings showed that the SFD diet was associated with improvements in cognitive performance. SFD diet mice also had superior cognitive performance compared to mice on an NC diet. Further to this, meta-regression analyses indicated a significant positive effect of study quality score and treatment duration on the effect size estimate for both the FD vs SFD analysis and the SFD vs NC analysis. Conclusion The findings of this meta-analysis demonstrate efficacy of SAM in acting as a cognitive performance-enhancing agent. As a corollary, SAM may be useful in improving spatial memory in patients suffering from many dementia forms including AD. PMID:25347725

  9. Identification of an intermediate methyl carrier in the radical S-adenosylmethionine methylthiotransferases RimO and MiaB.

    PubMed

    Landgraf, Bradley J; Arcinas, Arthur J; Lee, Kyung-Hoon; Booker, Squire J

    2013-10-16

    RimO and MiaB are radical S-adenosylmethionine (SAM) enzymes that catalyze the attachment of methylthio (-SCH3) groups to macromolecular substrates. RimO attaches a methylthio group at C3 of aspartate 89 of protein S12, a component of the 30S subunit of the bacterial ribosome. MiaB attaches a methylthio group at C2 of N(6)-(isopentenyl)adenosine, found at nucleotide 37 in several prokaryotic tRNAs. These two enzymes are prototypical members of a subclass of radical SAM enzymes called methylthiotransferases (MTTases). It had been assumed that the sequence of steps in MTTase reactions involves initial sulfur insertion into the organic substrate followed by capping of the inserted sulfur atom with a SAM-derived methyl group. In this work, however, we show that both RimO and MiaB from Thermotoga maritima catalyze methyl transfer from SAM to an acid/base labile acceptor on the protein in the absence of their respective macromolecular substrates. Consistent with the assignment of the acceptor as an iron-sulfur cluster, denaturation of the SAM-treated protein with acid results in production of methanethiol. When RimO or MiaB is first incubated with SAM in the absence of substrate and reductant and then incubated with excess S-adenosyl-l-[methyl-d3]methionine in the presence of substrate and reductant, production of the unlabeled product precedes production of the deuterated product, showing that the methylated species is chemically and kinetically competent to be an intermediate.

  10. Ursodeoxycholic Acid and S-adenosylmethionine for the Treatment of Intrahepatic Cholestasis of Pregnancy: A Meta-analysis

    PubMed Central

    Zhang, Yang; Lu, Linlin; Victor, David W; Xin, Yongning; Xuan, Shiying

    2016-01-01

    Context An optimal therapeutic strategy has not yet been identified for the pharmacological treatment of intrahepatic cholestasis of pregnancy (ICP). The aim of this study was to evaluate the efficacy and safety of ursodeoxycholic acid (UDCA) and S-adenosylmethionine (SAMe) in the treatment of ICP, both individually and in combination. Evidence Acquisition A meta-analysis of all randomized controlled trials (RCTs) comparing UDCA, SAMe, and combination therapy was performed. We carried out a literature search using pubmed, embase, the cochrane register of controlled trials, and the science citation index of web of science. The maternal clinical and biochemical responses, including pruritus scores, total bilirubin, total bile acids, alanine aminotransferase, and aspartate transaminase, were evaluated. Safety assessments, including preterm delivery, cesarean section, and meconium-stained amniotic fluid, were also analyzed. Results Five RCTs including 311 patients were evaluated. In comparison to SAMe, UDCA significantly reduced the pruritus score (OR = -0.45, 95% confidence interval [CI]: -0.66 to -0.25, P < 0.0001) and improved the levels of total bile acids (TBAs; OR = -0.59, 95% CI: -0.99 to –0.30, P < 0.0001) and alanine aminotransferase (ALT; OR = -0.38, 95% CI: -0.66 to -0.09, P = 0.01). UDCA was associated with significantly lower preterm delivery rates than SAMe (RR = 0.48, 95% CI: 0.32–0.72, P = 0.0004). Interestingly, combination therapy significantly reduced total bilirubin (TB; vs. SAMe, OR = -0.41, 95% CI, -0.74 to -0.08, P = 0.02), aspartate transaminase (AST; vs. UDCA, OR = -0.40, 95% CI, -0.74 to –0.06, P = 0.02), and the rate of preterm delivery (vs. SAMe, OR = 0.62, 95% CI, 0.42 - 0.91, P = 0.02), in comparison with either drug administered alone. Conclusions UDCA decreased the pruritus score, TBA, and ALT levels more effectively than SAMe, reducing the rate of preterm delivery for ICP. PMID:27799965

  11. S-Adenosylmethionine Regulates Dual-Specificity Mitogen-Activated Protein Kinase Phosphatase Expression in Mouse and Human Hepatocytes

    PubMed Central

    Tomasi, Maria Lauda; Ramani, Komal; Lopitz-Otsoa, Fernando; Rodríguez, Manuel S.; Li, Tony W. H.; Ko, Kwangsuk; Yang, Heping; Bardag-Gorce, Fawzia; Iglesias-Ara, Ainhoa; Feo, Francesco; Pascale, Maria Rosa; Mato, José M.; Lu, Shelly C.

    2010-01-01

    Increased mitogen-activated protein kinase (MAPK) activity correlates with a more malignant hepatocellular carcinoma (HCC) phenotype. There is a reciprocal regulation between p44/42 MAPK (extracellular signal-regulated kinase [ERK]1/2) and the dual-specificity MAPK phosphatase MKP-1/DUSP1. ERK phosphorylates DUSP1, facilitating its proteasomal degradation, whereas DUSP1 inhibits ERK activity. Methionine adenosyltransferase 1a (Mat1a) knockout (KO) mice express hepatic S-adenosylmethionine (SAM) deficiency and increased ERK activity and develop HCC. The aim of this study was to examine whether DUSP1 expression is regulated by SAM and if so, elucidate the molecular mechanisms. Studies were conducted using Mat1a KO mice livers, cultured mouse and human hepatocytes, and 20S and 26S proteasomes. DUSP1 messenger RNA (mRNA) and protein levels were reduced markedly in livers of Mat1a KO mice and in cultured mouse and human hepatocytes with protein falling to lower levels than mRNA. SAM treatment protected against the fall in DUSP1 mRNA and protein levels in mouse and human hepatocytes. SAM increased DUSP1 transcription, p53 binding to DUSP1 promoter, and stability of its mRNA and protein. Proteasomal chymotrypsin-like and caspase-like activities were increased in Mat1a KO livers and cultured hepatocytes, which was blocked by SAM treatment. SAM inhibited chymotrypsin-like and caspase-like activities by 40% and 70%, respectively, in 20S proteasomes and caused rapid degradation of some of the 26S proteasomal subunits, which was blocked by the proteasome inhibitor MG132. SAM treatment in Mat1a KO mice for 7 days raised SAM, DUSP1, mRNA and protein levels and lowered proteosomal and ERK activities. Conclusion DUSP1 mRNA and protein levels are lower in Mat1a KO livers and fall rapidly in cultured hepatocytes. SAM treatment increases DUSP1 expression through multiple mechanisms, and this may suppress ERK activity and malignant degeneration. PMID:20196119

  12. [Double-blind studies of the therapeutic action of S-Adenosylmethionine (SAMe) in oral administration, in liver cirrhosis and other chronic hepatitides].

    PubMed

    Miglio, F; Stefanini, G F; Corazza, G R; D'Ambro, A; Gasbarrini, G

    1975-05-02

    Six oral administrations per day of 30 mg S-adenosylmethionine (SAMe) for 30 days, in addition to 6000 gamma/day of Vitamine B12 induced marked improvements of biochemical parameters in 20 patients with hepatic cirrhosis or various chronic hepatites. Particularly, the protidemia, bilirubinemia and radial immunodiffusion have shown the highest favorable drug responses. These improvements were still lasting and even further increasing 30 days after the end of therapy. In another group of patients with similar diagnosis and under clinical conditions comparable to the previous group of twenty, the administration of Vitamine B12 alone, in the same doses as above, has not induced any alteration in the biochemical parameters.

  13. Low sulfide levels and a high degree of cystathionine β-synthase (CBS) activation by S-adenosylmethionine (SAM) in the long-lived naked mole-rat.

    PubMed

    Dziegelewska, Maja; Holtze, Susanne; Vole, Christiane; Wachter, Ulrich; Menzel, Uwe; Morhart, Michaela; Groth, Marco; Szafranski, Karol; Sahm, Arne; Sponholz, Christoph; Dammann, Philip; Huse, Klaus; Hildebrandt, Thomas; Platzer, Matthias

    2016-08-01

    Hydrogen sulfide (H2S) is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine β-synthase (CBS), an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration.

  14. The ferredoxin-like domain of the activating enzyme is required for generating a lasting glycyl radical in 4-hydroxyphenylacetate decarboxylase.

    PubMed

    Selvaraj, Brinda; Pierik, Antonio J; Bill, Eckhard; Martins, Berta M

    2014-12-01

    4-Hydroxyphenylacetate decarboxylase-activating enzyme (4Hpad-AE) uses S-adenosylmethionine (SAM or AdoMet) and a [4Fe-4S] ²⁺/⁺cluster (RS cluster) to generate a stable glycyl radical on the decarboxylase. 4Hpad-AE might bind up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert C-terminal to the RS cluster-binding motif. Except for the AEs of pyruvate formate-lyase and anaerobic ribonucleotide reductase, all glycyl radical-activating enzymes possess a similar ferredoxin-like domain, whose functional role is still poorly understood. To assess the role of the putative ferredoxin clusters from 4Hpad-AE, we combined biochemical and spectroscopic methods to characterize a truncated version of the protein (Δ66-AE) devoid of the ferredoxin-like domain. We found that Δ66-AE is stable, harbors a fully active RS cluster and can activate the decarboxylase. From the similar cleavage rates for S-adenosylmethionine of Δ66-AE and wild-type AE, we infer the reactivity of the RS cluster is unperturbed by the absence of the ferredoxin-like domain. Thus, the auxiliary clusters are not required as electron conduit to the RS cluster for effective reductive cleavage of SAM. The activation of the decarboxylase by Δ66-AE is almost as fast as with wild-type AE, but the generated glycyl radical is short living. We postulate that the ferredoxin-like domain is not required for SAM-dependent glycyl radical generation in the decarboxylase, but is necessary for producing a lasting glycyl radical.

  15. Allosteric regulation of an essential trypanosome polyamine biosynthetic enzyme by a catalytically dead homolog

    PubMed Central

    Willert, Erin K.; Fitzpatrick, Richard; Phillips, Margaret A.

    2007-01-01

    African sleeping sickness is a fatal disease that is caused by the protozoan parasite Trypanosoma brucei. Polyamine biosynthesis is an essential pathway in the parasite and is a validated drug target for treatment of the disease. S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in polyamine biosynthesis. Here, we show that trypanosomatids uniquely contain both a functional AdoMetDC and a paralog designated prozyme that has lost catalytic activity. The T. brucei prozyme forms a high-affinity heterodimer with AdoMetDC that stimulates its activity by 1,200-fold. Both genes are expressed in T. brucei, and analysis of AdoMetDC activity in T. brucei extracts supports the finding that the heterodimer is the functional enzyme in vivo. Thus, prozyme has evolved to be a catalytically dead but allosterically active subunit of AdoMetDC, providing an example of how regulators of multimeric enzymes can evolve through gene duplication and mutational drift. These data identify a distinct mechanism for regulating AdoMetDC in the parasite that suggests new strategies for the development of parasite-specific inhibitors of the polyamine biosynthetic pathway. PMID:17485680

  16. Reprogramming of gene expression during compression wood formation in pine: Coordinated modulation of S-adenosylmethionine, lignin and lignan related genes

    PubMed Central

    2012-01-01

    Background Transcript profiling of differentiating secondary xylem has allowed us to draw a general picture of the genes involved in wood formation. However, our knowledge is still limited about the regulatory mechanisms that coordinate and modulate the different pathways providing substrates during xylogenesis. The development of compression wood in conifers constitutes an exceptional model for these studies. Although differential expression of a few genes in differentiating compression wood compared to normal or opposite wood has been reported, the broad range of features that distinguish this reaction wood suggest that the expression of a larger set of genes would be modified. Results By combining the construction of different cDNA libraries with microarray analyses we have identified a total of 496 genes in maritime pine (Pinus pinaster, Ait.) that change in expression during differentiation of compression wood (331 up-regulated and 165 down-regulated compared to opposite wood). Samples from different provenances collected in different years and geographic locations were integrated into the analyses to mitigate the effects of multiple sources of variability. This strategy allowed us to define a group of genes that are consistently associated with compression wood formation. Correlating with the deposition of a thicker secondary cell wall that characterizes compression wood development, the expression of a number of genes involved in synthesis of cellulose, hemicellulose, lignin and lignans was up-regulated. Further analysis of a set of these genes involved in S-adenosylmethionine metabolism, ammonium recycling, and lignin and lignans biosynthesis showed changes in expression levels in parallel to the levels of lignin accumulation in cells undergoing xylogenesis in vivo and in vitro. Conclusions The comparative transcriptomic analysis reported here have revealed a broad spectrum of coordinated transcriptional modulation of genes involved in biosynthesis of

  17. Protective Effects of S-Adenosylmethionine and Its Combinations With Taurine and/or Betaine Against Lipopolysaccharide or Polyinosinic-polycytidylic Acid-induced Acute Hepatotoxicity

    PubMed Central

    Lee, Seo Yeon; Ko, Kwang Suk

    2016-01-01

    Background Several mechanisms for the pathogenesis of many liver diseases are related with oxidative stress, endotoxins, and infections by many microorganisms. These can lead to chronic hepatitis, cirrhosis, and even liver cancer. The aim of this study was to evaluate the effects of S-adenosylmethionine (SAMe) and its combinations with taurine and/or betaine against hepatotoxicites induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (polyI:C). Methods RAW 264.7 macrophage cells and seven-week-old male C57BL/6 mice were pretreated with SAMe (SAM or AdoMet), taurine, and/or betaine. In order to mimic hepatic injury like endotoxemia or viral infection, cells and mice were treated with LPS or polyI:C. Concentrations of glutathione (GSH), mRNA expressions of GSH synthesizing enzymes, and inflammatory markers were measured by biochemical assays and quantitative real-time PCR. Results In RAW 264.7 cells and mice, pretreatment of SAMe alone or SAMe with taurine and/or betaine attenuated the decrease in GSH levels and mRNA expressions of GSH synthesizing enzymes. In addition, pretreatment of SAMe with taurine and/or betaine prevented the excessive increase in inflammatory mediators produced by LPS or polyI:C treatment. Conclusions Treatment with SAMe in combination with taurine and betaine, would have anti-oxidant functions in addition to anti-inflammatory action against bacterial and/or viral inflammation. PMID:27722141

  18. Rescue of Early bace-1 and Global DNA Demethylation by S-Adenosylmethionine Reduces Amyloid Pathology and Improves Cognition in an Alzheimer’s Model

    PubMed Central

    Do Carmo, Sonia; Hanzel, Cecilia E.; Jacobs, Marie L.; Machnes, Ziv; Iulita, M. Florencia; Yang, Jingyun; Yu, Lei; Ducatenzeiler, Adriana; Danik, Marc; Breuillaud, Lionel S.; Bennett, David A.; Szyf, Moshe; Cuello, A. Claudio

    2016-01-01

    General DNA hypomethylation is associated with Alzheimer’s disease (AD), but it is unclear when DNA hypomethylation starts or plays a role in AD pathology or whether DNA re-methylation would rescue early amyloid-related cognitive impairments. In an APP transgenic mouse model of AD-like amyloid pathology we found that early intraneuronal amyloid beta build-up is sufficient to unleash a global and beta-site amyloid precursor protein cleaving enzyme 1 (bace-1) DNA demethylation in AD-vulnerable brain regions. S-adenosylmethionine administration at these early stages abolished this hypomethylation, diminished the amyloid pathology and restored cognitive capabilities. To assess a possible human significance of findings, we examined the methylation at 12 CpGs sites in the bace-1 promoter, using genome-wide DNA methylation data from 740 postmortem human brains. Thus, we found significant associations of bace-1 promoter methylation with β-amyloid load among persons with AD dementia, and PHFtau tangle density. Our results support a plausible causal role for the earliest amyloid beta accumulation to provoke DNA hypomethylation, influencing AD pathological outcomes. PMID:27681803

  19. S-Adenosylmethionine suppresses the expression of Smad3/4 in activated human hepatic stellate cells via Rac1 promoter methylation

    PubMed Central

    BIAN, KANGQI; ZHANG, FENG; WANG, TINGTING; ZOU, XIAOPING; DUAN, XUHONG; CHEN, GUANGXIA; ZHUGE, YUZHENG

    2016-01-01

    The aim of the present study was to investigate whether S-adenosylmethionine (SAM) was able to suppress activated human hepatic stellate cells (HSCs). Human LX-2 HSCs were cultured with SAM or NSC23766, and were transfected with plasmids encoding ras-related C3 botulinum toxin substrate 1 (Rac1) protein or an empty expression vector. Cell proliferation was detected by Cell Counting Kit-8. Cell migration and invasion were determined using the Transwell assay. The expression levels of Rac1 and Smad3/4 were detected by reverse transcription-quantitative polymerase chain reaction (PCR) or western blotting. The methylation status of Rac1 promoters was measured by methylation-specific PCR. The results demonstrated that SAM and NSC23766 suppressed the expression of Smad3/4 in LX-2 cells. The overexpression of Rac1 enhanced the proliferation, migration and invasion of LX-2 cells. In addition, compared with the control groups, a marked increase was observed in the protein expression levels of Smad3/4 in the LX-2 cells transfected with Rac1 plasmids. The methylation-specific PCR findings showed that SAM increased the methylation of Rac1 promoters. The results of the present study suggested that Rac1 enhanced the expression of Smad3/4 in activated HSCs; however, this increase may be suppressed by SAM-induced methylation of Rac1 promoters. PMID:26986629

  20. Analysis of trace degradation products (decarboxylated diastereoisomers) of S-adenosylmethionine by electrophoresis in capillaries with cationic coatings (N-methylpolyvinylpyridinium or divalent barium).

    PubMed

    Sebastiano, Roberto; Knob, Radim; Citterio, Attilio; Righetti, Pier Giorgio

    2010-10-01

    Commercial preparations of S-adenosylmethionine (SAM) when analyzed in uncoated capillaries show a minute impurity believed to be decarboxylated (dc) SAM. By using two types of cationic coatings, thus reducing the electro-endo-osmotic flow (EOF), it was possible to separate this impurity into two diastereoisomers of dcSAM. The coatings evaluated for this purpose were: (i) N-methylpolyvinylpyridinium, used under reversed EOF at acidic conditions (pH 4.0) and (ii) deposition of divalent barium at alkaline pH values (pH 9.4), providing reduced EOF. Under these conditions, it was possible to separate this impurity into two diastereoisomers, which by chemical synthesis were indeed proven to be dcSAM. It was further demonstrated that, in the alkylation of 5'-methylthioadenosine by 3-bromopropylamine in bromidric acid to dcSAM, another minute impurity was present, proven, via mass spectrometry, to consist of S-(5'-adenosyl)-3-thiopropylamine (decarboxylated and demethylated (dc-SAH)). The LOD for the two dcSAM diastereoisomers was assessed as 17.5 μg/mL and their LOQ as 25.5 μg/mL. By the barium-based protocol it was possible to quantify the dcSAM, present in a commercial sample of SAM, as a 0.1% impurity.

  1. The thiamine biosynthetic enzyme ThiC catalyzes multiple turnovers and is inhibited by S-adenosylmethionine (AdoMet) metabolites.

    PubMed

    Palmer, Lauren D; Downs, Diana M

    2013-10-18

    ThiC (4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase; EC 4.1.99.17) is a radical S-adenosylmethionine (AdoMet) enzyme that uses a [4Fe-4S](+) cluster to reductively cleave AdoMet to methionine and a 5'-deoxyadenosyl radical that initiates catalysis. In plants and bacteria, ThiC converts the purine intermediate 5-aminoimidazole ribotide to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate, an intermediate of thiamine pyrophosphate (coenzyme B1) biosynthesis. In this study, assay conditions were implemented that consistently generated 5-fold molar excess of HMP, demonstrating that ThiC undergoes multiple turnovers. ThiC activity was improved by in situ removal of product 5'-deoxyadenosine. The activity was inhibited by AdoMet metabolites S-adenosylhomocysteine, adenosine, 5'-deoxyadenosine, S-methyl-5'-thioadenosine, methionine, and homocysteine. Neither adenosine nor S-methyl-5'-thioadenosine had been shown to inhibit radical AdoMet enzymes, suggesting that ThiC is distinct from other family members. The parameters for improved ThiC activity and turnover described here will facilitate kinetic and mechanistic analyses of ThiC.

  2. Crystallization and preliminary X-ray crystallographic studies of CrArsM, an arsenic(III) S-adenosylmethionine methyltransferase from Chlamydomonas reinhardtii

    PubMed Central

    Packianathan, Charles; Pillai, Jitesh K.; Riaz, Ahmed; Kandavelu, Palani; Sankaran, Banumathi; Rosen, Barry P.

    2014-01-01

    Arsenic is one the most toxic environmental substances. Arsenic is ubiquitous in water, soil and food, and ranks first on the Environmental Protection Agency’s Superfund Priority List of Hazardous Substances. Arsenic(III) S-adenosylmethionine methyltransferases (AS3MT in animals and ArsM in microbes) are key enzymes of arsenic biotransformation, catalyzing the methylation of inorganic arsenite to give methyl, dimethyl and trimethyl products. Arsenic methyltransferases are found in members of every kingdom from bacteria to humans (EC 2.1.1.137). In the human liver, hAS3MT converts inorganic arsenic into more toxic and carcinogenic forms. CrArsM, an ortholog of hAS3MT from the eukaryotic green alga Chlamydomonas reinhardtii, was purified by chemically synthesizing the gene and expressing it in Escherichia coli. Synthetic purified CrArsM was crystallized in an unliganded form. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to space group R3:H, with unit-cell parameters a = b = 157.8, c = 95.4 Å, γ = 120° and two molecules in the asymmetric unit. Complete data sets were collected and processed to a resolution of 2.40 Å. PMID:25286945

  3. Coproporphyrin Excretion and Low Thiol Levels Caused by Point Mutation in the Rhodobacter sphaeroides S-Adenosylmethionine Synthetase Gene ▿ †

    PubMed Central

    Sabaty, Monique; Adryanczyk, Géraldine; Roustan, Chloë; Cuiné, Stephan; Lamouroux, Christine; Pignol, David

    2010-01-01

    A spontaneous mutant of Rhodobacter sphaeroides f. sp. denitrificans IL-106 was found to excrete a large amount of a red compound identified as coproporphyrin III, an intermediate in bacteriochlorophyll and heme synthesis. The mutant, named PORF, is able to grow under phototrophic conditions but has low levels of intracellular cysteine and glutathione and overexpresses the cysteine synthase CysK. The expression of molybdoenzymes such as dimethyl sulfoxide (DMSO) and nitrate reductases is also affected under certain growth conditions. Excretion of coproporphyrin and overexpression of CysK are not directly related but were both found to be consequences of a diminished synthesis of the key metabolite S-adenosylmethionine (SAM). The wild-type phenotype is restored when the gene metK encoding SAM synthetase is supplied in trans. The metK gene in the mutant strain has a mutation leading to a single amino acid change (H145Y) in the encoded protein. This point mutation is responsible for a 70% decrease in intracellular SAM content which probably affects the activities of numerous SAM-dependent enzymes such as coproporphyrinogen oxidase (HemN); uroporphyrinogen III methyltransferase (CobA), which is involved in siroheme synthesis; and molybdenum cofactor biosynthesis protein A (MoaA). We propose a model showing that the attenuation of the activities of SAM-dependent enzymes in the mutant could be responsible for the coproporphyrin excretion, the low cysteine and glutathione contents, and the decrease in DMSO and nitrate reductase activities. PMID:20038586

  4. Crystallization and preliminary X-ray crystallographic studies of CrArsM, an arsenic(III) S-adenosylmethionine methyltransferase from Chlamydomonas reinhardtii.

    PubMed

    Packianathan, Charles; Pillai, Jitesh K; Riaz, Ahmed; Kandavelu, Palani; Sankaran, Banumathi; Rosen, Barry P

    2014-10-01

    Arsenic is one the most toxic environmental substances. Arsenic is ubiquitous in water, soil and food, and ranks first on the Environmental Protection Agency's Superfund Priority List of Hazardous Substances. Arsenic(III) S-adenosylmethionine methyltransferases (AS3MT in animals and ArsM in microbes) are key enzymes of arsenic biotransformation, catalyzing the methylation of inorganic arsenite to give methyl, dimethyl and trimethyl products. Arsenic methyltransferases are found in members of every kingdom from bacteria to humans (EC 2.1.1.137). In the human liver, hAS3MT converts inorganic arsenic into more toxic and carcinogenic forms. CrArsM, an ortholog of hAS3MT from the eukaryotic green alga Chlamydomonas reinhardtii, was purified by chemically synthesizing the gene and expressing it in Escherichia coli. Synthetic purified CrArsM was crystallized in an unliganded form. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to space group R3:H, with unit-cell parameters a = b = 157.8, c = 95.4 Å, γ = 120° and two molecules in the asymmetric unit. Complete data sets were collected and processed to a resolution of 2.40 Å.

  5. COMMUNICATION: Folate and S-adenosylmethionine modulate synaptic activity in cultured cortical neurons: acute differential impact on normal and apolipoprotein-deficient mice

    NASA Astrophysics Data System (ADS)

    Serra, Michael; Chan, Amy; Dubey, Maya; Gilman, Vladimir; Shea, Thomas B.

    2008-12-01

    Folate deficiency is accompanied by a decline in the cognitive neurotransmitter acetylcholine and a decline in cognitive performance in mice lacking apolipoprotein E (ApoE-/- mice), a low-density lipoprotein that regulates aspects of lipid metabolism. One direct consequence of folate deficiency is a decline in S-adenosylmethionine (SAM). Since dietary SAM supplementation maintains acetylcholine levels and cognitive performance in the absence of folate, we examined herein the impact of folate and SAM on neuronal synaptic activity. Embryonic cortical neurons from mice expressing or lacking ApoE (ApoE+/+ or -/-, respectively) were cultured for 1 month on multi-electrode arrays, and signaling was recorded. ApoE+/+ cultures displayed significantly more frequent spontaneous signals than ApoE-/- cultures. Supplementation with 166 µm SAM (not normally present in culture medium) increased signal frequency and decreased signal amplitude in ApoE+/+ cultures. SAM also increased the frequency of tightly clustered signal bursts. Folate deprivation reversibly reduced signal frequency in ApoE+/+ cultures; SAM supplementation maintained signal frequency despite folate deprivation. These findings support the importance of dietary supplementation with folate and SAM on neuronal health. Supplementation with 166 µm SAM did not alter signaling in ApoE-/- cultures, which may be a reflection of the reduced SAM levels in ApoE-/- mice. The differential impact of SAM on ApoE+/+ and -/- neurons underscores the combined impact of nutritional and genetic deficiencies on neuronal homeostasis.

  6. Low sulfide levels and a high degree of cystathionine β-synthase (CBS) activation by S-adenosylmethionine (SAM) in the long-lived naked mole-rat

    PubMed Central

    Dziegelewska, Maja; Holtze, Susanne; Vole, Christiane; Wachter, Ulrich; Menzel, Uwe; Morhart, Michaela; Groth, Marco; Szafranski, Karol; Sahm, Arne; Sponholz, Christoph; Dammann, Philip; Huse, Klaus; Hildebrandt, Thomas; Platzer, Matthias

    2016-01-01

    Hydrogen sulfide (H2S) is a gaseous signalling molecule involved in many physiological and pathological processes. There is increasing evidence that H2S is implicated in aging and lifespan control in the diet-induced longevity models. However, blood sulfide concentration of naturally long-lived species is not known. Here we measured blood sulfide in the long-lived naked mole-rat and five other mammalian species considerably differing in lifespan and found a negative correlation between blood sulfide and maximum longevity residual. In addition, we show that the naked mole-rat cystathionine β-synthase (CBS), an enzyme whose activity in the liver significantly contributes to systemic sulfide levels, has lower activity in the liver and is activated to a higher degree by S-adenosylmethionine compared to other species. These results add complexity to the understanding of the role of H2S in aging and call for detailed research on naked mole-rat transsulfuration. PMID:26803480

  7. S-adenosylmethionine Administration Attenuates Low Brain-Derived Neurotrophic Factor Expression Induced by Chronic Cerebrovascular Hypoperfusion or Beta Amyloid Treatment.

    PubMed

    Li, Qian; Cui, Jing; Fang, Chen; Zhang, Xiaowen; Li, Liang

    2016-04-01

    Chronic cerebrovascular hypoperfusion is a high-risk factor for Alzheimer's disease (AD) as it is conducive to beta amyloid (Aβ) over-production. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family widely expressed in the central nervous system. The structure of the rat BDNF gene is complex, consisting of eight non-coding exons (I-VIII) and one coding exon (IX). The BDNF gene is transcribed from multiple promoters located upstream of different 5' non-coding exons to produce a heterogeneous population of BDNF mRNAs. S-adenosylmethionine (SAM) produced in the methionine cycle is the primary methyl donor and the precursor of glutathione. In this study, a cerebrovascular hypoperfusion rat model and an Aβ intrahippocampal injection rat model were used to explore the expression profiles of all BDNF transcripts in the hippocampus with chronic cerebrovascular hypoperfusion or Aβ injection as well as with SAM treatment. We found that the BDNF mRNAs and protein were down-regulated in the hippocampus undergoing chronic cerebrovascular hypoperfusion as well as Aβ treatment, and BDNF exons IV and VI played key roles. SAM improved the low BDNF expression following these insults mainly through exons IV and VI. These results suggest that SAM plays a neuroprotective role by increasing the expression of endogenous BDNF and could be a potential target for AD therapy.

  8. Twenty-four-hour changes of S-adenosylmethionine, S-adenosylhomocysteine adenosine and their metabolizing enzymes in rat liver; possible physiological significance in phospholipid methylation.

    PubMed

    Chagoya de Sánchez, V; Hernández-Muñoz, R; Sánchez, L; Vidrio, S; Yáñez, L; Suárez, J

    1991-01-01

    1. The metabolic control of adenosine concentration in the rat liver through the 24-hr cycle is related to the activity of adenosine-metabolizing enzymes [5'-nucleotidase (5'N), adenosine deaminase (A.D.), adenosine kinase (A.K.) and S-adenosylhomocysteine hydrolase (SAH-H)]. 2. Two peaks of adenosine were observed, one at 12:00 hr caused by high activity of 5'N and SAH-H, and the other at 02:00 hr, caused by a decrease in purine catabolism and purine utilization, low activity of SAH-H and de novo purine formation. 3. The similarity of the adenosine and S-adenosylmethionine (SAM) profiles through the 24-hr cycle suggests a role of adenosine in transmethylation reactions, because, during the night (02:00 hr), the metabolic conditions favor the formation and accumulation of S-adenosylhomocysteine (SAH), with consequent inhibition of transmethylation reactions. 4. In the 24-hr variation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the lowest ratio of PC/PE was observed at 24:00-02:00 hr when SAH concentration is high, whereas the highest PC/PE ratio occurs at the same time as one of the SAM/SAH ratio maxima.

  9. Polyamine homoeostasis as a drug target in pathogenic protozoa: peculiarities and possibilities.

    PubMed

    Birkholtz, Lyn-Marie; Williams, Marni; Niemand, Jandeli; Louw, Abraham I; Persson, Lo; Heby, Olle

    2011-09-01

    New drugs are urgently needed for the treatment of tropical and subtropical parasitic diseases, such as African sleeping sickness, Chagas' disease, leishmaniasis and malaria. Enzymes in polyamine biosynthesis and thiol metabolism, as well as polyamine transporters, are potential drug targets within these organisms. In the present review, the current knowledge of unique properties of polyamine metabolism in these parasites is outlined. These properties include prozyme regulation of AdoMetDC (S-adenosylmethionine decarboxylase) activity in trypanosomatids, co-expression of ODC (ornithine decarboxylase) and AdoMetDC activities in a single protein in plasmodia, and formation of trypanothione, a unique compound linking polyamine and thiol metabolism in trypanosomatids. Particularly interesting features within polyamine metabolism in these parasites are highlighted for their potential in selective therapeutic strategies.

  10. Polyamine homoeostasis as a drug target in pathogenic protozoa: peculiarities and possibilities

    PubMed Central

    Birkholtz, Lyn-Marie; Williams, Marni; Niemand, Jandeli; Louw, Abraham I.; Persson, Lo; Heby, Olle

    2011-01-01

    New drugs are urgently needed for the treatment of tropical and subtropical parasitic diseases, such as African sleeping sickness, Chagas' disease, leishmaniasis and malaria. Enzymes in polyamine biosynthesis and thiol metabolism, as well as polyamine transporters, are potential drug targets within these organisms. In the present review, the current knowledge of unique properties of polyamine metabolism in these parasites is outlined. These properties include prozyme regulation of AdoMetDC (S-adenosylmethionine decarboxylase) activity in trypanosomatids, co-expression of ODC (ornithine decarboxylase) and AdoMetDC activities in a single protein in plasmodia, and formation of trypanothione, a unique compound linking polyamine and thiol metabolism in trypanosomatids. Particularly interesting features within polyamine metabolism in these parasites are highlighted for their potential in selective therapeutic strategies. PMID:21834794

  11. S-adenosylmethionine prevents the up regulation of Toll-like receptor (TLR) signaling caused by chronic ethanol feeding in rats.

    PubMed

    Oliva, Joan; Bardag-Gorce, Fawzia; Li, Jun; French, Barbara A; French, Samuel W

    2011-06-01

    Toll-like receptors (TLR) play a role in mediating the proinflammatory response, fibrogenesis and carcinogenesis in chronic liver diseases such as alcoholic liver disease, non-alcoholic liver disease, hepatitis C and hepatocellular carcinoma. This is true in experimental models of these diseases. For this reason, we investigated the TLR proinflammatory response in the chronic intragastric tube feeding rat model of alcohol liver disease. The methyl donor S-adenosylmethionine was also fed to prevent the gene expression changes induced by ethanol. Ethanol feeding tended to increase the up regulation of the gene expression of TLR2 and TLR4. SAMe feeding prevented this. TLR4 and MyD88 protein levels were significantly increased by ethanol and this was prevented by SAMe. This is the first report where ethanol feeding induced TLR2 and SAMe prevented the induction by ethanol. CD34, FOS, interferon responsive factor 1 (IRF-1), Jun, TLR 1,2,3,4,6 and 7 and Traf-6 were found to be up regulated as seen by microarray analysis where rats were sacrificed at high blood alcohol levels compared to pair fed controls. Il-6, IL-10 and IFNγ were also up regulated by high blood levels of ethanol. The gene expression of CD14, MyD88 and TNFR1SF1 were not up regulated by ethanol but were down regulated by SAMe. The gene expression of IL-1R1 and IRF1 tended to be up regulated by ethanol and this was prevented by feeding SAMe. The results suggest that SAMe, fed chronically prevents the activation of TLR pathways caused by ethanol. In this way the proinflammatory response, fibrogenesis, cirrhosis and hepatocellular carcinoma formation due to alcohol liver disease could be prevented by SAMe.

  12. Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S-Adenosylmethionine:Sterol C-24 Methyltransferase (SAM:SMT)

    PubMed Central

    Kaneshiro, Edna S.; Johnston, Laura Q.; Nkinin, Stephenson W.; Romero, Becky I.; Giner, José-Luis

    2014-01-01

    The AIDS-associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The P. carinii S-adenosylmethionine:sterol C24-methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C-24 position of the sterol side chain producing both C28 and C29 24-alkylsterols in approximately the same proportions whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography (HPLC) and proton nuclear magnetic resonance spectroscopy (1H-NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C28 sterol ergosterol as the major sterol, and also resulted in low levels of C29 sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ24(28)-sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C29 sterols as that found in P. carinii. PMID:25230683

  13. Glucocorticoid-induced S-adenosylmethionine enhances the interferon signaling pathway by restoring STAT1 protein methylation in hepatitis B virus-infected cells.

    PubMed

    Bing, Yuntao; Zhu, Siying; Yu, Guozheng; Li, Ting; Liu, Weijun; Li, Changsheng; Wang, Yitao; Qi, Haolong; Guo, Tao; Yuan, Yufeng; He, Yueming; Liu, Zhisu; Liu, Quanyan

    2014-11-21

    Patients with chronic hepatitis B usually exhibit a low response to treatment with interferon α (IFN-α). An alternative approach to increase the response rate of IFN-α might be to immunologically stimulate the host with glucocorticoids (GCs) before treatment with IFN-α, but the underlying mechanism remains unclear. We hypothesized that the GCs enhance IFN signaling by inducing S-adenosylmethionine (AdoMet) when hepatitis B virus (HBV) replication was effectively suppressed by IFN-α. Here, we investigated the effect of GCs and IFN-α on AdoMet production and methionine adenosyltransferase 1A (MAT1A) expression in vitro. Furthermore, we determined whether post-transcriptional regulation is involved in HBV-repressed MAT1A expression and AdoMet production induced by dexamethasone (Dex). We found that AdoMet homeostasis was disrupted by Dex and that Dex directly regulated MAT1A expression by enhancing the binding of the glucocorticoid receptor (GR) to the glucocorticoid-response element (GRE) of the MAT1A promoter. HBV reduced AdoMet production by increasing methylation at GRE sites within the MAT1A promoter. The X protein of hepatitis B virus led to hypermethylation in the MAT1A promoter by recruiting DNA methyltransferase 1, and it inhibited GR binding to the GRE in the MAT1A promoter. Dex could increase an antiviral effect by inducing AdoMet production via a positive feedback loop when HBV is effectively suppressed by IFN-α, and the mechanism that involves Dex-induced AdoMet could increase STAT1 methylation rather than STAT1 phosphorylation. These findings provide a possible mechanism by which GC-induced AdoMet enhances the antiviral activity of IFN-α by restoring STAT1 methylation in HBV-infected cells.

  14. S-adenosylmethionine and S-adenosylhomocysteine in plasma and cerebrospinal fluid in Rett syndrome and the effect of folinic acid supplementation.

    PubMed

    Hagebeuk, Eveline E O; Duran, Marinus; Abeling, Nico G G M; Vyth, Arno; Poll-The, Bwee Tien

    2013-11-01

    Rett syndrome is a neurodevelopmental disorder characterized by cognitive and locomotor regression and stereotypic hand movements. The disorder is caused by mutations in the X chromosomal MECP2 a gene encoding methyl CpG-binding protein. It has been associated with disturbances of cerebral folate homeostasis, as well as with speculations on a compromised DNA-methylation. Folinic acid is the stable form of folate. Its derived intermediate 5-MTHF supports the conversion of homocysteine to methionine, the precursor of S-adenosylmethionine (SAM). This in turn donates its methyl group to various acceptors, including DNA, thereby being converted to S-adenosylhomocysteine (SAH). The SAM/SAH ratio reflects the methylation potential. The goal of our study was to influence DNA methylation processes and ameliorate the clinical symptoms in Rett syndrome. Therefore we examined the hypothesis that folinic acid supplementation, besides increasing cerebrospinal fluid (CSF) 5-MTHF (p = 0.003), influences SAM and SAH and their ratio. In our randomized, double-blind crossover study on folinic acid supplementation, ten female Rett patients received both folinic acid and placebo for 1 year each. It was shown that both SAM and SAH levels in the CSF remained unchanged following folinic acid administration (p = 0.202 and p = 0.097, respectively) in spite of a rise of plasma SAM and SAH (p = 0.007; p = 0.009). There was no significant change in the SAM/SAH ratio either in plasma or CSF. The apparent inability of Rett patients to upregulate SAM and SAH levels in the CSF may contribute to the biochemical anomalies of the Rett syndrome. Our studies warrant further attempts to promote DNA methylation in the true region of interest, i.e. the brain.

  15. Analysis of the liver mitochondrial proteome in response to ethanol and S-adenosylmethionine treatments: novel molecular targets of disease and hepatoprotection.

    PubMed

    Andringa, Kelly K; King, Adrienne L; Eccleston, Heather B; Mantena, Sudheer K; Landar, Aimee; Jhala, Nirag C; Dickinson, Dale A; Squadrito, Giuseppe L; Bailey, Shannon M

    2010-05-01

    S-adenosylmethionine (SAM) minimizes alcohol hepatotoxicity; however, the molecular mechanisms responsible for SAM hepatoprotection remain unknown. Herein, we use proteomics to determine whether the hepatoprotective action of SAM against early-stage alcoholic liver disease is linked to alterations in the mitochondrial proteome. For this, male rats were fed control or ethanol-containing liquid diets +/- SAM and liver mitochondria were prepared for proteomic analysis. Two-dimensional isoelectric focusing (2D IEF/SDS-PAGE) and blue native gel electrophoresis (BN-PAGE) were used to determine changes in matrix and oxidative phosphorylation (OxPhos) proteins, respectively. SAM coadministration minimized alcohol-dependent inflammation and preserved mitochondrial respiration. SAM supplementation preserved liver SAM levels in ethanol-fed rats; however, mitochondrial SAM levels were increased by ethanol and SAM treatments. With use of 2D IEF/SDS-PAGE, 30 proteins showed significant changes in abundance in response to ethanol, SAM, or both. Classes of proteins affected by ethanol and SAM treatments were chaperones, beta oxidation proteins, sulfur metabolism proteins, and dehydrogenase enzymes involved in methionine, glycine, and choline metabolism. BN-PAGE revealed novel changes in the levels of 19 OxPhos proteins in response to ethanol, SAM, or both. Ethanol- and SAM-dependent alterations in the proteome were not linked to corresponding changes in gene expression. In conclusion, ethanol and SAM treatment led to multiple changes in the liver mitochondrial proteome. The protective effects of SAM against alcohol toxicity are mediated, in part, through maintenance of proteins involved in key mitochondrial energy conserving and biosynthetic pathways. This study demonstrates that SAM may be a promising candidate for treatment of alcoholic liver disease.

  16. Effects of S-Adenosylmethionine and Its Combinations With Taurine and/or Betaine on Glutathione Homeostasis in Ethanol-induced Acute Hepatotoxicity

    PubMed Central

    Lee, Seo Yeon; Ko, Kwang Suk

    2016-01-01

    Background Exposure to ethanol abuse and severe oxidative stress are risk factors for hepatocarcinoma. The aim of this study was to evaluate the effects of S-adenosylmethionine (SAMe) and its combinations with taurine and/or betaine on the level of glutathione (GSH), a powerful antioxidant in the liver, in acute hepatotoxicity induced by ethanol. Methods To examine the effects of SAMe and its combinations with taurine and/or betaine on ethanol-induced hepatotoxicity, AML12 cells and C57BL/6 mice were pretreated with SAMe, taurine, and/or betaine, followed by ethanol challenge. Cell viability was detected with an MTT assay. GSH concentration and mRNA levels of GSH synthetic enzymes were measured using GSH reductase and quantitative real-time reverse transcriptase-PCR. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured with commercially available kits. Results Pretreatment of SAMe, with or without taurine and/or betaine, attenuated decreases in GSH levels and mRNA expression of the catalytic subunit of glutamate-cysteine ligase (GCL), the rate-limiting enzyme for GSH synthesis, in ethanol-treated cells and mice. mRNA levels of the modifier subunit of GCL and glutathione synthetase were increased in mice treated with SAMe combinations. SAMe, taurine, and/or betaine pretreatment restored serum ALT and AST levels to control levels in the ethanol-treated group. Conclusions Combinations of SAMe with taurine and/or betaine have a hepatoprotective effect against ethanol-induced liver injury by maintaining GSH homeostasis. PMID:27722142

  17. Expression of S-adenosylmethionine Hydrolase in Tissues Synthesizing Secondary Cell Walls Alters Specific Methylated Cell Wall Fractions and Improves Biomass Digestibility

    PubMed Central

    Eudes, Aymerick; Zhao, Nanxia; Sathitsuksanoh, Noppadon; Baidoo, Edward E. K.; Lao, Jeemeng; Wang, George; Yogiswara, Sasha; Lee, Taek Soon; Singh, Seema; Mortimer, Jenny C.; Keasling, Jay D.; Simmons, Blake A.; Loqué, Dominique

    2016-01-01

    Plant biomass is a large source of fermentable sugars for the synthesis of bioproducts using engineered microbes. These sugars are stored as cell wall polymers, mainly cellulose and hemicellulose, and are embedded with lignin, which makes their enzymatic hydrolysis challenging. One of the strategies to reduce cell wall recalcitrance is the modification of lignin content and composition. Lignin is a phenolic polymer of methylated aromatic alcohols and its synthesis in tissues developing secondary cell walls is a significant sink for the consumption of the methyl donor S-adenosylmethionine (AdoMet). In this study, we demonstrate in Arabidopsis stems that targeted expression of AdoMet hydrolase (AdoMetase, E.C. 3.3.1.2) in secondary cell wall synthesizing tissues reduces the AdoMet pool and impacts lignin content and composition. In particular, both NMR analysis and pyrolysis gas chromatography mass spectrometry of lignin in engineered biomass showed relative enrichment of non-methylated p-hydroxycinnamyl (H) units and a reduction of dimethylated syringyl (S) units. This indicates a lower degree of methylation compared to that in wild-type lignin. Quantification of cell wall-bound hydroxycinnamates revealed a reduction of ferulate in AdoMetase transgenic lines. Biomass from transgenic lines, in contrast to that in control plants, exhibits an enrichment of glucose content and a reduction in the degree of hemicellulose glucuronoxylan methylation. We also show that these modifications resulted in a reduction of cell wall recalcitrance, because sugar yield generated by enzymatic biomass saccharification was greater than that of wild-type plants. Considering that transgenic plants show no important diminution of biomass yields, and that heterologous expression of AdoMetase protein can be spatiotemporally optimized, this novel approach provides a valuable option for the improvement of lignocellulosic biomass feedstock. PMID:27486577

  18. S-adenosylmethionine blocks collagen I production by preventing transforming growth factor-beta induction of the COL1A2 promoter.

    PubMed

    Nieto, Natalia; Cederbaum, Arthur I

    2005-09-02

    To study the anti-fibrogenic mechanisms of S-adenosylmethionine (AdoMet), transgenic mice harboring the -17 kb to +54 bp of the collagen alpha2 (I) promoter (COL1A2) cloned upstream from the beta-gal reporter gene were injected with carbon tetrachloride (CCl4) to induce fibrosis and coadministered either AdoMet or saline. Control groups received AdoMet or mineral oil. AdoMet lowered the pathology in CCl4-treated mice as shown by transaminase levels, hematoxylin and eosin, Masson's trichrome staining, and collagen I expression. beta-Galactosidase activity indicated activation of the COL1A2 promoter in stellate cells from CCl4-treated mice and repression of such activation by AdoMet. Lipid peroxidation, transforming growth factor-beta (TGFbeta) expression, and decreases in glutathione levels were prevented by AdoMet. Incubation of primary stellate cells with AdoMet down-regulated basal and TGFbeta-induced collagen I and alpha-smooth muscle actin proteins. AdoMet metabolites down-regulated collagen I protein and mRNA levels. AdoMet repressed basal and TGFbeta-induced reporter activity in stellate cells transfected with COL1A2 promoter deletion constructs. AdoMet blocked TGFbeta induction of the -378 bp region of the COL1A2 promoter and prevented the phosphorylation of extracellular signal-regulated kinase 1/2 and the binding of Sp1 to the TGFbeta-responsive element. These observations unveil a novel mechanism by which AdoMet could ameliorate liver fibrosis.

  19. A Phase II Randomized, Controlled Trial of S-Adenosylmethionine in Reducing Serum α-Fetoprotein in Patients with Hepatitis C Cirrhosis and Elevated AFP.

    PubMed

    Morgan, Timothy R; Osann, Kathryn; Bottiglieri, Teodoro; Pimstone, Neville; Hoefs, John C; Hu, Ke-Qin; Hassanein, Tarek; Boyer, Thomas D; Kong, Lorene; Chen, Wen-Pin; Richmond, Ellen; Gonzalez, Rachel; Rodriguez, Luz M; Meyskens, Frank L

    2015-09-01

    In animal models of hepatocellular carcinoma (HCC), deficiency of S-adenosylmethionine (SAMe) increased the risk of HCC whereas administration of SAMe reduced HCC. The aim of this trial was to determine whether oral SAMe administration to patients with hepatitis C cirrhosis would decrease serum α-fetoprotein (AFP) level, a biomarker of HCC risk in hepatitis C. This was a prospective, randomized, placebo-controlled, double-blind trial of SAMe, up to 2.4 g/d, for 24 weeks as compared with placebo among subjects with hepatitis C cirrhosis and a mildly elevated serum AFP. Primary outcome was change in AFP between baseline and week 24. Secondary outcomes included changes in routine tests of liver function and injury, other biomarkers of HCC risk, SAMe metabolites, markers of oxidative stress, and quality of life. One hundred ten subjects were randomized and 87 (44 SAMe and 43 placebo) completed treatment. There was no difference in the change in AFP during 24 weeks among subjects receiving SAMe as compared with placebo. Changes in markers of liver function, liver injury, and hepatitis C viral level were not significantly different between groups. Similarly, SAMe did not change markers of oxidative stress or serum glutathione level. SAMe blood level increased significantly among subjects receiving SAMe. Changes in quality of life did not differ between groups. Overall, this trial did not find that SAMe treatment improved serum AFP in subjects with advanced hepatitis C cirrhosis and a mildly elevated AFP. SAMe did not improve tests of liver function or injury or markers of oxidative stress or antioxidant potential.

  20. Post-translational Modification of Ribosomal Proteins: Structural and Functional Characterization of RimO from Thermotoga maritima, a Radical S-adenosylmethionine methylthiotransferase

    SciTech Connect

    Arragain, S.; Latour, J; Forouhar, F; Neely, H; Montelione, G; Hunt, J; Mulliez, E; Fontecave, M; Atta, M; et al.

    2010-01-01

    Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826-1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 {angstrom} crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family.

  1. Post-translational Modification of Ribosomal Proteins - Structural and Functional Characterization of RimO from Thermotoga Maritima, A Radiacal S-Adenosylmethionine Methylthiotransferase

    SciTech Connect

    Arragain, S.; Garcia-Serres, R; Blondin, G; Douki, T; Clemancey, M; Latour, J; Forouhar, F; Neely, H; Montelione, G; et. al.

    2010-01-01

    Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826-1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 {angstrom} crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family.

  2. Altered growth and polyamine catabolism following exposure of the chocolate spot pathogen Botrytis fabae to the essential oil of Ocimum basilicum.

    PubMed

    Oxenham, Senga K; Svoboda, Katja P; Walters, Dale R

    2005-01-01

    Biomass of the fungal pathogen Botrytis fabae in liquid culture amended with two chemotypes of the essential oil of basil, Ocimum basilicum, was reduced significantly at concentrations of 50 ppm or less. The methyl chavicol chemotype oil increased the activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), but polyamine concentrations were not significantly altered. In contrast, the linalol chemotype oil decreased AdoMetDC activity in B. fabae, although again polyamine concentrations were not altered significantly. However activities of the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO) were increased significantly in B. fabae grown in the presence of the essential oil of the two chemotypes. It is suggested that the elevated activities of DAO and PAO may be responsible, in part, for the antifungal effects of the basil oil, possibly via the generation of hydrogen peroxide and the subsequent triggering of programmed cell death.

  3. Abscisic acid, H2O2 and nitric oxide interactions mediated cold-induced S-adenosylmethionine synthetase in Medicago sativa subsp. falcata that confers cold tolerance through up-regulating polyamine oxidation.

    PubMed

    Guo, Zhenfei; Tan, Jiali; Zhuo, Chunliu; Wang, Congying; Xiang, Bin; Wang, Zengyu

    2014-06-01

    S-adenosylmethionine synthetase (SAMS) is the key enzyme catalysing the formation of S-adenosylmethionine (SAM), a precursor of polyamines and ethylene. To investigate the potential role of SAMS in cold tolerance, we isolated MfSAMS1 from the cold-tolerant germplasm Medicago sativa subsp. falcata and analysed the association of SAM-derived polyamines with cold tolerance. The expression of MfSAMS1 in leaves was greatly induced by cold, abscisic acid (ABA), H2O2 and nitric oxide (NO). Our data revealed that ABA, H2O2 and NO interactions mediated the cold-induced MfSAMS1 expression and cold acclimation in falcata. SAM, putrescine, spermidine and spermine levels, ethylene production and polyamine oxidation were sequentially altered in response to cold, indicating that SAMS-derived SAM is preferentially used in polyamine synthesis and homeostasis during cold acclimation. Antioxidant enzyme activities were also induced in response to cold and showed correlation with polyamine oxidation. Overexpression of MfSAMS1 in tobacco resulted in elevated SAM levels, but polyamine levels and ethylene production in the transgenic plants were not significantly changed. Compared to the wild type, transgenic plants had increased levels of apoplastic H2O2, higher transcript levels of genes involved in polyamine synthesis and oxidation, and higher activities of polyamine oxidation and antioxidant enzymes. The results showed that overexpression of MfSAMS1 promoted polyamine synthesis and oxidation, which in turn improved H2 O2 -induced antioxidant protection, as a result enhanced tolerance to freezing and chilling stress in transgenic plants. This is the first report demonstrating that SAMS plays an important role in plant tolerance to cold via up-regulating polyamine oxidation.

  4. Characterization of the Candida albicans Amino Acid Permease Family: Gap2 Is the Only General Amino Acid Permease and Gap4 Is an S-Adenosylmethionine (SAM) Transporter Required for SAM-Induced Morphogenesis

    PubMed Central

    Kraidlova, Lucie; Schrevens, Sanne; Tournu, Hélène; Van Zeebroeck, Griet; Sychrova, Hana

    2016-01-01

    ABSTRACT Amino acids are key sources of nitrogen for growth of Candida albicans. In order to detect and take up these amino acids from a broad range of different and changing nitrogen sources inside the host, this fungus must be able to adapt via its expression of genes for amino acid uptake and further metabolism. We analyzed six C. albicans putative general amino acid permeases based on their homology to the Saccharomyces cerevisiae Gap1 general amino acid permease. We generated single- and multiple-deletion strains and found that, based on growth assays and transcriptional or posttranscriptional regulation, Gap2 is the functional orthologue to ScGap1, with broad substrate specificity. Expression analysis showed that expression of all GAP genes is under control of the Csy1 amino acid sensor, which is different from the situation in S. cerevisiae, where the expression of ScGAP1 is not regulated by Ssy1. We show that Gap4 is the functional orthologue of ScSam3, the only S-adenosylmethionine (SAM) transporter in S. cerevisiae, and we report that Gap4 is required for SAM-induced morphogenesis. IMPORTANCE Candida albicans is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the C. albicans genome encodes 6 orthologues of the Saccharomyces. cerevisiae general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that C. albicans Gap2 is the functional orthologue of ScGap1 and that C. albicans Gap4 is an orthologue of ScSam3, an S-adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of C. albicans. PMID

  5. Removing a bottleneck in the Bacillus subtilis biotin pathway: bioA utilizes lysine rather than S-adenosylmethionine as the amino donor in the KAPA-to-DAPA reaction.

    PubMed

    Van Arsdell, Scott W; Perkins, John B; Yocum, R Rogers; Luan, Linda; Howitt, C Linda; Chatterjee, Nilu Prasad; Pero, Janice G

    2005-07-05

    In biotin biosynthesis, DAPA aminotransferase encoded by the bioA gene catalyzes the formation of the intermediate 7,8-diaminopelargonic acid (DAPA) from 7-keto-8-aminopelargonic acid (KAPA). DAPA aminotransferases from Escherichia coli, Serratia marcescens, and Bacillus sphaericus use S-adenosylmethionine (SAM) as the amino donor. Our observation that SAM is not an amino donor for B. subtilis DAPA aminotransferase led to a search for an alternative amino donor for this enzyme. Testing of 26 possible amino acids in a cell-free extract assay revealed that only l-lysine was able to dramatically stimulate the in vitro conversion of KAPA to DAPA by the B. subtilis DAPA aminotransferase. The K(m) for lysine and KAPA was estimated to be between 2 and 25 mM, which is significantly higher than the K(m) of purified E. coli BioA for SAM (0.15 mM). This higher requirement for lysine resulted in accumulation of KAPA during fermentation of B. subtilis biotin producing strains. However, this pathway bottleneck could be relieved by either addition of exogenous lysine to the medium or by introduction of lysine deregulated mutations into the production strains.

  6. Effects of Chromosomal Integration of the Vitreoscilla Hemoglobin Gene (vgb) and S-Adenosylmethionine Synthetase Gene (metK) on ε-Poly-L-Lysine Synthesis in Streptomyces albulus NK660.

    PubMed

    Gu, Yanyan; Wang, Xiaomeng; Yang, Chao; Geng, Weitao; Feng, Jun; Wang, Yuanyuan; Wang, Shufang; Song, Cunjiang

    2016-04-01

    ε-Poly-L-lysine (ε-PL) is a widely used natural food preservative. To test the effects of the Vitreoscilla hemoglobin (VHb) and S-adenosylmethionine (SAM) on ε-PL synthesis in Streptomyces albulus NK660, the heterologous VHb gene (vgb) and SAM synthetase gene (metK) were inserted into the S. albulus NK660 chromosome under the control of the constitutive ermE* promoter. CO-difference spectrum analysis showed S. albulus NK660-VHb strain could express functional VHb. S. albulus NK660-VHb produced 26.67 % higher ε-PL and 14.57 % higher biomass than the wild-type control, respectively. Reversed-phase high-pressure liquid chromatography (RP-HPLC) results showed the overexpression of the metK gene resulted in increased intracellular SAM synthesis in S. albulus NK660-SAM, which caused increases of biomass as well as the transcription level of ε-PL synthetase gene (pls). Results indicated that the expression of vgb and metK gene improved on ε-PL synthesis and biomass for S. albulus NK660, respectively.

  7. Protective Effect of Tyrosol and S-Adenosylmethionine against Ethanol-Induced Oxidative Stress of Hepg2 Cells Involves Sirtuin 1, P53 and Erk1/2 Signaling.

    PubMed

    Stiuso, Paola; Bagarolo, Maria Libera; Ilisso, Concetta Paola; Vanacore, Daniela; Martino, Elisa; Caraglia, Michele; Porcelli, Marina; Cacciapuoti, Giovanna

    2016-04-26

    Oxidative stress plays a major role in ethanol-induced liver damage, and agents with antioxidant properties are promising as therapeutic opportunities in alcoholic liver disease. In the present work, we investigated the effect of S-adenosylmethionine (AdoMet), Tyrosol (Tyr), and their combination on HepG2 cells exposed to ethanol exploring the potential molecular mechanisms. We exposed HepG2 cells to 1 M ethanol for 4 and 48 h; thereafter, we recorded a decreased cell viability, increase of intracellular reactive oxygen species (ROS) and lipid accumulation, and the release into culture medium of markers of liver disease such as triacylglycerol, cholesterol, transaminases, albumin, ferritin, and homocysteine. On the other hand, AdoMet and Tyrosol were able to attenuate or antagonize these adverse changes induced by acute exposure to ethanol. The protective effects were paralleled by increased Sirtuin 1 protein expression and nuclear translocation and increased ERK1/2 phosphorylation that were both responsible for the protection of cells from apoptosis. Moreover, AdoMet increased p53 and p21 expression, while Tyrosol reduced p21 expression and enhanced the expression of uncleaved caspase 3 and 9, suggesting that its protective effect may be related to the inhibition of the apoptotic machinery. Altogether, our data show that AdoMet and Tyrosol exert beneficial effects in ethanol-induced oxidative stress in HepG2 cells and provide a rationale for their potential use in combination in the prevention of ethanol-induced liver damage.

  8. Ethanol exposure modulates hepatic S-adenosylmethionine and S-adenosylhomocysteine levels in the isolated perfused rat liver through changes in the redox state of the NADH/NAD+ system

    PubMed Central

    Watson, Walter H.; Song, Zhenyuan; Kirpich, Irina A.; Deaciuc, Ion V.; Chen, Theresa; McClain, Craig J.

    2013-01-01

    Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD+ ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high-performance liquid chromatography. The increase in the NADH/NAD+ ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD+-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD+ ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD+ ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD+ redox couple and the effects of ethanol on methionine metabolism in the liver. PMID:21296661

  9. Genetics Home Reference: malonyl-CoA decarboxylase deficiency

    MedlinePlus

    ... deficiency of malonyl-CoA decarboxylase malonic aciduria malonyl-coenzyme A decarboxylase deficiency MCD deficiency Related Information How ... molecular characterization of nine new patients with malonyl-coenzyme A decarboxylase deficiency. J Inherit Metab Dis. 2007 ...

  10. Genetics Home Reference: aromatic l-amino acid decarboxylase deficiency

    MedlinePlus

    ... l-amino acid decarboxylase deficiency aromatic l-amino acid decarboxylase deficiency Enable Javascript to view the expand/ ... Open All Close All Description Aromatic l-amino acid decarboxylase (AADC) deficiency is an inherited disorder that ...

  11. Cfr and RlmN contain a single [4Fe-4S] cluster, which directs two distinct reactivities for S-adenosylmethionine: methyl transfer by SN2 displacement and radical generation.

    PubMed

    Grove, Tyler L; Radle, Matthew I; Krebs, Carsten; Booker, Squire J

    2011-12-14

    The radical SAM (RS) proteins RlmN and Cfr catalyze methylation of carbons 2 and 8, respectively, of adenosine 2503 in 23S rRNA. Both reactions are similar in scope, entailing the synthesis of a methyl group partially derived from S-adenosylmethionine (SAM) onto electrophilic sp(2)-hybridized carbon atoms via the intermediacy of a protein S-methylcysteinyl (mCys) residue. Both proteins contain five conserved Cys residues, each required for turnover. Three cysteines lie in a canonical RS CxxxCxxC motif and coordinate a [4Fe-4S]-cluster cofactor; the remaining two are at opposite ends of the polypeptide. Here we show that each protein contains only the one "radical SAM" [4Fe-4S] cluster and the two remaining conserved cysteines do not coordinate additional iron-containing species. In addition, we show that, while wild-type RlmN bears the C355 mCys residue in its as-isolated state, RlmN that is either engineered to lack the [4Fe-4S] cluster by substitution of the coordinating cysteines or isolated from Escherichia coli cultured under iron-limiting conditions does not bear a C355 mCys residue. Reconstitution of the [4Fe-4S] cluster on wild-type apo RlmN followed by addition of SAM results in rapid production of S-adenosylhomocysteine (SAH) and the mCys residue, while treatment of apo RlmN with SAM affords no observable reaction. These results indicate that in Cfr and RlmN, SAM bound to the unique iron of the [4Fe-4S] cluster displays two reactivities. It serves to methylate C355 of RlmN (C338 of Cfr), or to generate the 5'-deoxyadenosyl 5'-radical, required for substrate-dependent methyl synthase activity.

  12. Cfr and RlmN Contain a Single [4Fe-4S] Cluster, which Directs Two Distinct Reactivities for S-Adenosylmethionine: Methyl Transfer by SN2 Displacement and Radical Generation

    PubMed Central

    Grove, Tyler L.; Radle, Matthew I.; Krebs, Carsten; Booker, Squire J.

    2012-01-01

    The radical SAM (RS) proteins RlmN and Cfr catalyze methylation of carbons 2 and 8, respectively, of adenosine 2503 in 23S rRNA. Both reactions are similar in scope, entailing the synthesis of a methyl group partially derived from S-adenosylmethionine (SAM) onto electrophilic sp2-hybridized carbon atoms via the intermediacy of a protein S-methylcysteinyl (mCys) residue. Both proteins contain five conserved Cys residues, each of which is required for turnover. Three cysteines lie in a canonical RS CxxxCxxC motif and coordinate a [4Fe–4S]-cluster cofactor. The remaining two cysteines are at opposite ends of the polypeptide. Herein we show that each protein contains only the one “radical SAM” [4Fe–4S] cluster, and that the two remaining conserved cysteines do not coordinate additional iron-containing species. In addition, we show that while wild-type RlmN bears the C355 mCys residue in its as-isolated state, RlmN that is either engineered to lack the [4Fe–4S] cluster by substitution of the coordinating cysteines, or isolated from Escherichia coli cultured under iron-limiting conditions, does not bear a C355 mCys residue. Reconstitution of the [4Fe–4S] cluster on wild-type apo RlmN followed by addition of SAM results in rapid production of S-adenosylhomocysteine (SAH) and the mCys residue, while treatment of apo RlmN with SAM affords no observable reaction. These results indicate that in Cfr and RlmN, SAM bound to the unique iron of the [4Fe–4S] cluster displays two reactivities. It serves to methylate C355 of RlmN (C338 of Cfr), or it serves to generate the 5’-deoxyadenosyl 5’-radical, required for substrate-dependent methyl synthase activity. PMID:21916495

  13. Protective Effect of Tyrosol and S-Adenosylmethionine against Ethanol-Induced Oxidative Stress of Hepg2 Cells Involves Sirtuin 1, P53 and Erk1/2 Signaling

    PubMed Central

    Stiuso, Paola; Bagarolo, Maria Libera; Ilisso, Concetta Paola; Vanacore, Daniela; Martino, Elisa; Caraglia, Michele; Porcelli, Marina; Cacciapuoti, Giovanna

    2016-01-01

    Oxidative stress plays a major role in ethanol-induced liver damage, and agents with antioxidant properties are promising as therapeutic opportunities in alcoholic liver disease. In the present work, we investigated the effect of S-adenosylmethionine (AdoMet), Tyrosol (Tyr), and their combination on HepG2 cells exposed to ethanol exploring the potential molecular mechanisms. We exposed HepG2 cells to 1 M ethanol for 4 and 48 h; thereafter, we recorded a decreased cell viability, increase of intracellular reactive oxygen species (ROS) and lipid accumulation, and the release into culture medium of markers of liver disease such as triacylglycerol, cholesterol, transaminases, albumin, ferritin, and homocysteine. On the other hand, AdoMet and Tyrosol were able to attenuate or antagonize these adverse changes induced by acute exposure to ethanol. The protective effects were paralleled by increased Sirtuin 1 protein expression and nuclear translocation and increased ERK1/2 phosphorylation that were both responsible for the protection of cells from apoptosis. Moreover, AdoMet increased p53 and p21 expression, while Tyrosol reduced p21 expression and enhanced the expression of uncleaved caspase 3 and 9, suggesting that its protective effect may be related to the inhibition of the apoptotic machinery. Altogether, our data show that AdoMet and Tyrosol exert beneficial effects in ethanol-induced oxidative stress in HepG2 cells and provide a rationale for their potential use in combination in the prevention of ethanol-induced liver damage. PMID:27128904

  14. PqqD Is a Novel Peptide Chaperone That Forms a Ternary Complex with the Radical S-Adenosylmethionine Protein PqqE in the Pyrroloquinoline Quinone Biosynthetic Pathway*

    PubMed Central

    Latham, John A.; Iavarone, Anthony T.; Barr, Ian; Juthani, Prerak V.; Klinman, Judith P.

    2015-01-01

    Pyrroloquinoline quinone (PQQ) is a product of a ribosomally synthesized and post-translationally modified pathway consisting of five conserved genes, pqqA-E. PqqE is a radical S-adenosylmethionine (RS) protein with a C-terminal SPASM domain, and is proposed to catalyze the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the peptide substrate PqqA. PqqD is a 10-kDa protein with an unknown function, but is essential for PQQ production. Recently, in Klebsiella pneumoniae (Kp), PqqD and PqqE were shown to interact; however, the stoichiometry and KD were not obtained. Here, we show that the PqqE and PqqD interaction transcends species, also occurring in Methylobacterium extorquens AM1 (Me). The stoichiometry of the MePqqD and MePqqE interaction is 1:1 and the KD, determined by surface plasmon resonance spectroscopy (SPR), was found to be ∼12 μm. Moreover, using SPR and isothermal calorimetry techniques, we establish for the first time that MePqqD binds MePqqA tightly (KD ∼200 nm). The formation of a ternary MePqqA-D-E complex was captured by native mass spectrometry and the KD for the MePqqAD-MePqqE interaction was found to be ∼5 μm. Finally, using a bioinformatic analysis, we found that PqqD orthologues are associated with the RS-SPASM family of proteins (subtilosin, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin), all of which modify either peptides or proteins. In conclusion, we propose that PqqD is a novel peptide chaperone and that PqqD orthologues may play a similar role in peptide modification pathways that use an RS-SPASM protein. PMID:25817994

  15. Zymographic detection of cinnamic acid decarboxylase activity.

    PubMed

    Prim, Núria; Pastor, F I Javier; Diaz, Pilar

    2002-11-01

    The manuscript includes a concise description of a new, fast and simple method for detection of cinnamic acid decarboxylase activity. The method is based on a color shift caused a by pH change and may be an excellent procedure for large screenings of samples from natural sources, as it involves no complex sample processing or purification. The method developed can be used in preliminary approaches to biotransformation processes involving detection of hydroxycinnamic acid decarboxylase activity.

  16. Up-regulation of spermidine/spermine N1-acetyltransferase (SSAT) expression is a part of proliferative but not anabolic response of mouse kidney.

    PubMed

    Dudkowska, Magdalena; Stachurska, Agnieszka; Grzelakowska-Sztabert, Barbara; Manteuffel-Cymborowska, Małgorzata

    2002-01-01

    A differential expression pattern of spermidine/spermine N(1)-acetyltransferase (SSAT), the enzyme critical to proper homeostasis of cellular polyamines, is reported in mouse kidney undergoing hyperplasia and hypertrophy. We have shown that SSAT activity and SSAT mRNA are significantly induced by antifolate CB 3717 and folate that evoke a drug-injury-dependent hyperplasia. In contrast, SSAT activity is down-regulated in the testosterone-induced hypertrophic kidney, while SSAT mRNA is positively controlled by this androgen. Catecholamine depletion evoked by reserpine drastically decreases the folate-induced activity of S-adenosylmethionine decarboxylase (AdoMetDC), which limits polyamine biosynthesis, but has no effect on SSAT activity augmented by CB 3717. Our results document that the increased SSAT expression solely accompanies the proliferative response of mouse kidney, and suggest the importance of post-transcriptional regulation to the control of SSAT activity in both hyperplastic and hypertrophic experimental models.

  17. Induction of aromatic-L-amino acid decarboxylase by decarboxylase inhibitors in idiopathic parkinsonism.

    PubMed

    Boomsma, F; Meerwaldt, J D; Man in 't Veld, A J; Hovestadt, A; Schalekamp, M A

    1989-06-01

    We evaluated the effect of administration of L-dopa, alone or in combination with a peripheral decarboxylase inhibitor, on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD). After single-dose administration of L-dopa plus benserazide (Madopar) in healthy subjects and in chronically treated patients with parkinsonism, plasma ALAAD followed for 2 to 3 hours fell, but returned to predosing levels within 90 minutes. Four groups of patients with idiopathic parkinsonism were studied during chronic treatment: Group I, no L-dopa treatment (n = 31); Group II, L-dopa alone (n = 15); Group III, L-dopa plus benserazide (n = 28); and Group IV, L-dopa plus carbidopa (Sinemet, n = 30). Plasma ALAAD 2 hours after dosing was normal in Groups I and II. ALAAD was increased threefold in Groups III and IV, suggesting induction of ALAAD by the coadministration of a peripheral decarboxylase inhibitor. In a study of 3 patients in whom L-dopa/benserazide was started, plasma ALAAD rose gradually over 3 to 4 weeks. Further detailed pharmacokinetic studies of L-dopa, dopamine, and ALAAD in plasma and cerebrospinal fluid are required to determine if the apparent ALAAD induction by a peripheral decarboxylase inhibitor may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena.

  18. Arginine Decarboxylase Is Localized in Chloroplasts.

    PubMed Central

    Borrell, A.; Culianez-Macia, F. A.; Altabella, T.; Besford, R. T.; Flores, D.; Tiburcio, A. F.

    1995-01-01

    Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC. PMID:12228631

  19. Dopa decarboxylase activity of the living human brain

    SciTech Connect

    Gjedde, A.; Reith, J.; Dyve, S.; Leger, G.; Guttman, M.; Diksic, M.; Evans, A.; Kuwabara, H. )

    1991-04-01

    Monoaminergic neurons use dopa decarboxylase to form dopamine from L-3,4-dihydroxyphenylalanine (L-dopa). We measured regional dopa decarboxylase activity in brains of six healthy volunteers with 6-({sup 18}F)fluoro-L-dopa and positron emission tomography. We calculated the enzyme activity, relative to its Km, with a kinetic model that yielded the relative rate of conversion of 6-({sup 18}F)fluoro-L-dopa to ({sup 18}F)fluorodopamine. Regional values of relative dopa decarboxylase activity ranged from nil in occipital cortex to 1.9 h-1 in caudate nucleus and putamen, in agreement with values obtained in vitro.

  20. Three Distinct Glutamate Decarboxylase Genes in Vertebrates

    PubMed Central

    Grone, Brian P.; Maruska, Karen P.

    2016-01-01

    Gamma-aminobutyric acid (GABA) is a widely conserved signaling molecule that in animals has been adapted as a neurotransmitter. GABA is synthesized from the amino acid glutamate by the action of glutamate decarboxylases (GADs). Two vertebrate genes, GAD1 and GAD2, encode distinct GAD proteins: GAD67 and GAD65, respectively. We have identified a third vertebrate GAD gene, GAD3. This gene is conserved in fishes as well as tetrapods. We analyzed protein sequence, gene structure, synteny, and phylogenetics to identify GAD3 as a homolog of GAD1 and GAD2. Interestingly, we found that GAD3 was lost in the hominid lineage. Because of the importance of GABA as a neurotransmitter, GAD3 may play important roles in vertebrate nervous systems. PMID:27461130

  1. Keto-isovalerate decarboxylase enzymes and methods of use thereof

    DOEpatents

    McElvain, Jessica; O'Keefe, Daniel P.; Paul, Brian James; Payne, Mark S.; Rothman, Steven Cary; He, Hongxian

    2016-01-19

    Provided herein are polypeptides and polynucleotides encoding such polypeptides which have ketoisovalerate decarboxylase activity. Also provided are recombinant host cells comprising such polypeptides and polynucleotides and methods of use thereof.

  2. Mapping of glutamic acid decarboxylase (GAD) genes

    SciTech Connect

    Edelhoff, S.; Adler, D.A.; Disteche, C.M.; Grubin, C.E.; Karlsen, A.E.; Lernmark, A.; Foster, D. )

    1993-07-01

    Glutamic acid decarboxylase (GAD) catalyzes the synthesis of [gamma]-aminobutyric acid (GABA), which is known as a major inhibitory neurotransmitter in the central nervous system (CNS), but is also present outside the CNS. Recent studies showed that GAD is the major target of autoantibodies associated with the development of insulin-dependent diabetes mellitus and of the rare stiff man syndrome. Studies of GAD expression have demonstrated multiple transcripts, suggesting several isoforms of GAD. In this study, three different genes were mapped by in situ hybridization to both human and mouse chromosomes. The GAD1 gene was mapped to human chromosome 2q31 and to mouse chromosome 2D in a known region of conservation between human and mouse. GAD2, previously mapped to human chromosome 10p11.2-p12, was mapped to mouse chromosome 2A2-B, which identifies a new region of conservation between human and mouse chromosomes. A potential GAD3 transcript was mapped to human chromosome 22q13 and to mouse chromosome 15E in a known region of conservation between human and mouse. It is concluded that the GAD genes may form a family with as many as three related members. 30 refs., 5 figs.

  3. Post-transcriptional regulation of ornithine decarboxylase

    PubMed Central

    Nowotarski, Shannon L.; Origanti, Sofia; Shantz, Lisa M.

    2013-01-01

    Activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC), and intracellular levels of ODC protein are controlled very tightly. Numerous studies have described ODC regulation at the levels of transcription, translation and protein degradation in normal cells, and dysregulation of these processes in response to oncogenic stimuli. Although post-transcriptional regulation of ODC has been well-documented, the RNA binding proteins (RBPs) that interact with ODC mRNA and control synthesis of the ODC protein have not been defined. Using Ras-transformed rat intestinal epithelial cells (Ras12V cells) as a model, we have begun identifying the RBPs that associate with the ODC transcript. Binding of RBPs could potentially regulate ODC synthesis by either changing mRNA stability or rate of mRNA translation. Techniques for measuring RBP binding and translation initiation are described here. Targeting control of ODC translation or mRNA decay could be a valuable method of limiting polyamine accumulation and subsequent tumor development in a variety of cancers. PMID:21318880

  4. Localization of arginine decarboxylase in tobacco plants.

    PubMed

    Bortolotti, Cristina; Cordeiro, Alexandra; Alcázar, Rubén; Borrell, Antoni; Culiañez-Macià, Francisco A.; Tiburcio, Antonio F.; Altabella, Teresa

    2004-01-01

    The lack of knowledge about the tissue and subcellular distribution of polyamines (PAs) and the enzymes involved in their metabolism remains one of the main obstacles in our understanding of the biological role of PAs in plants. Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We have characterized a cDNA coding for ADC from Nicotiana tabacum L. cv. Petit Havana SR1. The deduced ADC polypeptide had 721 amino acids and a molecular mass of 77 kDa. The ADC cDNA was overexpressed in Escherichia coli, and the ADC fusion protein obtained was used to produce polyclonal antibodies. Using immunological methods, we demonstrate the presence of the ADC protein in all plant organs analysed: flowers, seeds, stems, leaves and roots. Moreover, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplast. In photosynthetic tissues, ADC is located mainly in chloroplasts, whereas in non-photosynthetic tissues the protein appears to be located in nuclei. The different compartmentation of ADC may be related to distinct functions of the protein in different cell types.

  5. A Porphodimethene Chemical Inhibitor of Uroporphyrinogen Decarboxylase

    PubMed Central

    Yip, Kenneth W.; Zhang, Zhan; Sakemura-Nakatsugawa, Noriko; Huang, Jui-Wen; Vu, Nhu Mai; Chiang, Yi-Kun; Lin, Chih-Lung; Kwan, Jennifer Y. Y.; Yue, Shijun; Jitkova, Yulia; To, Terence; Zahedi, Payam; Pai, Emil F.; Schimmer, Aaron D.; Lovell, Jonathan F.; Sessler, Jonathan L.; Liu, Fei-Fei

    2014-01-01

    Uroporphyrinogen decarboxylase (UROD) catalyzes the conversion of uroporphyrinogen to coproporphyrinogen during heme biosynthesis. This enzyme was recently identified as a potential anticancer target; its inhibition leads to an increase in reactive oxygen species, likely mediated by the Fenton reaction, thereby decreasing cancer cell viability and working in cooperation with radiation and/or cisplatin. Because there is no known chemical UROD inhibitor suitable for use in translational studies, we aimed to design, synthesize, and characterize such a compound. Initial in silico-based design and docking analyses identified a potential porphyrin analogue that was subsequently synthesized. This species, a porphodimethene (named PI-16), was found to inhibit UROD in an enzymatic assay (IC50 = 9.9 µM), but did not affect porphobilinogen deaminase (at 62.5 µM), thereby exhibiting specificity. In cellular assays, PI-16 reduced the viability of FaDu and ME-180 cancer cells with half maximal effective concentrations of 22.7 µM and 26.9 µM, respectively, and only minimally affected normal oral epithelial (NOE) cells. PI-16 also combined effectively with radiation and cisplatin, with potent synergy being observed in the case of cisplatin in FaDu cells (Chou-Talalay combination index <1). This work presents the first known synthetic UROD inhibitor, and sets the foundation for the design, synthesis, and characterization of higher affinity and more effective UROD inhibitors. PMID:24587102

  6. Properties of oxaloacetate decarboxylase from Veillonella parvula.

    PubMed Central

    Ng, S K; Wong, M; Hamilton, I R

    1982-01-01

    Oxaloacetate decarboxylase was purified to 136-fold from the oral anaerobe Veillonella parvula. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity of the enzyme was exhibited at pH 7.0 for both carboxylation and decarboxylation. At this pH, the Km values for oxaloacetate and Mg2+ were at 0.06 and 0.17 mM, respectively, whereas the Km values for pyruvate, CO2, and Mg2+ were 3.3, 1.74, and 1.85 mM, respectively. Hyperbolic kinetics were observed with all of the aforementioned compounds. The Keq' was 2.13 X 10(-3) mM-1 favoring the decarboxylation of oxaloacetate. In the carboxylation step, avidin, acetyl coenzyme A, biotin, and coenzyme A were not required. ADP and NADH had no effect on either the carboxylation or decarboxylation step, but ATP inhibited the carboxylation step competitively and the decarboxylation step noncompetitively. These types of inhibition fitted well with the overall lactate metabolism of the non-carbohydrate-fermenting anaerobe. PMID:7076619

  7. Self Sacrifice in Radical S-adenosylmethionine Proteins

    PubMed Central

    Booker, Squire J.; Cicchillo, Robert M.; Grove, Tyler L.

    2007-01-01

    The radical SAM superfamily of metalloproteins catalyze the reductive cleavage of S-adenosyl-l-methionine to generate a 5′-deoxyadenosyl radical (5′-dA•) intermediate that is obligate for turnover. The 5′-dA• acts as a potent oxidant, initiating turnover by abstracting a hydrogen atom from an appropriate substrate. A special class of these enzymes use this strategy to functionalize unactivated C–H bonds by insertion of sulfur atoms. This review will describe the characterization of three members of this class—biotin synthase, lipoyl synthase, and MiaB protein—each of which has been shown to cannibalize itself during turnover. PMID:17936058

  8. Characterization of a second lysine decarboxylase isolated from Escherichia coli.

    PubMed Central

    Kikuchi, Y; Kojima, H; Tanaka, T; Takatsuka, Y; Kamio, Y

    1997-01-01

    We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in lambda Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated Idc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli. PMID:9226257

  9. Nucleotide sequence of the pyruvate decarboxylase gene from Zymomonas mobilis.

    PubMed

    Neale, A D; Scopes, R K; Wettenhall, R E; Hoogenraad, N J

    1987-02-25

    Pyruvate decarboxylase (EC 4.1.1.1), the penultimate enzyme in the alcoholic fermentation pathway of Zymomonas mobilis, converts pyruvate to acetaldehyde and carbon dioxide. The complete nucleotide sequence of the structural gene encoding pyruvate decarboxylase from Zymomonas mobilis has been determined. The coding region is 1704 nucleotides long and encodes a polypeptide of 567 amino acids with a calculated subunit mass of 60,790 daltons. The amino acid sequence was confirmed by comparison with the amino acid sequence of a selection of tryptic fragments of the enzyme. The amino acid composition obtained from the nucleotide sequence is in good agreement with that obtained experimentally.

  10. Comparison between activation of ornithine decarboxylase and histidine decarboxylase in rat stomach.

    PubMed

    Ding, X Q; Chen, D; Rosengren, E; Persson, L; Hakanson, R

    1996-03-01

    We compared the responses of rat stomach ornithine decarboxylase (ODC) and histidine decarboxylase (HDC) to food intake, oral treatment with antisecretagogues, NaHCO3, and hypertonic NaCl, antrectomy, intravenous infusion of gastrin-17, the selective cholecystokinin (CCK)-B/gastrin receptor antagonist L-365,260, and the somatostatin analogue RC-160. The serum gastrin concentration and oxyntic mucosal ODC and HDC activities were higher in freely fed rats than in fasted rats. Food intake in fasted rats raised the serum gastrin concentration and the ODC and HDC activities. Ranitidine, omeprazole, and NaHCO3 raised the serum gastrin concentration and activated ODC and HDC. Hypertonic NaCl raised the ODC activity 200-fold, whereas circulating gastrin and HDC activity were increased only moderately. Infusion of gastrin-17 activated HDC but not ODC. L-365,260 prevented the activation of HDC but not of ODC in response to food intake and treatment with omeprazole, NaHCO3, or hypertonic NaCl. Antrectomy prevented the food- and omeprazole-evoked rise in oxyntic mucosal HDC activity but not the rise in ODC activity. RC-160 suppressed HDC activity after food intake and treatment with omeprazole, NaHCO3, or NaCl. In contrast, RC-160 suppressed omeprazole- and NaHCO3-evoked ODC activation but not that evoked by food intake or NaCl. The results support the view that HDC in the oxyntic mucosa is activated by gastrin and suppressed by somatostatin. The induction of ODC is not mediated by gastrin; ODC activation appears to be related to acid inhibition per se or to mucosal maintenance and repair; somatostatin, or rather the lack of it, might contribute to the induction of ODC after acid blockade. The mechanism behind the activation of rat stomach ODC seems to differ depending on the type of stimulus.

  11. 3-Aminooxy-1-Aminopropane and Derivatives Have an Antiproliferative Effect on Cultured Plasmodium falciparum by Decreasing Intracellular Polyamine Concentrations

    PubMed Central

    Das Gupta, Robin; Krause-Ihle, Tanja; Bergmann, Bärbel; Müller, Ingrid B.; Khomutov, Alex R.; Müller, Sylke; Walter, Rolf D.; Lüersen, Kai

    2005-01-01

    The intraerythrocytic development of Plasmodium falciparum correlates with increasing levels of the polyamines putrescine, spermidine, and spermine in the infected red blood cells; and compartmental analyses revealed that the majority is associated with the parasite. Since depletion of cellular polyamines is a promising strategy for inhibition of parasite proliferation, new inhibitors of polyamine biosynthesis were tested for their antimalarial activities. The ornithine decarboxylase (ODC) inhibitor 3-aminooxy-1-aminopropane (APA) and its derivatives CGP 52622A and CGP 54169A as well as the S-adenosylmethionine decarboxlyase (AdoMetDC) inhibitors CGP 40215A and CGP 48664A potently affected the bifunctional P. falciparum ODC-AdoMetDC, with Ki values in the low nanomolar and low micromolar ranges, respectively. Furthermore, the agents were examined for their in vitro plasmodicidal activities in 48-h incubation assays. APA, CGP 52622A, CGP 54169A, and CGP 40215A were the most effective, with 50% inhibitory concentrations below 3 μM. While the effects of the ODC inhibitors were completely abolished by the addition of putrescine, growth inhibition by the AdoMetDC inhibitor CGP 40215A could not be antagonized by putrescine or spermidine. Moreover, CGP 40215A did not affect the cellular polyamine levels, indicating a mechanism of action against P. falciparum independent of polyamine synthesis. In contrast, the ODC inhibitors led to decreased cellular putrescine and spermidine levels in P. falciparum, supporting the fact that they exert their antimalarial activities by inhibition of the bifunctional ODC-AdoMetDC. PMID:15980361

  12. Lotus hairy roots expressing inducible arginine decarboxylase activity.

    PubMed

    Chiesa, María A; Ruiz, Oscar A; Sánchez, Diego H

    2004-05-01

    Biotechnological uses of plant cell-tissue culture usually rely on constitutive transgene expression. However, such expression of transgenes may not always be desirable. In those cases, the use of an inducible promoter could be an alternative approach. To test this hypothesis, we developed two binary vectors harboring a stress-inducible promoter from Arabidopsis thaliana, driving the beta-glucuronidase reporter gene and the oat arginine decarboxylase. Transgenic hairy roots of Lotus corniculatus were obtained with osmotic- and cold-inducible beta-glucuronidase and arginine decarboxylase activities. The increase in the activity of the latter was accompanied by a significant rise in total free polyamines level. Through an organogenesis process, we obtained L. corniculatus transgenic plants avoiding deleterious phenotypes frequently associated with the constitutive over-expression of arginine decarboxylation and putrescine accumulation.

  13. Arginine decarboxylase as the source of putrescine for tobacco alkaloids

    NASA Technical Reports Server (NTRS)

    Tiburcio, A. F.; Galston, A. W.

    1986-01-01

    The putrescine which forms a part of nicotine and other pyrrolidine alkaloids is generally assumed to arise through the action of ornithine decarboxylase (ODC). However, we have previously noted that changes in the activity of arginine decarboxylase (ADC), an alternate source of putrescine, parallel changes in tissue alkaloids, while changes in ODC activity do not. This led us to undertake experiments to permit discrimination between ADC and ODC as enzymatic sources of putrescine destined for alkaloids. Two kinds of evidence presented here support a major role for ADC in the generation of putrescine going into alkaloids: (a) A specific 'suicide inhibitor' of ADC effectively inhibits the biosynthesis of nicotine and nornicotine in tobacco callus, while the analogous inhibitor of ODC is less effective, and (b) the flow of 14C from uniformly labelled arginine into nicotine is much more efficient than that from ornithine.

  14. Ornithine decarboxylase activity and: [125I]iododeoxyuridine incorporation in rat prostate.

    PubMed Central

    Fuller, D J; Donaldson, L J; Thomas, G H

    1975-01-01

    The relationship between ornithine decarboxylase activity and [125I]iododexyuridine incorporation was studied in prostates from castrated rats (aged 5, 26 and 80 weeks) injected daily with testosterone for up to 10 days. The results suggest that ornithine decarboxylase activity is a parameter of secretory activity, rather than growth, in the ventral prostate. In the dorsolateral prostate, ornithine decarboxylase activity tends to parallel [125I]iododeoxyuridine incorporation. PMID:1212206

  15. Relief of autoinhibition by conformational switch explains enzyme activation by a catalytically dead paralog

    PubMed Central

    Volkov, Oleg A; Kinch, Lisa; Ariagno, Carson; Deng, Xiaoyi; Zhong, Shihua; Grishin, Nick; Tomchick, Diana R; Chen, Zhe; Phillips, Margaret A

    2016-01-01

    Catalytically inactive enzyme paralogs occur in many genomes. Some regulate their active counterparts but the structural principles of this regulation remain largely unknown. We report X-ray structures of Trypanosoma brucei S-adenosylmethionine decarboxylase alone and in functional complex with its catalytically dead paralogous partner, prozyme. We show monomeric TbAdoMetDC is inactive because of autoinhibition by its N-terminal sequence. Heterodimerization with prozyme displaces this sequence from the active site through a complex mechanism involving a cis-to-trans proline isomerization, reorganization of a β-sheet, and insertion of the N-terminal α-helix into the heterodimer interface, leading to enzyme activation. We propose that the evolution of this intricate regulatory mechanism was facilitated by the acquisition of the dimerization domain, a single step that can in principle account for the divergence of regulatory schemes in the AdoMetDC enzyme family. These studies elucidate an allosteric mechanism in an enzyme and a plausible scheme by which such complex cooperativity evolved. DOI: http://dx.doi.org/10.7554/eLife.20198.001 PMID:27977001

  16. Cell biology, physiology and enzymology of phosphatidylserine decarboxylase.

    PubMed

    Di Bartolomeo, Francesca; Wagner, Ariane; Daum, Günther

    2017-01-01

    Phosphatidylethanolamine is one of the most abundant phospholipids whose major amounts are formed by phosphatidylserine decarboxylases (PSD). Here we provide a comprehensive description of different types of PSDs in the different kingdoms of life. In eukaryotes, type I PSDs are mitochondrial enzymes, whereas other PSDs are localized to other cellular compartments. We describe the role of mitochondrial Psd1 proteins, their function, enzymology, biogenesis, assembly into mitochondria and their contribution to phospholipid homeostasis in much detail. We also discuss briefly the cellular physiology and the enzymology of Psd2. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

  17. Resolution of brewers' yeast pyruvate decarboxylase into two isozymes.

    PubMed

    Kuo, D J; Dikdan, G; Jordan, F

    1986-03-05

    A novel purification method was developed for brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) that for the first time resolved the enzyme into two isozymes on DEAE-Sephadex chromatography. The isozymes were found to be distinct according to sodium dodecyl sulfate polyacrylamide gel electrophoresis: the first one to be eluted gave rise to one band, the second to two bands. The isozymes were virtually the same so far as specific activity, KM, inhibition kinetics and irreversible binding properties by the mechanism-based inhibitor (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid are concerned. This finding resolves a longstanding controversy concerning the quaternary structure of this enzyme.

  18. Vector-mediated chromosomal integration of the glutamate decarboxylase gene in streptococcus thermophilus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The integrative vector pINTRS was used to transfer glutamate decarboxylase (GAD) activity to Streptococcus thermophilus ST128, thus allowing for the production of '-aminobutyric acid (GABA). In pINTRS, the gene encoding glutamate decarboxylase, gadB, was flanked by DNA fragments homologous to a S. ...

  19. Molecular and functional analyses of amino acid decarboxylases involved in cuticle tanning in Tribolium castaneum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspartate 1-decarboxylase (ADC) and dopa decarboxylase (DDC) provide b–alanine and dopamine used in insect cuticle tanning. Beta-alanine is conjugated with dopamine to yield N-b-alanyldopamine (NBAD), a substrate for the phenoloxidase laccase that catalyzes the synthesis of cuticle protein cross-li...

  20. Functionally diverse biotin-dependent enzymes with oxaloacetate decarboxylase activity.

    PubMed

    Lietzan, Adam D; St Maurice, Martin

    2014-02-15

    Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N' of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO2 in order to power the export of sodium ions from the cytoplasm. A homologous carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii. Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.

  1. Tyrosine decarboxylase from Lactobacillus brevis: soluble expression and characterization.

    PubMed

    Zhang, Kai; Ni, Ye

    2014-02-01

    Tyrosine decarboxylase (TDC, EC 4.1.1.25) is an enzyme that catalyzes the decarboxylation of l-tyrosine to produce tyramine and CO2. In this study, a 1881-bp tdc gene from Lactobacillus brevis was cloned and heterologously expressed in Escherichia coli BL21 (DE3). Glucose was discovered to play an important role in the soluble expression of rLbTDC. After optimization, recombinant TDC (rLbTDC) was achieved in excellent solubility and a yield of 224mg rLbTDC/L broth. The C-terminal His-Tagged rLbTDC was one-step purified with 90% recovery. Based on SDS-PAGE and gel filtration analysis, rLbTDC is a dimer composed of two identical subunits of approximately 70kDa. Using l-tyrosine as substrate, the specific activity of rLbTDC was determined to be 133.5U/mg in the presence of 0.2mM pyridoxal-5'-phosphate at 40°C and pH 5.0. The Km and Vmax values of rLbTDC were 0.59mM and 147.1μmolmin(-1)mg(-1), respectively. In addition to l-tyrosine, rLbTDC also exhibited decarboxylase activity towards l-DOPA. This study has demonstrated, for the first time, the soluble expression of tdc gene from L. brevis in heterologous host.

  2. Crystal structure of pyruvate decarboxylase from Zymobacter palmae

    PubMed Central

    Buddrus, Lisa; Andrews, Emma S. V.; Leak, David J.; Danson, Michael J.; Arcus, Vickery L.; Crennell, Susan J.

    2016-01-01

    Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg2+ ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15 Å resolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55 Å and R r.i.m. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were R work = 0.186 (0.271 in the highest resolution bin) and R free = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions. PMID:27599861

  3. Uncovering the Lactobacillus plantarum WCFS1 Gallate Decarboxylase Involved in Tannin Degradation

    PubMed Central

    Jiménez, Natalia; Curiel, José Antonio; Reverón, Inés; de las Rivas, Blanca

    2013-01-01

    Lactobacillus plantarum is a lactic acid bacterium able to degrade tannins by the subsequent action of tannase and gallate decarboxylase enzymes. The gene encoding tannase had previously been identified, whereas the gene encoding gallate decarboxylase is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of gallic-acid induced L. plantarum extracts showed a 54-kDa protein which was absent in the uninduced cells. This protein was identified as Lp_2945, putatively annotated UbiD. Homology searches identified ubiD-like genes located within three-gene operons which encoded the three subunits of nonoxidative aromatic acid decarboxylases. L. plantarum is the only bacterium in which the lpdC (lp_2945) gene and the lpdB and lpdD (lp_0271 and lp_0272) genes are separated in the chromosome. Combination of extracts from recombinant Escherichia coli cells expressing the lpdB, lpdC, and lpdC genes demonstrated that LpdC is the only protein required to yield gallate decarboxylase activity. However, the disruption of these genes in L. plantarum revealed that the lpdB and lpdC gene products are essential for gallate decarboxylase activity. Similar to L. plantarum tannase, which exhibited activity only in esters derived from gallic and protocatechuic acids, purified His6-LpdC protein from E. coli showed decarboxylase activity against gallic and protocatechuic acids. In contrast to the tannase activity, gallate decarboxylase activity is widely present among lactic acid bacteria. This study constitutes the first genetic characterization of a gallate decarboxylase enzyme and provides new insights into the role of the different subunits of bacterial nonoxidative aromatic acid decarboxylases. PMID:23645198

  4. [Inhibitory effect of essential oils, food additives, peracetic acid and detergents on bacterial histidine decarboxylase].

    PubMed

    Kamii, Eri; Terada, Gaku; Akiyama, Jyunki; Isshiki, Kenji

    2011-01-01

    The aim of this study is to examine whether various essential oils, food additives, peracetic acid and detergents inhibit bacterial histidine decarboxylase. Crude extract of Morganella morganii NBRC3848 was prepared and incubated with various agents. Histidine decarboxylase activity was significantly inhibited (p<0.05) by 26 of 45 compounds tested. Among the 26 agents, sodium hypochlorite completely decomposed both histidine and histamine, while peracetic acid caused slight decomposition. Histidine and histamine were stable in the presence of the other 24 agents. These results indicated that 25 of the agents examined were inhibitors of histidine decarboxylase.

  5. Glutamic acid decarboxylase autoimmunity in Batten disease and other disorders.

    PubMed

    Pearce, David A; Atkinson, Mark; Tagle, Danilo A

    2004-12-14

    Degenerative diseases of the CNS, such as stiff-person syndrome (SPS), progressive cerebellar ataxia, and Rasmussen encephalitis, have been characterized by the presence of autoantibodies. Recent findings in individuals with Batten disease and in animal models for the disorder indicate that this condition may be associated with autoantibodies against glutamic acid decarboxylase (GAD), an enzyme that converts the excitatory neurotransmitter glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Anti-GAD autoantibodies could result in excess excitatory neurotransmitters, leading to the seizures and other symptoms observed in patients with Batten disease. The pathogenic potential of GAD autoantibodies is examined in light of what is known for other autoimmune disorders, such as multiple sclerosis, SPS, Rasmussen encephalitis, and type 1 diabetes, and may have radical implications for diagnosis and management of Batten disease.

  6. Levodopa combined with peripheral decarboxylase inhibition in Parkinson's disease

    PubMed Central

    Barbeau, André; Mars, Harold; Botez, Mihai I.; Joubert, Marie

    1972-01-01

    The authors report their experience, over a 26-month period, in the management of 60 parkinsonian patients with the combination of levodopa and an inhibitor of peripheral dopa-decarboxylase, Ro 4-4602. This approach to Parkinson's disease is useful, safe, and at least as effective as levodopa alone. To date there have been no recognizable toxic effects attributable to Ro 4-4602. This agent appears to prolong the duration of action of levodopa, smoothing out its therapeutic effects. The percentage of patients obtaining a very good and excellent response is slightly increased. There is a possible diminution in the late-occurring bradykinetic and hypotonic freezing episodes. Nausea and cardiac arrhythmias are lessened, as are the incidence and severity of hypotension. Abnormal involuntary movements remain the limiting adverse side effect. PMID:5034697

  7. An endosymbiont positively modulates ornithine decarboxylase in host trypanosomatids

    SciTech Connect

    Frossard, Mariana Lins; Seabra, Sergio Henrique; Matta, Renato Augusto da; Souza, Wanderley de; Garcia de Mello, Fernando; Motta, Maria Cristina Machado . E-mail: motta@biof.ufrj.br

    2006-05-05

    Summary: Some trypanosomatids, such as Crithidia deanei, are endosymbiont-containing species. Aposymbiotic strains are obtained after antibiotic treatment, revealing interesting aspects of this symbiotic association. Ornithine decarboxylase (ODC) promotes polyamine biosynthesis and contributes to cell proliferation. Here, we show that ODC activity is higher in endosymbiont-bearing trypanosomatids than in aposymbiotic cells, but isolated endosymbionts did not display this enzyme activity. Intriguingly, expressed levels of ODC were similar in both strains, suggesting that ODC is positively modulated in endosymbiont-bearing cells. When the aposymbiotic strain was grown in conditioned medium, obtained after cultivation of the endosymbiont-bearing strain, cellular proliferation as well as ODC activity and localization were similar to that observed in the endosymbiont-containing trypanosomatids. Furthermore, dialyzed-heated medium and trypsin treatment reduced ODC activity of the aposymbiont strain. Taken together, these data indicate that the endosymbiont can enhance the protozoan ODC activity by providing factors of protein nature, which increase the host polyamine metabolism.

  8. Altered subcellular localization of ornithine decarboxylase in Alzheimer's disease brain

    SciTech Connect

    Nilsson, Tatjana . E-mail: Tatjana.Nilsson@ki.se; Bogdanovic, Nenad; Volkman, Inga; Winblad, Bengt; Folkesson, Ronnie; Benedikz, Eirikur

    2006-06-02

    The amyloid precursor protein can through ligand-mimicking induce expression of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine biosynthesis. We report here the regional distribution and cellular localization of ODC immunoreactivity in Alzheimer's disease (AD) brains. In frontal cortex and hippocampus of control cases, the most pronounced ODC immunoreactivity was found in the nucleus. In possible and definite AD the immunoreactivity had shifted to the cytoplasm. In cerebellum of control cases, ODC staining was found in a small portion of Purkinje cells, mostly in the nucleus. In AD, both possible and definite, the number of stained Purkinje cells increased significantly and immunoreactivity was shifted to the cytoplasm, even though it was still prominent in the nucleus. In conclusion, our study reveals an early shift of the ODC immunoreactivity in AD from the nuclear compartment towards the cytoplasm.

  9. Kinetic challenges facing oxalate, malonate, acetoacetate, and oxaloacetate decarboxylases.

    PubMed

    Wolfenden, Richard; Lewis, Charles A; Yuan, Yang

    2011-04-20

    To compare the powers of the corresponding enzymes as catalysts, the rates of uncatalyzed decarboxylation of several aliphatic acids (oxalate, malonate, acetoacetate, and oxaloacetate) were determined at elevated temperatures and extrapolated to 25 °C. In the extreme case of oxalate, the rate of the uncatalyzed reaction at pH 4.2 was 1.1 × 10(-12) s(-1), implying a 2.5 × 10(13)-fold rate enhancement by oxalate decarboxylase. Whereas the enzymatic decarboxylation of oxalate requires O(2) and Mn(II), the uncatalyzed reaction is unaffected by the presence of these cofactors and appears to proceed by heterolytic elimination of CO(2).

  10. Mouse ornithine decarboxylase gene: cloning, structure, and expression.

    PubMed Central

    Brabant, M; McConlogue, L; van Daalen Wetters, T; Coffino, P

    1988-01-01

    We used molecular cloning to isolate a functional gene for mouse ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) from a cell line in which that gene had been selectively amplified. The position of the 5' terminus of the mRNA was identified, and the coding sequence was shown to be preceded by a 312- or 313-nucleotide (nt) untranslated leader. The latter is highly G + C rich, particularly in its 5'-most portion. The leader can be anticipated to have extensive and stable secondary structure. The transcription unit of the gene is of relatively small size, approximately equal to 6.2 kilobases (kb) from the start site to the proximal site of polyadenylylation. Sequence analysis of DNA near the transcription start position demonstrated the presence of a "TATA" box, but no "CAAT" box. Functional properties of the cloned gene were tested by transfecting it into cultured cells. Expression of the putative full-length gene efficiently conferred ornithine decarboxylase activity on recipient mutant cells deficient in that activity. To assess the function and strength of the OrnDCase promoter region and to delimit its boundaries, we used a transient expression assay. Upstream of a bacterial chloramphenicol acetyltransferase gene was placed a portion of the OrnDCase gene, including the presumed promoter region, spanning a region from approximately equal to 3.0 kb 5' of the site of transcription initiation to the first 250 nt of the transcript. When expressed in mouse NIH 3T3 cells, this OrnDCase genomic element was comparable in strength to the Rous sarcoma virus long terminal repeat promoter. A similar construct, truncated so as to retain only 264 base pairs of the OrnDCase gene 5' to the site of transcription start, yielded undiminished levels of expression. Images PMID:3353375

  11. Coenzyme A biosynthesis: steric course of 4'-phosphopantothenoyl-L-cysteine decarboxylase.

    PubMed

    Aberhart, D J; Ghoshal, P K; Cotting, J A; Russell, D J

    1985-12-03

    4'-Phosphopantothenoyl-L-cysteine decarboxylase (PPC decarboxylase) was partially purified from rat liver. 4'-Phosphopantothenoyl[2-2H1]-L-cysteine was synthesized and converted by PPC decarboxylase to 4'-phosphol[1-2H1]pantetheine. The product was degraded by reduction with Raney nickel followed by acidic hydrolysis to [1-2H1]ethylamine. The latter was converted to the (-)-camphanamide derivative, NMR studies of which revealed that the deuterium was located in the pro-1S position. Also, unlabeled 4'-phosphopantothenoyl-L-cysteine was incubated with PPC decarboxylase in D2O, giving, after degradation, the (-)-camphanamide of (1R)-[1-2H1]ethylamine. The results show that the decarboxylation takes place with retention of configuration. These results are discussed in terms of possible mechanisms for the decarboxylation.

  12. A Liquid-Based Colorimetric Assay of Lysine Decarboxylase and Its Application to Enzymatic Assay.

    PubMed

    Kim, Yong Hyun; Sathiyanarayanan, Ganesan; Kim, Hyun Joong; Bhatia, Shashi Kant; Seo, Hyung-Min; Kim, Jung-Ho; Song, Hun-Seok; Kim, Yun-Gon; Park, Kyungmoon; Yang, Yung-Hun

    2015-12-28

    A liquid-based colorimetric assay using a pH indicator was introduced for high-throughput monitoring of lysine decarboxylase activity. The assay is based on the color change of bromocresol purple, measured at 595 nm in liquid reaction mixture, due to an increase of pH by the production of cadaverine. Bromocresol purple was selected as the indicator because it has higher sensitivity than bromothymol blue and pheonol red within a broad range and shows good linearity within the applied pH. We applied this for simple determination of lysine decarboxylase reusability using 96-well plates, and optimization of conditions for enzyme overexpression with different concentrations of IPTG on lysine decarboxylase. This assay is expected to be applied for monitoring and quantifying the liquid-based enzyme reaction in biotransformation of decarboxylase in a high-throughput way.

  13. Structural Basis of the Substrate Specificity and Enzyme Catalysis of a Papaver somniferum Tyrosine Decarboxylase.

    PubMed

    Guan, Huai; Song, Shuaibao; Robinson, Howard; Liang, Jing; Ding, Haizhen; Li, Jianyong; Han, Qian

    2017-01-01

    Tyrosine decarboxylase (TyDC), a type II pyridoxal 5'-phosphate decarboxylase, catalyzes the decarboxylation of tyrosine. Due to a generally high sequence identity to other aromatic amino acid decarboxylases (AAADs), primary sequence information is not enough to understand substrate specificities with structural information. In this study, we selected a typical TyDC from Papaver somniferum as a model to study the structural basis of AAAD substrate specificities. Analysis of the native P. somniferum TyDC crystal structure and subsequent molecular docking and dynamics simulation provide some structural bases that explain substrate specificity for tyrosine. The result confirmed the previous proposed mechanism for the enzyme selectivity of indolic and phenolic substrates. Additionally, this study yields the first crystal structure for a plant type II pyridoxal-5'-phosphate decarboxylase.

  14. Structural Basis of the Substrate Specificity and Enzyme Catalysis of a Papaver somniferum Tyrosine Decarboxylase

    PubMed Central

    Guan, Huai; Song, Shuaibao; Robinson, Howard; Liang, Jing; Ding, Haizhen; Li, Jianyong; Han, Qian

    2017-01-01

    Tyrosine decarboxylase (TyDC), a type II pyridoxal 5′-phosphate decarboxylase, catalyzes the decarboxylation of tyrosine. Due to a generally high sequence identity to other aromatic amino acid decarboxylases (AAADs), primary sequence information is not enough to understand substrate specificities with structural information. In this study, we selected a typical TyDC from Papaver somniferum as a model to study the structural basis of AAAD substrate specificities. Analysis of the native P. somniferum TyDC crystal structure and subsequent molecular docking and dynamics simulation provide some structural bases that explain substrate specificity for tyrosine. The result confirmed the previous proposed mechanism for the enzyme selectivity of indolic and phenolic substrates. Additionally, this study yields the first crystal structure for a plant type II pyridoxal-5'-phosphate decarboxylase. PMID:28232911

  15. Glutamic acid decarboxylase isoform distribution in transgenic mouse septum: an anti-GFP immunofluorescence study.

    PubMed

    Verimli, Ural; Sehirli, Umit S

    2016-09-01

    The septum is a basal forebrain region located between the lateral ventricles in rodents. It consists of lateral and medial divisions. Medial septal projections regulate hippocampal theta rhythm whereas lateral septal projections are involved in processes such as affective functions, memory formation, and behavioral responses. Gamma-aminobutyric acidergic neurons of the septal region possess the 65 and 67 isoforms of the enzyme glutamic acid decarboxylase. Although data on the glutamic acid decarboxylase isoform distribution in the septal region generally appears to indicate glutamic acid decarboxylase 67 dominance, different studies have given inconsistent results in this regard. The aim of this study was therefore to obtain information on the distributions of both of these glutamic acid decarboxylase isoforms in the septal region in transgenic mice. Two animal groups of glutamic acid decarboxylase-green fluorescent protein knock-in transgenic mice were utilized in the experiment. Brain sections from the region were taken for anti-green fluorescent protein immunohistochemistry in order to obtain estimated quantitative data on the number of gamma-aminobutyric acidergic neurons. Following the immunohistochemical procedures, the mean numbers of labeled cells in the lateral and medial septal nuclei were obtained for the two isoform groups. Statistical analysis yielded significant results which indicated that the 65 isoform of glutamic acid decarboxylase predominates in both lateral and medial septal nuclei (unpaired two-tailed t-test p < 0.0001 for LS, p < 0.01 for MS). This study is the first to reveal the dominance of glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice.

  16. Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes.

    PubMed

    Bassez, T; Paris, J; Omilli, F; Dorel, C; Osborne, H B

    1990-11-01

    The level at which ornithine decarboxylase expression is regulated in growing oocytes has been investigated. Immunoprecipitation of the in vivo labelled proteins showed that ornithine decarboxylase accumulated less rapidly in stage IV oocytes than in previtellogenic stage I + II oocytes. Quantitative Northern analysis showed that ornithine decarboxylase mRNA is abundant in oocytes (about 8 x 10(8) transcripts/cell) and this number does not significantly change during oogenesis. Polysome analysis showed that this mRNA is present in polysomes in stage I + II oocytes but has passed into puromycin-insensitive mRNP particles by stage IV of oogenesis. Therefore, during the growth phase of oogenesis, ornithine decarboxylase expression is regulated at a translational level. These results are discussed relative to the temporal expression of ornithine decarboxylase and of other proteins whose expression also decreases during oogenesis. In order to perform these experiments, the cDNA (XLODC1) corresponding to Xenopus laevis ornithine decarboxylase mRNA was cloned and sequenced.

  17. Polyamine homoeostasis.

    PubMed

    Persson, Lo

    2009-11-04

    The polyamines are essential for a variety of functions in the mammalian cell. Although their specific effects have not been fully elucidated, it is clear that the cellular polyamines have to be kept within certain levels for normal cell function. Polyamine homoeostasis in mammalian cells is achieved by a complex network of regulatory mechanisms affecting synthesis and degradation, as well as membrane transport of polyamines. The two key enzymes in the polyamine biosynthetic pathway, ODC (ornithine decarboxylase) and AdoMetDC (S-adenosylmethionine decarboxylase), are strongly regulated by feedback mechanisms at several levels, including transcriptional, translational and post-translational. Some of these mechanisms have been shown to be truly unique and include upstream reading frames and ribosomal frameshifting, as well as ubiquitin-independent proteasomal degradation. SSAT (spermidine/spermine N1-acetyltransferase), which is a crucial enzyme for degradation and efflux of polyamines, is also highly regulated by polyamines. A cellular excess of polyamines rapidly induces SSAT, resulting in increased degradation/efflux of the polyamines. The polyamines appear to induce both transcription and translation of the SSAT mRNA. However, the major part of the polyamine-induced increase in SSAT is caused by a marked stabilization of the enzyme against degradation by the 26S proteasome. In addition, active transport of extracellular polyamines into the cell contributes to cellular polyamine homoeostasis. Depletion of cellular polyamines rapidly induces an increased uptake of exogenous polyamines, whereas an excess of polyamines down-regulates the polyamine transporter(s). However, the protein(s) involved in polyamine transport and the exact mechanisms by which the polyamines regulate the transporter(s) are not yet known.

  18. Targeting polyamine metabolism for finding new drugs against leishmaniasis: a review.

    PubMed

    Ilari, Andrea; Fiorillo, Annarita; Baiocco, Paola; Poser, Elena; Angiulli, Gabriella; Colotti, Gianni

    2015-01-01

    Leishmaniasis is a neglected disease affecting more than 12 million people worldwide. The most used drugs are pentavalent antimonials that are very toxic and display the problem of drug resistance, especially in endemic regions such as Bihar in India. For this reason, it is urgent to find new and less toxic drugs against leishmaniasis. To this end, the understanding of pathways affecting parasite survival is of prime importance for targeted drug discovery. The parasite survival inside the macrophage is strongly dependent on polyamine metabolism. Polyamines are, in fact, very important for cell growth and proliferation. In particular, spermidine (Spd), the final product of the polyamine biosynthesis pathway, serves as a precursor for trypanothione (N1,N8- bis(glutathionyl)spermidine, T(SH)2) and hypusine (N(ε)-(4-amino-2-hydroxybutyl)lysine). T(SH)2 is a key molecule for parasite defense against the hydrogen peroxide produced by macrophages during the infection. Hypusination is a posttranslational modification occurring exclusively in the eukaryotic initiation factor 5A (eIF5A), which has an important role in avoiding the ribosome stalling during the biosynthesis of protein containing polyprolines sequences. The enzymes, belonging to the spermidine metabolism, i.e. arginase (ARG), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetDC), spermidine synthase (SpdS), trypanothione synthetase (TryS or TSA), trypanothione reductase (TryR or TR), tryparedoxin peroxidase (TXNPx), deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) are promising targets for the development of new drugs against leishmaniasis. This minireview furnishes a picture of the structural, functional and inhibition studies on polyamine metabolism enzymes that could guide the discovery of new drugs against leishmaniasis.

  19. Anti-glutamic acid decarboxylase antibody positive neurological syndromes

    PubMed Central

    Tohid, Hassaan

    2016-01-01

    A rare kind of antibody, known as anti-glutamic acid decarboxylase (GAD) autoantibody, is found in some patients. The antibody works against the GAD enzyme, which is essential in the formation of gamma aminobutyric acid (GABA), an inhibitory neurotransmitter found in the brain. Patients found with this antibody present with motor and cognitive problems due to low levels or lack of GABA, because in the absence or low levels of GABA patients exhibit motor and cognitive symptoms. The anti-GAD antibody is found in some neurological syndromes, including stiff-person syndrome, paraneoplastic stiff-person syndrome, Miller Fisher syndrome (MFS), limbic encephalopathy, cerebellar ataxia, eye movement disorders, and epilepsy. Previously, excluding MFS, these conditions were called ‘hyperexcitability disorders’. However, collectively, these syndromes should be known as “anti-GAD positive neurological syndromes.” An important limitation of this study is that the literature is lacking on the subject, and why patients with the above mentioned neurological problems present with different symptoms has not been studied in detail. Therefore, it is recommended that more research is conducted on this subject to obtain a better and deeper understanding of these anti-GAD antibody induced neurological syndromes. PMID:27356651

  20. Multiple roles of the active site lysine of Dopa decarboxylase.

    PubMed

    Bertoldi, Mariarita; Voltattorni, Carla Borri

    2009-08-15

    The pyridoxal 5'-phosphate dependent-enzyme Dopa decarboxylase, responsible for the irreversible conversion of l-Dopa to dopamine, is an attractive drug target. The contribution of the pyridoxal-Lys303 to the catalytic mechanisms of decarboxylation and oxidative deamination is analyzed. The K303A variant binds the coenzyme with a 100-fold decreased apparent equilibrium binding affinity with respect to the wild-type enzyme. Unlike the wild-type, K303A in the presence of l-Dopa displays a parallel progress course of formation of both dopamine and 3,4-dihydroxyphenylacetaldehyde (plus ammonia) with a burst followed by a linear phase. Moreover, the finding that the catalytic efficiencies of decarboxylation and of oxidative deamination display a decrease of 1500- and 17-fold, respectively, with respect to the wild-type, is indicative of a different impact of Lys303 mutation on these reactions. Kinetic analyses reveal that Lys303 is involved in external aldimine formation and hydrolysis as well as in product release which affects the rate-determining step of decarboxylation.

  1. Studies on uroporphyrinogen decarboxylase from Chlorella kessleri (Trebouxiophyceae, Chlorophyta).

    PubMed

    Juárez, Angela B; Aldonatti, Carmen; Vigna, María S; Ríos de Molina, María Del C

    2007-02-01

    Uroporphyrinogen decarboxylase (UroD) (EC 4.1.1.37) is an enzyme from the tetrapyrrole biosynthetic pathway, in which chlorophyll is the main final product in algae. This is the first time that a study on UroD activity has been performed in a green alga (Chlorella). We isolated and partially purified the enzyme from a Chlorella kessleri (Trebouxiophyceae, Chlorophyta) strain (Copahue, Neuquén, Argentina), and describe for the first time some of its properties. In C. kessleri, the decarboxylation of uroporphyrinogen III occurs in two stages, via 7 COOH and then 6 and 5 COOH intermediates, with the decarboxylation of the 7 COOH compound being the rate-limiting step for the reaction. Cultures in the exponential growth phase showed the highest specific activity values. The most suitable conditions to measure UroD activity in C. kessleri were as follows: 0.23-0.3 mg protein/mL, approximately 6-8 micromol/L uroporphyrinogen III, and 20 min incubation time. Gel filtration chromatography and Western blot assays indicated that UroD from C. kessleri is a dimer of approximately 90 kDa formed by species of lower molecular mass, which conserves enzymatic activity.

  2. Chemical modification of oxalate decarboxylase to improve adsorption capacity.

    PubMed

    Lin, Rihui; He, Junbin; Wu, Jia; Cai, Xinghua; Long, Han; Chen, Shengfeng; Liu, Haiqian

    2017-02-03

    In order to enhance the adsorption capacity of oxalate decarboxylase (Oxdc) on calcium oxalate monohydrate crystals and improve the application performance of Oxdc, chemical modification of Oxdc with ethylenediaminetetraacetic dianhydride (EDTAD) was investigated in this work. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography tandem mass spectrometry (LC/MS) analysis results demonstrated that Oxdc and EDTAD have been covalently bound, and suggested that the chemical modification occurred at the free amino of the side chain and the α-amine of the N-terminus of Oxdc. Fluorescene and circular dichroic measurement showed that the structure and conformation of Oxdc were tinily altered after modification by EDTAD. The optimum pH of EDTAD-modified Oxdc was shifted to the alkaline side about 1.5 unit and it has a higher thermostability. The analysis of kinetic parameters indicated that the EDTAD-modified Oxdc showed a higher affinity towards the substrate. Through modification the adsorption capacity of Oxdc onto CaOx monohydrate crystals was increased by 42.42%.

  3. Glutamate decarboxylase from Lactobacillus brevis: activation by ammonium sulfate.

    PubMed

    Hiraga, Kazumi; Ueno, Yoshie; Oda, Kohei

    2008-05-01

    In this study, the glutamate decarboxylase (GAD) gene from Lactobacillus brevis IFO12005 (Biosci. Biotechnol. Biochem., 61, 1168-1171 (1997)), was cloned and expressed. The deduced amino acid sequence showed 99.6% and 53.1% identity with GAD of L. brevis ATCC367 and L. lactis respectively. The His-tagged recombinant GAD showed an optimum pH of 4.5-5.0, and 54 kDa on SDS-PAGE. The GAD activity and stability was significantly dependent on the ammonium sulfate concentration, as observed in authentic GAD. Gel filtration showed that the inactive form of the GAD was a dimer. In contrast, the ammonium sulfate-activated form was a tetramer. CD spectral analyses at pH 5.5 revealed that the structures of the tetramer and the dimer were similar. Treatment of the GAD with high concentrations of ammonium sulfate and subsequent dilution with sodium glutamate was essential for tetramer formation and its activation. Thus the biochemical properties of the GAD from L. brevis IFO12005 were significantly different from those from other sources.

  4. Molecular cloning and expression of the mouse ornithine decarboxylase gene.

    PubMed Central

    McConlogue, L; Gupta, M; Wu, L; Coffino, P

    1984-01-01

    We used mRNA from a mutant S49 mouse lymphoma cell line that produces ornithine decarboxylase (OrnDCase) as its major protein product to synthesize and clone cDNA. Plasmids containing OrnDCase cDNA were identified by hybrid selection of OrnDCase mRNA and in vitro translation. The two of these with the largest inserts together span 2.05 kilobases of cDNA. Southern blot analysis of DNA from wild-type or mutant S49 cells, cleaved with EcoRI or with BamHI, revealed multiple bands homologous to OrnD-Case cDNA, only one of which was amplified in the mutant cells. RNA transfer blot analysis showed that the major OrnD-Case mRNA in the mouse lymphoma cells is 2.0 kilobases long. A similar size mRNA was found in mouse kidney and was more abundant in the kidneys of mice treated with testosterone, an inducer of OrnDCase activity in that tissue. Images PMID:6582509

  5. Expression of ornithine decarboxylase in precancerous and cancerous gastric lesions

    PubMed Central

    Miao, Xin-Pu; Li, Jian-Sheng; Li, Hui-Yan; Zeng, Shi-Ping; Zhao, Ye; Zeng, Jiang-Zheng

    2007-01-01

    AIM: To investigate the expression of ornithine decarboxylase (ODC) in precancerous and cancerous gastric lesions. METHODS: We studied the expression of ODC in gastric mucosa from patients with chronic superficial gastritis (CSG, n = 32), chronic atrophic gastritis [CAG, n = 43; 15 with and 28 without intestinal metaplasia (IM)], gastric dysplasia (DYS, n = 11) and gastric cancer (GC, n = 48) tissues using immunohistochemical staining. All 134 biopsy specimens of gastric mucosa were collected by gastroscopy. METHODS: The positive rate of ODC expression was 34.4%, 42.9%, 73.3%, 81.8% and 91.7% in cases with CSG, CAG without IM, CAG with IM, DYS and GC, respectively (P < 0.01), The positive rate of ODC expression increased in the order of CSG < CAG (without IM) < CAG (with IM) < DYS and finally, GC. In addition, ODC positive immunostaining rate was lower in well-differentiated GC than in poorly-differentiated GC (P < 0.05). CONCLUSION: The expression of ODC is positively correlated with the degree of malignity of gastric mucosa and development of gastric lesions. This finding indicates that ODC may be used as a good biomarker in the screening and diagnosis of precancerous lesions. PMID:17569126

  6. Ornithine decarboxylase as a marker for premalignancy in the stomach.

    PubMed Central

    Patchett, S E; Alstead, E M; Butruk, L; Przytulski, K; Farthing, M J

    1995-01-01

    Assessment of mucosal ornithine decarboxylase (ODC) activity in the human large bowel may be of value as a marker of potential malignant risk. Its value as a marker of premalignancy in the upper gastrointestinal tract is less clear. Using a [14C]-ornithine bioassay, gastric mucosal ODC activity was measured in 32 normal subjects and 22 patients with confirmed gastric cancer. These results were compared with 47 patients at increased risk of upper gastrointestinal malignancy, (32 patients with partial gastric resection, 15 patients with familial adenomatous polyposis). Median ODC activity in normal subjects was 371 pmol/mg protein/h, (interquartile range (IQR), 230-617). There was no variation with age or sex and no relation to Helicobacter pylori status. Normal subjects had significantly lower ODC activity than patients with a gastric resection or confirmed gastric cancer, but similar to patients with familial adenomatous polyposis. Furthermore, no difference in activity was identified between patients with a gastric resection and established gastric cancer. ODC activity was, however, significantly increased in areas of gastric atrophy or intestinal metaplasia, regardless of the clinical group from which the samples were obtained. It is concluded that measurement of mucosal ODC activity does not provide additional predictive information of malignant risk in the stomach and investigation of other potential biomarkers of malignancy is warranted. PMID:7672662

  7. Histidine Decarboxylase Deficiency Prevents Autoimmune Diabetes in NOD Mice

    PubMed Central

    Alkan, Manal; Machavoine, François; Rignault, Rachel; Dam, Julie; Dy, Michel; Thieblemont, Nathalie

    2015-01-01

    Recent evidence has highlighted the role of histamine in inflammation. Since this monoamine has also been strongly implicated in the pathogenesis of type-1 diabetes, we assessed its effect in the nonobese diabetic (NOD) mouse model. To this end, we used mice (inactivated) knocked out for the gene encoding histidine decarboxylase, the unique histamine-forming enzyme, backcrossed on a NOD genetic background. We found that the lack of endogenous histamine in NOD HDC−/− mice decreased the incidence of diabetes in relation to their wild-type counterpart. Whereas the proportion of regulatory T and myeloid-derived suppressive cells was similar in both strains, histamine deficiency was associated with increased levels of immature macrophages, as compared with wild-type NOD mice. Concerning the cytokine pattern, we found a decrease in circulating IL-12 and IFN-γ in HDC−/− mice, while IL-6 or leptin remained unchanged, suggesting that histamine primarily modulates the inflammatory environment. Paradoxically, exogenous histamine given to NOD HDC−/− mice provided also protection against T1D. Our study supports the notion that histamine is involved in the pathogenesis of diabetes, thus providing additional evidence for its role in the regulation of the immune response. PMID:26090474

  8. Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

    SciTech Connect

    Kanerva, Kristiina; Maekitie, Laura T.; Baeck, Nils; Andersson, Leif C.

    2010-07-01

    Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.

  9. Interaction of NAP-22 with brain glutamic acid decarboxylase (GAD).

    PubMed

    Maekawa, Shohei; Kobayashi, Yuumi; Odagaki, Sin-Ichi; Makino, Midori; Kumanogoh, Haruko; Nakamura, Shun; Morita, Mitsuhiro; Hayashi, Fumio

    2013-03-14

    NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched protein localized mainly in the synaptic vesicles and the synaptic plasma membrane. Biochemically, it is recovered in the lipid raft fraction. In order to understand the physiological function of the neuronal lipid raft, NAP-22 binding proteins were screened with a pull-down assay. Glutamic acid decarboxylase (GAD) was detected through LC-MS/MS, and Western blotting using a specific antibody confirmed the result. Two isoforms of GAD, GAD65 and GAD67, were expressed in bacteria as GST-fusion forms and the interaction with NAP-22 was confirmed in vitro. Partial co-localization of NAP-22 with GAD65 and GAD67 was also observed in cultured neurons. The binding showed no effect on the enzymatic activity of GAD65 and GAD67. These results hence suggest that NAP-22 could participate in the transport of GAD65 and GAD67 to the presynaptic termini and their retention on the synaptic vesicles as an anchoring protein.

  10. Reactivation of substrate-inactivated brain glutamate decarboxylase.

    PubMed

    Meeley, M P; Martin, D L

    1983-03-01

    The effects of ATP and inorganic phosphate (Pi) on the reactivation of glutamate apodecarboxylase by its cofactor pyridoxal-5'-phosphate (pyridoxal-P) was studied. Apoenzyme was prepared by preincubation with glutamate. Apoenzyme prepared with glutamate alone was reactivated slowly and incompletely by adding a saturating concentration of pyridoxal-P (20 microM). Reactivation was slightly enhanced by 1-10 mM Pi. Reactivation by pyridoxal-P plus Pi was greatly enhanced by the presence of low concentrations (less than 100 microM) of ATP during the preparation of apoenzyme with glutamate. Reactivation was much lower if Pi was omitted. Enhancement of reactivation by ATP was due to its effect during apoenzyme formation, since ATP did not enhance reactivation if added only during reactivation and since the enhancing effect persisted after the removal of free ATP by chromatography on Sephadex G-25 after apoenzyme preparation and before reactivation. Reactivation was inhibited by high concentrations of ATP (greater than 100 microM), possibly by competition of ATP for the cofactor binding site. Four factors (glutamate, pyridoxal-P, ATP, and Pi) control a cycle of inactivation and reactivation that appears to be important in the regulation of brain glutamate decarboxylase.

  11. Expression of arginine decarboxylase in brain regions and neuronal cells

    PubMed Central

    Iyo, Abiye H.; Zhu, Meng-Yang; Ordway, Gregory A.; Regunathan, Soundar

    2010-01-01

    After our initial report of a mammalian gene for arginine decarboxylase, an enzyme for the synthesis of agmatine from arginine, we have determined the regional expression of ADC in rat. We have analyzed the expression of ADC in rat brain regions by activity, protein and mRNA levels, and the regulation of expression in neuronal cells by RNA interference. In rat brain, ADC was widely expressed in major brain regions, with a substantial amount in hypothalamus, followed by cortex, and with least amounts in locus coeruleus and medulla. ADC mRNA was detected in primary astrocytes and C6 glioma cells. While no ADC message was detected in fresh neurons (3 days old), significant message appeared in differentiated neurons (3 weeks old). PC12 cells, treated with nerve growth factor, had higher ADC mRNA compared with naive cells. The siRNA mixture directed towards the N-terminal regions of ADC cDNA down-regulated the levels of mRNA and protein in cultured neurons/C6 glioma cells and these cells produced lower agmatine. Thus, this study demonstrates that ADC message is expressed in rat brain regions, that it is regulated in neuronal cells and that the down-regulation of ADC activity by specific siRNA leads to lower agmatine production. PMID:16445852

  12. Arginase, Arginine Decarboxylase, Ornithine Decarboxylase, and Polyamines in Tomato Ovaries (Changes in Unpollinated Ovaries and Parthenocarpic Fruits Induced by Auxin or Gibberellin).

    PubMed Central

    Alabadi, D.; Aguero, M. S.; Perez-Amador, M. A.; Carbonell, J.

    1996-01-01

    Arginase (EC 3.5.3.1) activity has been found in the ovaries and Young fruits of tomato (Lycopersicon esculentum Mill. cv Rutgers).Changes in arginase, arginine decarboxylase (EC 4.1.1.19), and ornithine decarboxylase activity (EC 4.1.1.17) and levels of free and conjugated putrescine, spermidine, and spermine were determined in unpollinated ovaries and in parthenocarpic fruits during the early stages of development induced by 2,4-dichlorophenoxyacetic acid (2,4-D) or gibberellic acid (GA3). Levels of arginase, free spermine, and conjugates of the three polyamines were constant in unpollinated ovaries and characteristic of a presenescent step. A marked decrease in arginase activity, free spermine, and polyamine conjugates was associated with the initiation of fruit growth due to cell division, and when cell expansion was initiated, the absence of arginase indicated a redirection of nitrogen metabolism to the synthesis of arginine. A transient increase in arginine decarboxylase and ornithine decarboxylase was also observed in 2,4-D-induced fruits. In general, 2,4-D treatments produced faster changes than GA3, and without treatment, unpollinated ovaries developed only slightly and senescence was hardly visible. Sensitivity to 2,4-D and GA3 treatment remained for at least 2 weeks postanthesis. PMID:12226441

  13. Nucleotide sequence and expression of the Enterobacter aerogenes alpha-acetolactate decarboxylase gene in brewer's yeast.

    PubMed Central

    Sone, H; Fujii, T; Kondo, K; Shimizu, F; Tanaka, J; Inoue, T

    1988-01-01

    The nucleotide sequence of a 1.4-kilobase DNA fragment containing the alpha-acetolactate decarboxylase gene of Enterobacter aerogenes was determined. The sequence contains an entire protein-coding region of 780 nucleotides which encodes an alpha-acetolactate decarboxylase of 260 amino acids. The DNA sequence coding for alpha-acetolactate decarboxylase was placed under the control of the alcohol dehydrogenase I promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of autonomous replication in both S. cerevisiae and Escherichia coli. Brewer's yeast cells transformed by this plasmid showed alpha-acetolactate decarboxylase activity and were used in laboratory-scale fermentation experiments. These experiments revealed that the diacetyl concentration in wort fermented by the plasmid-containing yeast strain was significantly lower than that in wort fermented by the parental strain. These results indicated that the alpha-acetolactate decarboxylase activity produced by brewer's yeast cells degraded alpha-acetolactate and that this degradation caused a decrease in diacetyl production. PMID:3278689

  14. Hepatic ornithine decarboxylase induction by potato glycoalkaloids in rats.

    PubMed

    Caldwell, K A; Grosjean, O K; Henika, P R; Friedman, M

    1991-08-01

    The induction of hepatic ornithine decarboxylase (ODC) activity in rat livers by the potato glycoalkaloids alpha-solanine, alpha-chaconine, and their aglycone solanidine, has been studied. Ip administration of alpha-solanine at 7.5, 15 and 30 mg/kg body weight produced markedly elevated enzyme activity at 4 hr after treatment, with a linear dose response. The increase was four-fold at the lowest dose administered to 12-fold at the highest. ODC activity was measured at 1, 2, 3, 4, 5, 6, 8, and 24hr after alpha-solanine was given. A statistically significant increase in enzyme activity was evident at 3 hr after treatment; maximal activity occurred at 5 hr and was approximately 12 times greater than the dimethylsulphoxide (DMSO) control level. Elevated activities persisted for several hours, decreasing to about one-third of the maximal level at 8 hr. The relative effects of alpha-solanine, alpha-chaconine and solanidine on ODC activities were studied at 4 hr using an equimolar dose of 17 mM/kg body weight. ODC activity induced by alpha-chaconine was higher than that induced by alpha-solanine; the latter activity was two-thirds that of the former. The aglycone solanidine did not induce any increase in activity compared with the DMSO control. ODC activity with dexamethasone, a glucocorticoid, at 4 mg/kg body weight, followed a pattern similar to that of alpha-solanine. However, maximal activity occurred slightly earlier at 4 hr after treatment. The results show that the extent of induced ODC activity depends on the structure of the potato alkaloid.

  15. Bacopa monniera recombinant mevalonate diphosphate decarboxylase: Biochemical characterization.

    PubMed

    Abbassi, Shakeel J; Vishwakarma, Rishi K; Patel, Parth; Kumari, Uma; Khan, Bashir M

    2015-08-01

    Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.33) is an important enzyme in the mevalonic acid pathway catalyzing the Mg(2+)-ATP dependant decarboxylation of mevalonate 5-diphosphate (MVAPP) to isopentenyl diphosphate (IPP). Bacopa monniera recombinant MDD (BmMDD) protein was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Km and Vmax for MVAPP were 144 μM and 52 U mg(-1) respectively. The values of turnover (kcat) and kcat/Km for mevalonate 5-diphosphate were determined to be 40s(-1) and 2.77×10(5) M(-1) s(-1) and kcat and kcat/Km values for ATP were found to be 30 s(-1) and 2.20×10(4) M(-1) s(-1), respectively. pH activity profile indicated the involvement of carboxylate ion, lysine and arginine for the activity of enzyme. The apparent activation energy for the BmMDD catalyzed reaction was 12.7 kJ mol(-1). Optimum pH and temperature for the forward reaction was found to be 8.0 and 45 °C. The enzyme was most stable at pH 7 at 20 °C with the deactivation rate constant (Kd(*)) of 1.69×10(-4) and half life (t1/2) of 68 h. The cation studies suggested that BmMDD is a cation dependant enzyme and optimum activity was achieved in the presence of Mg(2+).

  16. Purification and properties of diaminopimelate decarboxylase from Escherichia coli

    PubMed Central

    White, P. J.; Kelly, Bridget

    1965-01-01

    1. Diaminopimelate decarboxylase from a soluble extract of Escherichia coli A.T.C.C. 9637 was purified 200-fold by precipitation of nucleic acids, fractionation with acetone and then with ammonium sulphate, adsorption on calcium phosphate gel and chromatography on DEAE-cellulose or DEAE-Sephadex. 2. The purified enzyme showed only one component in the ultracentrifuge, with a sedimentation coefficient of 5·4s. One major peak and three much smaller peaks were observed on electrophoresis of the enzyme at pH8·9. 3. The mol.wt. of the enzyme was approx. 200000. The catalytic constant was 2000mol. of meso-diaminopimelic acid decomposed/min./mol. of enzyme, at 37°. The relative rates of decarboxylation at 25°, 37° and 45° were 0·17:1·0:1·6. At 37° the Michaelis constant was 1·7mm and the optimum pH was 6·7–6·8. 4. There was an excess of acidic amino acids over basic amino acids in the enzyme, which was bound only on basic cellulose derivatives at pH6·8. 5. The enzyme had an absolute requirement for pyridoxal phosphate as a cofactor; no other derivative of pyridoxine had activity. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was also needed as an activator. 6. In the presence of 2,3-dimercaptopropan-1-ol (1mm), heavy-metal ions (Cu2+, Hg2+) did not inhibit the enzyme, but there was inhibition by several amino acids with analogous structures to diaminopimelate, generally at high concentrations relative to the substrate. Penicillamine was inhibitory at relatively low concentrations; its action was prevented by pyridoxal phosphate. PMID:14343156

  17. Cloning and nucleotide sequence of wild type and a mutant histidine decarboxylase from Lactobacillus 30a.

    PubMed

    Vanderslice, P; Copeland, W C; Robertus, J D

    1986-11-15

    Prohistidine decarboxylase from Lactobacillus 30a is a protein that autoactivates to histidine decarboxylase by cleaving its peptide chain between serines 81 and 82 and converting Ser-82 to a pyruvoyl moiety. The pyruvoyl group serves as the prosthetic group for the decarboxylation reaction. We have cloned and determined the nucleotide sequence of the gene for this enzyme from a wild type strain and from a mutant with altered autoactivation properties. The nucleotide sequence modifies the previously determined amino acid sequence of the protein. A tripeptide missed in the chemical sequence is inserted, and three other amino acids show conservative changes. The activation mutant shows a single change of Gly-58 to an Asp. Sequence analysis up- and downstream from the gene suggests that histidine decarboxylase is part of a polycistronic message, and that the transcriptional promotor region is strongly homologous to those of other Gram-positive organisms.

  18. The effect of a high fat diet on pyruvate decarboxylase deficiency without central nervous system involvement.

    PubMed

    Kodama, S; Yagi, R; Ninomiya, M; Goji, K; Takahashi, T; Morishita, Y; Matsuo, T

    1983-01-01

    A nine-year-old Japanese boy with low pyruvate decarboxylase activity in fibroblasts showed no central nervous symptoms except for muscle fatigue. The pyruvate decarboxylase activities in fibroblasts of the patient and two control subjects were 0.407 +/- 0.083, 1.029 +/- 0.137 and 1.607 +/- 0.096 mumoles/g protein/30 min, respectively. The Michaelis-Menten constant (Km) was the same in the patient and controls. There was no inhibitor of pyruvate decarboxylase in the patient's fibroblasts. A high fat diet has been given to the patient for five years. At present he does not complain of any kind of muscle fatigue, except after severe exercise. Mental and physiological development of the patient are within the normal ranges. However, trials of orally administered thiamine hydrochloride or thiamine hydrochloride combined with lipoamide did not improve his muscle fatigue.

  19. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a...

  20. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme... FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus subtilis. The food additive...

  1. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a...

  2. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a...

  3. Ornithine Decarboxylase Activity Is Required for Prostatic Budding in the Developing Mouse Prostate

    PubMed Central

    Gamat, Melissa; Malinowski, Rita L.; Parkhurst, Linnea J.; Steinke, Laura M.; Marker, Paul C.

    2015-01-01

    The prostate is a male accessory sex gland that produces secretions in seminal fluid to facilitate fertilization. Prostate secretory function is dependent on androgens, although the mechanism by which androgens exert their effects is still unclear. Polyamines are small cationic molecules that play pivotal roles in DNA transcription, translation and gene regulation. The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase, which is encoded by the gene Odc1. Ornithine decarboxylase mRNA decreases in the prostate upon castration and increases upon administration of androgens. Furthermore, testosterone administered to castrated male mice restores prostate secretory activity, whereas administering testosterone and the ornithine decarboxylase inhibitor D,L-α-difluromethylornithine (DFMO) to castrated males does not restore prostate secretory activity, suggesting that polyamines are required for androgens to exert their effects. To date, no one has examined polyamines in prostate development, which is also androgen dependent. In this study, we showed that ornithine decarboxylase protein was expressed in the epithelium of the ventral, dorsolateral and anterior lobes of the adult mouse prostate. Ornithine decarboxylase protein was also expressed in the urogenital sinus (UGS) epithelium of the male and female embryo prior to prostate development, and expression continued in prostatic epithelial buds as they emerged from the UGS. Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens. DFMO also significantly decreased the expression of developmental regulatory gene Notch1. Other genes implicated in prostatic development including Sox9, Wif1 and Srd5a2 were unaffected by DFMO. Together these results indicate that Odc1 and polyamines are required for androgens to exert their effect in mediating

  4. Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis1[OPEN

    PubMed Central

    Bunsupa, Somnuk; Hanada, Kousuke; Maruyama, Akira; Aoyagi, Kaori; Komatsu, Kana; Ueno, Hideki; Yamashita, Madoka; Sasaki, Ryosuke; Oikawa, Akira; Yamazaki, Mami

    2016-01-01

    Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs are potentially useful against Alzheimer’s disease, schizophrenia, and myasthenia gravis. Here, we cloned the bifunctional lysine/ornithine decarboxylase (L/ODC), the first gene involved in LA biosynthesis, from the LA-producing plants Lycopodium clavatum and Huperzia serrata. We describe the in vitro and in vivo functional characterization of the L. clavatum L/ODC (LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of l-Lys over l-ornithine (l-Orn) by about 5 times. Transient expression of LcL/ODC fused with the amino or carboxyl terminus of green fluorescent protein, in onion (Allium cepa) epidermal cells and Nicotiana benthamiana leaves, showed LcL/ODC localization in the cytosol. Transgenic tobacco (Nicotiana tabacum) hairy roots and Arabidopsis (Arabidopsis thaliana) plants expressing LcL/ODC enhanced the production of a Lys-derived alkaloid, anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In addition, we present an example of the convergent evolution of plant Lys decarboxylase that resulted in the production of Lys-derived alkaloids in Leguminosae (legumes) and Lycopodiaceae (clubmosses). This convergent evolution event probably occurred via the promiscuous functions of the ancestral Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The positive selection sites were detected by statistical analyses using phylogenetic trees and were confirmed by site-directed mutagenesis, suggesting the importance of those sites in granting the promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. This study contributes to a better understanding of LA biosynthesis and the molecular evolution of plant Lys decarboxylase. PMID:27303024

  5. Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor

    DOEpatents

    Maupin-Furlow, Julie A [Gainesville, FL; Talarico, Lee Ann [Gainesville, FL; Raj, Krishnan Chandra [Tamil Nadu, IN; Ingram, Lonnie O [Gainesville, FL

    2008-02-05

    The invention provides isolated nucleic acids molecules which encode pyruvate decarboxylase enzymes having improved decarboxylase activity, substrate affinity, thermostability, and activity at different pH. The nucleic acids of the invention also have a codon usage which allows for high expression in a variety of host cells. Accordingly, the invention provides recombinant expression vectors containing such nucleic acid molecules, recombinant host cells comprising the expression vectors, host cells further comprising other ethanologenic enzymes, and methods for producing useful substances, e.g., acetaldehyde and ethanol, using such host cells.

  6. Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity.

    PubMed

    Hwang, In-Wook; Makishima, Yu; Suzuki, Tomohiro; Kato, Tatsuya; Park, Sungjo; Terzic, Andre; Chung, Shin-Kyo; Park, Enoch Y

    2015-11-01

    Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is a vital catalytic reaction of lipid metabolism. While it is established that phosphorylation of MCD modulates the enzymatic activity, the specific phosphorylation sites associated with the catalytic function have not been documented due to lack of sufficient production of MCD with proper post-translational modifications. Here, we used the silkworm-based Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system to express human MCD (hMCD) and mapped phosphorylation effects on enzymatic function. Purified MCD from silkworm displayed post-translational phosphorylation and demonstrated coherent enzymatic activity with high yield (-200 μg/silkworm). Point mutations in putative phosphorylation sites, Ser-204 or Tyr-405 of hMCD, identified by bioinformatics and proteomics analyses reduced the catalytic activity, underscoring the functional significance of phosphorylation in modulating decarboxylase-based catalysis. Identified phosphorylated residues are distinct from the decarboxylation catalytic site, implicating a phosphorylation-induced global conformational change of MCD as responsible in altering catalytic function. We conclude that phosphorylation of Ser-204 and Tyr-405 regulates the decarboxylase function of hMCD leveraging the silkworm-based BmNPV bacmid expression system that offers a fail-safe eukaryotic production platform implementing proper post-translational modification such as phosphorylation.

  7. Structural and Mechanistic Studies on Klebsiella pneumoniae 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline Decarboxylase

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2010-11-12

    The stereospecific oxidative degradation of uric acid to (S)-allantoin was recently shown to proceed via three enzymatic steps. The final conversion is a decarboxylation of the unstable intermediate 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and is catalyzed by OHCU decarboxylase. Here we present the structures of Klebsiella pneumoniae OHCU decarboxylase in unliganded form and with bound allantoin. These structures provide evidence that ligand binding organizes the active site residues for catalysis. Modeling of the substrate and intermediates provides additional support for this hypothesis. In addition we characterize the steady state kinetics of this enzyme and report the first OHCU decarboxylase inhibitor, allopurinol, a structural isomer of hypoxanthine. This molecule is a competitive inhibitor of K. pneumoniae OHCU decarboxylase with a K{sub i} of 30 {+-} 2 {micro}m. Circular dichroism measurements confirm structural observations that this inhibitor disrupts the necessary organization of the active site. Our structural and biochemical studies also provide further insights into the mechanism of catalysis of OHCU decarboxylation.

  8. Detection and transfer of the glutamate decarboxylase gene in Streptococcus thermophilus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    GABA (gamma-aminobutyric acid) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermen...

  9. Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110

    Technology Transfer Automated Retrieval System (TEKTRAN)

    GABA ('-aminobutyric acid) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermented da...

  10. Inhibition of pyruvate decarboxylase from Z. mobilis by novel analogues of thiamine pyrophosphate: investigating pyrophosphate mimics.

    PubMed

    Erixon, Karl M; Dabalos, Chester L; Leeper, Finian J

    2007-03-07

    Replacement of the thiazolium ring of thiamine pyrophosphate with a triazole gives extremely potent inhibitors of pyruvate decarboxylase from Z. mobilis, with K(I) values down to 20 pM; this system was used to explore pyrophosphate mimics and several effective analogues were discovered.

  11. The Degradation of 14C-Glutamic Acid by L-Glutamic Acid Decarboxylase.

    ERIC Educational Resources Information Center

    Dougherty, Charles M; Dayan, Jean

    1982-01-01

    Describes procedures and semi-micro reaction apparatus (carbon dioxide trap) to demonstrate how a particular enzyme (L-Glutamic acid decarboxylase) may be used to determine the site or sites of labeling in its substrate (carbon-14 labeled glutamic acid). Includes calculations, solutions, and reagents used. (Author/SK)

  12. Presentation of opsoclonus myoclonus ataxia syndrome with glutamic acid decarboxylase antibodies.

    PubMed

    Bhandari, Hanul Srinivas

    2012-08-08

    In this rare case, the patient presented with opsoclonus, myoclonus and ataxia. Serological and imaging studies revealed high glutamic acid decarboxylase antibody (GAD-Ab) levels. High-dose corticosteroids were of no benefit and subsequent intravenous immunoglobulin (IVIg) administration proved resolution of the condition. Levetiracetam proved useful in symptomatically controlling the myoclonus. Follow-up GAD-Ab levels were within normal limits.

  13. The enzymatic activities of the Escherichia coli basic aliphatic amino acid decarboxylases exhibit a pH zone of inhibition.

    PubMed

    Kanjee, Usheer; Gutsche, Irina; Ramachandran, Shaliny; Houry, Walid A

    2011-11-01

    The stringent response regulator ppGpp has recently been shown by our group to inhibit the Escherichia coli inducible lysine decarboxylase, LdcI. As a follow-up to this observation, we examined the mechanisms that regulate the activities of the other four E. coli enzymes paralogous to LdcI: the constitutive lysine decarboxylase LdcC, the inducible arginine decarboxylase AdiA, the inducible ornithine decarboxylase SpeF, and the constitutive ornithine decarboxylase SpeC. LdcC and SpeC are involved in cellular polyamine biosynthesis, while LdcI, AdiA, and SpeF are involved in the acid stress response. Multiple mechanisms of regulation were found for these enzymes. In addition to LdcI, LdcC and SpeC were found to be inhibited by ppGpp; AdiA activity was found to be regulated by changes in oligomerization, while SpeF and SpeC activities were regulated by GTP. These findings indicate the presence of multiple mechanisms regulating the activity of this important family of decarboxylases. When the enzyme inhibition profiles are analyzed in parallel, a "zone of inhibition" between pH 6 and pH 8 is observed. Hence, the data suggest that E. coli utilizes multiple mechanisms to ensure that these decarboxylases remain inactive around neutral pH possibly to reduce the consumption of amino acids at this pH.

  14. Substrate specificity of thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases in Saccharomyces cerevisiae.

    PubMed

    Romagnoli, Gabriele; Luttik, Marijke A H; Kötter, Peter; Pronk, Jack T; Daran, Jean-Marc

    2012-11-01

    Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1Δ pdc5Δ pdc6Δ aro10Δ thi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols.

  15. Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae

    PubMed Central

    Romagnoli, Gabriele; Luttik, Marijke A. H.; Kötter, Peter; Pronk, Jack T.

    2012-01-01

    Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1Δ pdc5Δ pdc6Δ aro10Δ thi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols. PMID:22904058

  16. Immunohistochemical evidence for the coexistence of histidine decarboxylase-like and glutamate decarboxylase-like immunoreactivities in nerve cells of the magnocellular nucleus of the posterior hypothalamus of rats.

    PubMed Central

    Takeda, N; Inagaki, S; Shiosaka, S; Taguchi, Y; Oertel, W H; Tohyama, M; Watanabe, T; Wada, H

    1984-01-01

    Immunohistochemical staining of alternate consecutive sections revealed numerous histidine decarboxylase (L-histidine carboxy-lyase, EC 4.1.1.22)-like immunoreactive neurons that also contained glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15)-like immunoreactive structures in the tuberal magnocellular nucleus, the caudal magnocellular nucleus, and the postmammillary caudal magnocellular nucleus of the posterior hypothalamus of rats. Furthermore, in immunohistochemical double-staining procedures, almost all neurons in the magnocellular nuclei had both histidine decarboxylase-like and glutamate decarboxylase-like immunoreactivities. These results suggest the coexistence of histamine and gamma-aminobutyric acid in single neurons in these nuclei. Images PMID:6594708

  17. Radical S-adenosylmethionine enzyme catalyzed thioether bond formation in sactipeptide biosynthesis.

    PubMed

    Flühe, Leif; Marahiel, Mohamed A

    2013-08-01

    Sactipeptides represent a new emerging class of ribosomally assembled and posttranslationally modified peptides that show diverse bioactivities. Their common hallmark is an intramolecular thioether bond that crosslink the sulfur atom of a cysteine residue with the α-carbon of an acceptor amino acid. This review summarizes recent achievements concerning the biosynthesis of sactipeptides in general and with special focus on the common enzymatic radical SAM mechanism leading to the thioether linkage formation. In addition this mechanism is compared to the mechanism of thioether bond formation during lanthipeptide biosynthesis and to other radical based thioether bond forming reactions.

  18. [Parenteral S-adenosylmethionine compared to placebos in the treatment of alcoholic liver diseases].

    PubMed

    Diaz Belmont, A; Dominguez Henkel, R; Uribe Ancira, F

    1996-01-01

    The improvements in the knowledge of the action of ethanol over the hepatic cell, its direct action over the cell, and the intracytoplasmatic structures membranes, point out the possibilities of use of sulfo-adenosil-L-metionina (SAMe); as an util drug inn the treatment of the altered metilation reactions, that take place in those membranes, facilitating their physiological functions. The primary end point in this study was to demonstrate the therapeutic worth os SAMe, by parenteral route in 45 patients with alcoholic liver disease, which were determined by clinical laboratory and hepatic function test, label qith 32 points or more of the discriminatory function index. Divided into two groups, placebo-SAMe, randomized, double blind. As well as total plasmatic and reduced glutation and lipoperoxidation index, indirect form as malondehaldehyde. Were determined at the first visit anf after 8 and 15 days of treatment. Comparing the results of both groups there were a significative favorable results for the group treatment with SAMe and this confirms the utility of this drug in the treatment of patients with alcoholic liver disease with a discriminatory function index (Maddrey index), of 32 points or more.

  19. Identification of an S-adenosylmethionine (SAM) dependent arsenic methyltransferase in Danio rerio

    SciTech Connect

    Hamdi, Mohamad; Yoshinaga, Masafumi; Packianathan, Charles; Qin, Jie; Hallauer, Janell; McDermott, Joseph R.; Yang, Hung-Chi; Tsai, Kan-Jen; Liu, Zijuan

    2012-07-15

    Arsenic methylation is an important cellular metabolic process that modulates arsenic toxicity and carcinogenicity. Biomethylation of arsenic produces a series of mono-, di- and tri-methylated arsenic metabolites that can be detected in tissues and excretions. Here we report that zebrafish exposed to arsenite (As{sup III}) produces organic arsenicals, including MMA{sup III}, MMA{sup V} and DMA{sup V} with characteristic tissue ratios, demonstrating that an arsenic methylation pathway exists in zebrafish. In mammals, cellular inorganic arsenic is methylated by a SAM-dependent arsenic methyltransferase, AS3MT. A zebrafish arsenic methyltransferase homolog, As3mt, was identified by sequence alignment. Western blotting analysis showed that As3mt was universally expressed in zebrafish tissues. Prominent expression in liver and intestine correlated with methylated arsenic metabolites detected in those tissues. As3mt was expressed in and purified from Escherichia coli for in vitro functional studies. Our results demonstrated that As3mt methylated As{sup III} to DMA{sup V} as an end product and produced MMA{sup III} and MMA{sup V} as intermediates. The activity of As3mt was inhibited by elevated concentrations of the substrate As{sup III} as well as the metalloid selenite, which is a well-known antagonistic micronutrient of arsenic toxicity. The activity As3mt was abolished by substitution of either Cys160 or Cys210, which corresponds to conserved cysteine residues in AS3MT homologs, suggesting that they are involved in catalysis. Expression in zebrafish of an enzyme that has a similar function to human and rodent orthologs in catalyzing intracellular arsenic biomethylation validates the applicability of zebrafish as a valuable vertebrate model for understanding arsenic-associated diseases in humans. -- Highlights: ► Zebrafish methylated As{sup III} to MMA{sup III}, MMA{sup V} and DMA{sup V}. ► A zebrafish arsenic methyltransferase (As3mt) was purified in E. coli. ► As3mt catalyzed biomethylation of As{sup III} to DMA{sup V} and produced toxic intermediates. ► As3mt activity is inhibited by elevated substrate concentrations and selenite. ► C160 and C165 are predicted as As{sup III} binding sites.

  20. A decrease in S-adenosylmethionine synthetase activity increases the probability of spontaneous sporulation.

    PubMed Central

    Ochi, K; Freese, E

    1982-01-01

    Starting with a relaxed (relA) strain, mutants with reduced activity of adenosine triphosphate:L-methionine S-adenosyl transferase (EC 2.5.1.6; SAM synthetase) were isolated in Bacillus subtilis. One such mutant (gene symbol metE1) had only 3% of the normal SAM synthetase activity but grew almost as well as the parent strain. Another mutant was isolated (gene symbol spdC1) as being able to sporulate continually at a high frequency; it had one-half the normal SAM synthetase activity at 33 degrees C. Both mutants continually and spontaneously entered spore development at a higher frequency than the parent strain in a medium containing excess glucose, ammonium ions, and phosphate. Sporulation was prevented by a high concentration of SAM (1 mM or more) or by the combination of adenosine and methionine (0.5 mM or more each), both of which are precursors of SAM. In contrast to this continual increase in the spore titer, addition of decoyinine, an inhibitor of GMP synthetase, rapidly initiated massive sporulation. Various amino acid analogs also induced sporulation in the relA strain, the methionine analogs ethionine and selenomethionine being most effective. PMID:6811558

  1. Effect of the hexapeptide dalargin on ornithine decarboxylase activity in the duodenal mucosa of rats with experimental duodenal ulcer

    SciTech Connect

    Yarygin, K.N.; Shitin, A.G.; Polonskii, V.M.; Vinogradov, V.A.

    1987-08-01

    The authors study the effect of dalargin on ornithine decarboxylase in homogenates of the duodenal ulcer from rats with experimental duodenal ulcer induced by cysteamine. Activity of the enzyme was expressed in pmoles /sup 14/CO/sub 2//mg protein/h. Protein was determined by Lowry's method. The findings indicate that stimulation of ornithine decarboxylase and the antiulcerative effect of dalargin may be due to direct interaction of the peptide with cells of the intestinal mucosa and with enterocytes.

  2. Unusual space-group pseudo symmetry in crystals of human phosphopantothenoylcysteine decarboxylase

    SciTech Connect

    Manoj, N.; Ealick, S.E.

    2010-12-01

    Phosphopantothenoylcysteine (PPC) decarboxylase is an essential enzyme in the biosynthesis of coenzyme A and catalyzes the decarboxylation of PPC to phosphopantetheine. Human PPC decarboxylase has been expressed in Escherichia coli, purified and crystallized. The Laue class of the diffraction data appears to be {bar 3}m, suggesting space group R32 with two monomers per asymmetric unit. However, the crystals belong to the space group R3 and the asymmetric unit contains four monomers. The structure has been solved using molecular replacement and refined to a current R factor of 29%. The crystal packing can be considered as two interlaced lattices, each consistent with space group R32 and with the corresponding twofold axes parallel to each other but separated along the threefold axis. Thus, the true space group is R3 with four monomers per asymmetric unit.

  3. A coenzyme-independent decarboxylase/oxygenase cascade for the efficient synthesis of vanillin.

    PubMed

    Furuya, Toshiki; Miura, Misa; Kino, Kuniki

    2014-10-13

    Vanillin is one of the most widely used flavor compounds in the world as well as a promising versatile building block. The biotechnological production of vanillin from plant-derived ferulic acid has attracted much attention as a new alternative to chemical synthesis. One limitation of the known metabolic pathway to vanillin is its requirement for expensive coenzymes. Here, we developed a novel route to vanillin from ferulic acid that does not require any coenzymes. This artificial pathway consists of a coenzyme-independent decarboxylase and a coenzyme-independent oxygenase. When Escherichia coli cells harboring the decarboxylase/oxygenase cascade were incubated with ferulic acid, the cells efficiently synthesized vanillin (8.0 mM, 1.2 g L(-1) ) via 4-vinylguaiacol in one pot, without the generation of any detectable aromatic by-products. The efficient method described here might be applicable to the synthesis of other high-value chemicals from plant-derived aromatics.

  4. HemQ: An iron-coproporphyrin oxidative decarboxylase for protoheme synthesis in Firmicutes and Actinobacteria

    SciTech Connect

    Dailey, Harry A.; Gerdes, Svetlana

    2015-02-21

    Genes for chlorite dismutase-like proteins are found widely among heme-synthesizing bacteria and some Archaea. It is now known that among the Firmicutes and Actinobacteria these proteins do not possess chlorite dismutase activity but instead are essential for heme synthesis. These proteins, named HemQ, are ironcoproporphyrin (coproheme) decarboxylases that catalyze the oxidative decarboxylation of coproheme III into protoheme IX. As purified, HemQs do not contain bound heme, but readily bind exogeneously supplied heme with low micromolar affinity. We find that the heme-bound form of HemQ has low peroxidase activity and in the presence of peroxide the bound heme may be destroyed. Furthermore, it is possible that HemQ may serve a dual role as a decarboxylase in heme biosynthesis and a regulatory protein in heme homeostasis.

  5. HemQ: an iron-coproporphyrin oxidative decarboxylase for protoheme synthesis in Firmicutes and Actinobacteria

    PubMed Central

    Dailey, Harry A.; Gerdes, Svetlana

    2015-01-01

    Genes for chlorite dismutase-like proteins are found widely among hemesynthesizing bacteria and some Archaea. It is now known that among the Firmicutes and Actinobacteria these proteins do not possess chlorite dismutase activity but instead are essential for heme synthesis. These proteins, named HemQ, are ironcoproporphyrin (coproheme) decarboxylases that catalyze the oxidative decarboxylation of coproheme III into protoheme IX. As purified, HemQs do not contain bound heme, but readily bind exogeneously supplied heme with low micromolar affinity. The heme-bound form of HemQ has low peroxidase activity and in the presence of peroxide the bound heme may be destroyed. Thus, it is possible that HemQ may serve a dual role as a decarboxylase in heme biosynthesis and a regulatory protein in heme homeostasis. PMID:25711532

  6. Observation of Superoxide Production During Catalysis of Bacillus subtilis Oxalate Decarboxylase at pH4

    PubMed Central

    Twahir, Umar T.; Stedwell, Corey N.; Lee, Cory T.; Richards, Nigel G. J.; Polfer, Nicolas C.; Angerhofer, Alexander

    2015-01-01

    This contribution describes the trapping of the hydroperoxyl radical at a pH of 4 during turnover of wild-type oxalate decarboxylase and its T165V mutant using the spin trap BMPO. Radicals were detected and identified by a combination of EPR and mass spectrometry. Superoxide, or its conjugate acid, the hydroperoxyl radical, is expected as an intermediate in the decarboxylation and oxidation reactions of the oxalate monoanion both of which are promoted by oxalate decarboxylase. Another intermediate, the carbon dioxide radical anion was also observed. The quantitative yields of superoxide trapping is similar in the wild type and the mutant while it is significantly different for the trapping of the carbon dioxide radical anion. This suggests that the two radicals are released from different sites of the protein. PMID:25526893

  7. Increase of histidine decarboxylase activity in mice hypothalamus after intracerebroventricular administration of lipopolysaccharide.

    PubMed

    Niimi, M; Mochizuki, T; Cacabelos, R; Yamatodani, A

    1993-10-01

    The effect of intracerebroventricular (icv) administration of lipopolysaccharide on histidine decarboxylase activity and histamine content in the hypothalamus were investigated in male mice of ddY strain in vivo. Two-fold increase in histidine decarboxylase activity (HDC) was observed 4 h after administration of 50 mcg lipopolysaccharide, and HDC activity returned to the basal level within 12 h after injection. Furthermore, histamine contents showed a slight decrease at 1 and 2 h and a mild increase at 12 h after administration. However, changes in histamine content were not statistically significant. These results suggest that the increase of HDC activity in the hypothalamus by lipopolysaccharide may be involved in the central neuroimmune responses.

  8. Identification of FAH Domain-containing Protein 1 (FAHD1) as Oxaloacetate Decarboxylase*

    PubMed Central

    Pircher, Haymo; von Grafenstein, Susanne; Diener, Thomas; Metzger, Christina; Albertini, Eva; Taferner, Andrea; Unterluggauer, Hermann; Kramer, Christian; Liedl, Klaus R.; Jansen-Dürr, Pidder

    2015-01-01

    Fumarylacetoacetate hydrolase (FAH) domain-containing proteins occur in both prokaryotes and eukaryotes, where they carry out diverse enzymatic reactions, probably related to structural differences in their respective FAH domains; however, the precise relationship between structure of the FAH domain and the associated enzyme function remains elusive. In mammals, three FAH domain-containing proteins, FAHD1, FAHD2A, and FAHD2B, are known; however, their enzymatic function, if any, remains to be demonstrated. In bacteria, oxaloacetate is subject to enzymatic decarboxylation; however, oxaloacetate decarboxylases (ODx) were so far not identified in eukaryotes. Based on molecular modeling and subsequent biochemical investigations, we identified FAHD1 as a eukaryotic ODx enzyme. The results presented here indicate that dedicated oxaloacetate decarboxylases exist in eukaryotes. PMID:25575590

  9. HemQ: An iron-coproporphyrin oxidative decarboxylase for protoheme synthesis in Firmicutes and Actinobacteria

    DOE PAGES

    Dailey, Harry A.; Gerdes, Svetlana

    2015-02-21

    Genes for chlorite dismutase-like proteins are found widely among heme-synthesizing bacteria and some Archaea. It is now known that among the Firmicutes and Actinobacteria these proteins do not possess chlorite dismutase activity but instead are essential for heme synthesis. These proteins, named HemQ, are ironcoproporphyrin (coproheme) decarboxylases that catalyze the oxidative decarboxylation of coproheme III into protoheme IX. As purified, HemQs do not contain bound heme, but readily bind exogeneously supplied heme with low micromolar affinity. We find that the heme-bound form of HemQ has low peroxidase activity and in the presence of peroxide the bound heme may be destroyed.more » Furthermore, it is possible that HemQ may serve a dual role as a decarboxylase in heme biosynthesis and a regulatory protein in heme homeostasis.« less

  10. Perturbation of the Monomer-Monomer Interfaces of the Benzoylformate Decarboxylase Tetramer

    SciTech Connect

    Andrews, Forest H.; Rogers, Megan P.; Paul, Lake N.; McLeish, Michael J.

    2014-08-14

    The X-ray structure of benzoylformate decarboxylase (BFDC) from Pseudomonas putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from Lactococcus lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from Saccharomyces cerevisiae that indicated that it might be active as a dimer. To investigate this possibility in BFDC, we sought to shift the equilibrium toward dimer formation. Point mutations were made in the noncatalytic monomer–monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer–tetramer equilibrium and to provide an ~450-fold decrease in kcat. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site and the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.

  11. Cell density-correlated induction of pyruvate decarboxylase under aerobic conditions in the yeast Pichia stipitis.

    PubMed

    Mergler, M; Klinner, U

    2001-01-01

    During the aerobic batch cultivation of P. stipitis CBS 5776 with glucose, pyruvate decarboxylase was activated in a cell number-correlated manner. Activation started when a cell number between 7 x 10(7) and x 10(8) cells ml(-1) was reached and the enzyme activity increased during further cultivation. This induction might have been triggered either by an unknown quorum sensing system or by a shortage of cytoplasmic acetyl-CoA.

  12. Arginine and lysine decarboxylases and the acid tolerance response of Salmonella Typhimurium.

    PubMed

    Alvarez-Ordóñez, Avelino; Fernández, Ana; Bernardo, Ana; López, Mercedes

    2010-01-01

    Salmonella Typhimurium CECT 443 inactivation at pH 2.5 in Mineral Medium (MM) and MM supplemented with 0.01% (w/v) arginine, lysine or glutamic acid was studied using stationary-phase cells grown in buffered BHI pH 7.0 (non-acid adapted cells) and acidified BHI up to pH 4.5 with acetic, citric, lactic and hydrochloric acids (acid adapted cells). In all cases, acid adapted cells, with D-values ranging from 23.34 to 86.90 min, showed a significantly higher acid resistance than non-acid adapted cells, with D-values between 8.90 and 10.29 min. Whereas the conditions used for acid adaptation did not exert a significant effect on the acid resistance of the S. Typhimurium CECT 443 resulting cells, the inclusion of lysine and arginine in the challenge medium protected them against acid inactivation, reaching D-values of about 2 and 3 times higher, respectively, than those found in MM or MM supplemented with glutamic acid. None of these three amino acids significantly modified the acid resistance of non-acid adapted cells. The relative expression level of adiA (encoding the arginine decarboxylase), adiY (encoding the transcriptional activator of adiA), cadA (encoding the lysine decarboxylase) and cadB (encoding the lysine/cadaverine transport protein) was examined by quantitative PCR. Acid adapted cells showed higher relative expression levels for both systems, arginine decarboxylase and lysine decarboxylase, which demonstrates that the induction of specialized pH-homeostatic systems plays an important role in S. Typhimurium CECT 443 protection against acid stress. However, the increased acid resistance showed by acid adapted cells challenged in MM arginine or lysine free suggests the existence of other microbial survival strategies.

  13. Autoradiographic measurement of relative changes in ornithine decarboxylase in axotomized superior cervical ganglion neurons

    SciTech Connect

    Wells, M.R.

    1986-05-01

    An autoradiographic method is described for detecting changes in ornithine decarboxylase in axotomized superior cervical ganglion neurons of rats using (3H)difluoromethylornithine. An increase in binding to neurons was seen at 12 h and 1 day after crushing the postganglionic nerves. Binding returned to control values between 3 and 5 days postoperation. The patterns found using this method were in general agreement with prior reports of enzymatic changes in whole ganglia.

  14. Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA.

    PubMed

    Michael, A J; Furze, J M; Rhodes, M J; Burtin, D

    1996-02-15

    A cDNA for a plant ornithine decarboxylase (ODC), a key enzyme in putrescine and polyamine biosynthesis, has been isolated from root cultures of the solanaceous plant Datura stramonium. Reverse transcription-PCR employing degenerate oligonucleotide primers representing conserved motifs from other eukaryotic ODCs was used to isolate the cDNA. The longest open reading frame potentially encodes a peptide of 431 amino acids and exhibits similarity to other eukaryotic ODCs, prokaryotic and eukaryotic arginine decarboxylases (ADCs), prokaryotic meso-diaminopimelate decarboxylases and the product of the tabA gene of Pseudomonas syringae cv. tabaci. Residues involved at the active site of the mouse ODC are conserved in the plant enzyme. The plant ODC does not possess the C-terminal extension found in the mammalian enzyme, implicated in rapid turnover of the protein, suggesting that the plant ODC may have a longer half-life. Expression of the plant ODC in Escherichia coli and demonstration of ODC activity confirmed that the cDNA encodes an active ODC enzyme. This is the first description of the primary structure of a eukaryotic ODC isolated from an organism where the alternative ADC routine to putrescine is present.

  15. Experimental Evidence and In Silico Identification of Tryptophan Decarboxylase in Citrus Genus.

    PubMed

    De Masi, Luigi; Castaldo, Domenico; Pignone, Domenico; Servillo, Luigi; Facchiano, Angelo

    2017-02-11

    Plant tryptophan decarboxylase (TDC) converts tryptophan into tryptamine, precursor of indolealkylamine alkaloids. The recent finding of tryptamine metabolites in Citrus plants leads to hypothesize the existence of TDC activity in this genus. Here, we report for the first time that, in Citrus x limon seedlings, deuterium labeled tryptophan is decarboxylated into tryptamine, from which successively deuterated N,N,N-trimethyltryptamine is formed. These results give an evidence of the occurrence of the TDC activity and the successive methylation pathway of the tryptamine produced from the tryptophan decarboxylation. In addition, with the aim to identify the genetic basis for the presence of TDC, we carried out a sequence similarity search for TDC in the Citrus genomes using as a probe the TDC sequence reported for the plant Catharanthus roseus. We analyzed the genomes of both Citrus clementina and Citrus sinensis, available in public database, and identified putative protein sequences of aromatic l-amino acid decarboxylase. Similarly, 42 aromatic l-amino acid decarboxylase sequences from 23 plant species were extracted from public databases. Potential sequence signatures for functional TDC were then identified. With this research, we propose for the first time a putative protein sequence for TDC in the genus Citrus.

  16. Different mRNAs code for dopa decarboxylase in tissues of neuronal and nonneuronal origin

    SciTech Connect

    Krieger, M.; Coge, F.; Gros, F.; Thibault, J. )

    1991-03-15

    A cDNA clone for dopa decarboxylase has been isolated from a rat pheochromocytoma cDNA library and the cDNA sequence has been determined. It corresponds to an mRNA of 2094 nucleotides. The length of the mRNA was measured by primer-extension of rat pheochromocytoma RNA and the 5{prime} end of the sequence of the mRNA was confirmed by the PCR. A probe spanning the translation initiation site of the mRNA was used to hybridize with mRNAs from various organs of the rat. S1 nuclease digestion of the mRNAs annealed with this probe revealed two classes of mRNAs. The comparison of the cDNA sequence and published sequences for rat liver, human pheochromocytoma, and Droxophila dopa decarboxylase supported the conclusion that two mRNAs are produced: one is specific for tissue of neuronal origin and the other is specific for tissues of nonneuronal (mesodermal or endodermal) origin. The neuronal mRNA contains a 5{prime} untranslated sequence that is highly conserved between human and rat pheochromocytoma including a GA stretch. The coding sequence and the 3{prime} untranslated sequence of mRNAs from rat liver and pheochromocytoma are identical. The rat mRNA differs only in the 5{prime} untranslated region. Thus a unique gene codes for dopa decarboxylase and this gene gives rise to at least two transcripts presumably in response to different signals during development.

  17. EPR Spin Trapping of an Oxalate-Derived Free Radical in the Oxalate Decarboxylase Reaction

    PubMed Central

    Imaram, Witcha; Saylor, Benjamin T.; Centonze, Christopher P.; Richards, Nigel G. J.; Angerhofer, Alexander

    2011-01-01

    EPR spin trapping experiments on bacterial oxalate decarboxylase from Bacillus subtilis under turn-over conditions are described. The use of doubly 13C-labeled oxalate leads to a characteristic splitting of the observed radical adducts using the spin trap N-tert-butyl-α-phenylnitrone linking them directly to the substrate. The radical was identified as the carbon dioxide radical anion which is a key intermediate in the hypothetical reaction mechanism of both decarboxylase and oxidase activities. X-ray crystallography had identified a flexible loop, SENS161-4, which acts as a lid to the putative active site. Site directed mutagenesis of the hinge amino acids, S161 and T165 was explored and showed increased radical trapping yields compared to the wild type. In particular, T165V shows approximately ten times higher radical yields while at the same time its decarboxylase activity was reduced by about a factor of ten. This mutant lacks a critical H-bond between T165 and R92 resulting in compromised control over its radical chemistry allowing the radical intermediate to leak into the surrounding solution. PMID:21277974

  18. Overexpression, purification, crystallization and preliminary structural studies of p-coumaric acid decarboxylase from Lactobacillus plantarum

    SciTech Connect

    Rodríguez, Héctor; Rivas, Blanca de las; Muñoz, Rosario; Mancheño, José M.

    2007-04-01

    The enzyme p-coumaric acid decarboxylase (PDC) from L. plantarum has been recombinantly expressed, purified and crystallized. The structure has been solved at 2.04 Å resolution by the molecular-replacement method. The substrate-inducible p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum has been overexpressed in Escherichia coli, purified and confirmed to possess decarboxylase activity. The recombinant His{sub 6}-tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a solution containing 20%(w/v) PEG 4000, 12%(w/v) 2-propanol, 0.2 M sodium acetate, 0.1 M Tris–HCl pH 8.0 with 0.1 M barium chloride as an additive. Diffraction data were collected in-house to 2.04 Å resolution. Crystals belonged to the tetragonal space group P4{sub 3}, with unit-cell parameters a = b = 43.15, c = 231.86 Å. The estimated Matthews coefficient was 2.36 Å{sup 3} Da{sup −1}, corresponding to 48% solvent content, which is consistent with the presence of two protein molecules in the asymmetric unit. The structure of PDC has been determined by the molecular-replacement method. Currently, the structure of PDC complexed with substrate analogues is in progress, with the aim of elucidating the structural basis of the catalytic mechanism.

  19. 3,4-Dihydroxyphenylalanine (dopa) decarboxylase activity in the arthropod nervous system.

    PubMed

    Murdock, L L; Wirtz, R A; Köhler, G

    1973-04-01

    1. When homogenates of brains from mature adult locusts (Locusta migratoria) were incubated with l-3-(3,4-dihydroxyphenyl)[3-(14)C]alanine the major radioactive metabolite was dopamine, suggesting the presence of a dopa (3,4-dihydroxyphenylalanine) decarboxylase. 2. Decarboxylation of l-dopa by this tissue, measured under optimum conditions by a radiochemical method, was 21mumol of CO(2)/h per g wet wt. Apparent decarboxylation of l-tyrosine proceeded at 0.34mumol of CO(2)/h per g wet wt. There was no detectable decarboxylation of l-tryptophan, l-histidine or l-phenylalanine. 3. Dopa decarboxylase activity was found in all major regions of the ventral nerve cord of the mature locust (range: 4-7mumol of CO(2)/h per g wet wt.) but was low or absent in thoracic peripheral nerve. 4. Marked decarboxylation of l-dopa was found in homogenates of brains of four other species of insects, and in brain and ventral nerve cord, but not in the claw nerve, of the crayfish. 5. The activity of the locust brain enzyme may be slightly lower at the time of imaginal ecdysis than during the mature period. By contrast, the dopa decarboxylase that produces dopamine as an intermediate in cuticle biosynthesis is known to be high in activity at the time of ecdysis and low in activity during the intermoult stages.

  20. Isolation and sequence of the gene encoding ornithine decarboxylase, SPE1, from Candida albicans by complementation of a spe1 delta strain of Saccharomyces cerevisiae.

    PubMed

    McNemar, M D; Gorman, J A; Buckley, H R

    1997-11-01

    The gene encoding ornithine decarboxylase, SPE1, from the pathogenic yeast Candida albicans has been isolated by complementation of an ornithine decarboxylase-negative (spe1 delta) strain of Saccharomyces cerevisiae. Four transformants, three of which contain plasmids with the SPE1 gene, were isolated by selection on polyamine-free medium. The C. albicans ornithine decarboxylase (ODC) showed high homology with other eukaryotic ODCs at both the amino acid and nucleic acid levels.

  1. Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity

    SciTech Connect

    Johnson, Christopher W.; Salvachua, Davinia; Khanna, Payal; Smith, Holly; Peterson, Darren J.; Beckham, Gregg T.

    2016-04-22

    The conversion of biomass-derived sugars and aromatic molecules to cis,cis-muconic acid (referred to hereafter as muconic acid or muconate) has been of recent interest owing to its facile conversion to adipic acid, an important commodity chemical. Metabolic routes to produce muconate from both sugars and many lignin-derived aromatic compounds require the use of a decarboxylase to convert protocatechuate (PCA, 3,4-dihydroxybenzoate) to catechol (1,2-dihydroxybenzene), two central aromatic intermediates in this pathway. Several studies have identified the PCA decarboxylase as a metabolic bottleneck, causing an accumulation of PCA that subsequently reduces muconate production. A recent study showed that activity of the PCA decarboxylase is enhanced by co-expression of two genetically associated proteins, one of which likely produces a flavin-derived cofactor utilized by the decarboxylase. Using entirely genome-integrated gene expression, we have engineered Pseudomonas putida KT2440-derived strains to produce muconate from either aromatic molecules or sugars and demonstrate in both cases that co-expression of these decarboxylase associated proteins reduces PCA accumulation and enhances muconate production relative to strains expressing the PCA decarboxylase alone. In bioreactor experiments, co-expression increased the specific productivity (mg/g cells/h) of muconate from the aromatic lignin monomer p-coumarate by 50% and resulted in a titer of >15 g/L. In strains engineered to produce muconate from glucose, co-expression more than tripled the titer, yield, productivity, and specific productivity, with the best strain producing 4.92+/-0.48 g/L muconate. Furthermore, this study demonstrates that overcoming the PCA decarboxylase bottleneck can increase muconate yields from biomass-derived sugars and aromatic molecules in industrially relevant strains and cultivation conditions.

  2. Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity

    DOE PAGES

    Johnson, Christopher W.; Salvachua, Davinia; Khanna, Payal; ...

    2016-04-22

    The conversion of biomass-derived sugars and aromatic molecules to cis,cis-muconic acid (referred to hereafter as muconic acid or muconate) has been of recent interest owing to its facile conversion to adipic acid, an important commodity chemical. Metabolic routes to produce muconate from both sugars and many lignin-derived aromatic compounds require the use of a decarboxylase to convert protocatechuate (PCA, 3,4-dihydroxybenzoate) to catechol (1,2-dihydroxybenzene), two central aromatic intermediates in this pathway. Several studies have identified the PCA decarboxylase as a metabolic bottleneck, causing an accumulation of PCA that subsequently reduces muconate production. A recent study showed that activity of the PCAmore » decarboxylase is enhanced by co-expression of two genetically associated proteins, one of which likely produces a flavin-derived cofactor utilized by the decarboxylase. Using entirely genome-integrated gene expression, we have engineered Pseudomonas putida KT2440-derived strains to produce muconate from either aromatic molecules or sugars and demonstrate in both cases that co-expression of these decarboxylase associated proteins reduces PCA accumulation and enhances muconate production relative to strains expressing the PCA decarboxylase alone. In bioreactor experiments, co-expression increased the specific productivity (mg/g cells/h) of muconate from the aromatic lignin monomer p-coumarate by 50% and resulted in a titer of >15 g/L. In strains engineered to produce muconate from glucose, co-expression more than tripled the titer, yield, productivity, and specific productivity, with the best strain producing 4.92+/-0.48 g/L muconate. Furthermore, this study demonstrates that overcoming the PCA decarboxylase bottleneck can increase muconate yields from biomass-derived sugars and aromatic molecules in industrially relevant strains and cultivation conditions.« less

  3. Carbon Dioxide Effects on Ethanol Production, Pyruvate Decarboxylase, and Alcohol Dehydrogenase Activities in Anaerobic Sweet Potato Roots 1

    PubMed Central

    Chang, Ling A.; Hammett, Larry K.; Pharr, David M.

    1983-01-01

    The effect of varied anaerobic atmospheres on the metabolism of sweet potato (Ipomoea batatas [L.] Lam.) roots was studied. The internal gas atmospheres of storage roots changed rapidly when the roots were submerged under water. O2 and N2 gases disappeared quickly and were replaced by CO2. There were no appreciable differences in gas composition among the four cultivars that were studied. Under different anaerobic conditions, ethanol concentration in the roots was highest in a CO2 environment, followed by submergence and a N2 environment in all the cultivars except one. A positive relationship was found between ethanol production and pyruvate decarboxylase activity from both 100% CO2-treated and 100% N2-treated roots. CO2 atmospheres also resulted in higher pyruvate decarboxylase activity than did N2 atmospheres. Concentrations of CO2 were higher within anaerobic roots than those in the ambient anaerobic atmosphere. The level of pyruvate decarboxylase and ethanol in anaerobic roots was proportional to the ambient CO2 concentration. The measurable activity of pyruvate decarboxylase that was present in the roots was about 100 times less than that of alcohol dehydrogenase. Considering these observations, it is suggested that the rate-limiting enzyme for ethanol biosynthesis in sweet potato storage roots under anoxia is likely to be pyruvate decarboxylase rather than alcohol dehydrogenase. PMID:16662798

  4. An archaeal glutamate decarboxylase homolog functions as an aspartate decarboxylase and is involved in β-alanine and coenzyme A biosynthesis.

    PubMed

    Tomita, Hiroya; Yokooji, Yuusuke; Ishibashi, Takuya; Imanaka, Tadayuki; Atomi, Haruyuki

    2014-03-01

    β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5'-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of β-alanine, 4'-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the β-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for β-alanine biosynthesis in these organisms.

  5. An Archaeal Glutamate Decarboxylase Homolog Functions as an Aspartate Decarboxylase and Is Involved in β-Alanine and Coenzyme A Biosynthesis

    PubMed Central

    Tomita, Hiroya; Yokooji, Yuusuke; Ishibashi, Takuya; Imanaka, Tadayuki

    2014-01-01

    β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5′-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of β-alanine, 4′-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the β-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for β-alanine biosynthesis in these organisms. PMID:24415726

  6. Kinetic, Mutational, and Structural Analysis of Malonate Semialdehyde Decarboxylase from Coryneform bacterium strain FG41: Mechanistic Implications for the Decarboxylase and Hydratase Activities

    PubMed Central

    Guo, Youzhong; Serrano, Hector; Poelarends, Gerrit J.; Johnson, William H.; Hackert, Marvin L.; Whitman, Christian P.

    2013-01-01

    Malonate semialdehyde decarboxylase from Pseudomonas pavonaceae 170 (designated Pp MSAD) is in a bacterial catabolic pathway for the nematicide 1,3-dichloropropene. MSAD has two known activities: it catalyzes the metal-ion independent decarboxylation of malonate semialdehyde to produce acetaldehyde and carbon dioxide, as well as a low-level hydration of 2-oxo-3-pentynoate to yield acetopyruvate. The latter activity is not known to be biologically relevant. Previous studies identified Pro-1, Asp-37, and a pair of arginines (Arg-73 and Arg-75) as critical residues in these activities. MSAD from Coryneform bacterium strain FG41 (designated FG41 MSAD) shares 38% pairwise sequence identity with the Pseudomonas enzyme including Pro-1 and Asp-37. However, Gln-73 replaces Arg-73, and the second arginine is shifted to Arg-76 by the insertion of a glycine. In order to determine how these changes relate to the activities of FG41 MSAD, the gene was cloned and the enzyme expressed and characterized. The enzyme has a comparable decarboxylase activity, but a significantly reduced hydratase activity. Mutagenesis along with crystal structures of the native enzyme (2.0 Å resolution) and the enzyme modified by a 3-oxopropanoate moiety (resulting from the incubation of enzyme and 3-bromopropiolate) (2.2 Å resolution) provided a structural basis. The roles of Pro-1 and Asp-37 are likely the same as those proposed for MSAD. However, the side chains of Thr-72, Gln-73, and Tyr-123 replace those of Arg-73 and Arg-75 in the mechanism and play a role in binding and catalysis. The structures also show that Arg-76 is likely too distant to play a direct role in the mechanism. FG41 MSAD is the second functionally annotated homologue in the MSAD family of the tautomerase superfamily and could represent a new subfamily. PMID:23781927

  7. Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.

    PubMed

    Hayashi, Masaya; Okada, Akane; Yamamoto, Kumiko; Okugochi, Tomomi; Kusaka, Chika; Kudou, Daizou; Nemoto, Michiko; Inagaki, Junko; Hirose, Yuu; Okajima, Toshihide; Tamura, Takashi; Soda, Kenji; Inagaki, Kenji

    2016-12-21

    l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

  8. Biochemical Evaluation of the Decarboxylation and Decarboxylation-Deamination Activities of Plant Aromatic Amino Acid Decarboxylases*

    PubMed Central

    Torrens-Spence, Michael P.; Liu, Pingyang; Ding, Haizhen; Harich, Kim; Gillaspy, Glenda; Li, Jianyong

    2013-01-01

    Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site

  9. Fluorimetric assay for ornithine decarboxylase by high-performance liquid chromatography.

    PubMed

    Haraguchi, K; Kai, M; Kohashi, K; Ohkura, Y

    1980-12-05

    A highly sensitive method for the assay of ornithine decarboxylase in sample solutions prepared from rat tissue homogenate is described which employs high-performance liquid chromatography with fluorescence detection. Putrescine formed from ornithine under the optimal conditions for the enzyme reaction is treated by Cellex P column chromatography for clean-up and converted into the fluorescamine derivative in the presence of cupric ion which inhibits the reaction of interfering amines with fluorescamine. The derivative is separated by reversed-phase chromatography on LiChrosorb RP-18 with linear gradient elution. The lower limit of detection for putrescine formed enzymatically is 5 pmol.

  10. Endogenous Inactivators of Arginase, l-Arginine Decarboxylase, and Agmatine Amidinohydrolase in Evernia prunastri Thallus 1

    PubMed Central

    Legaz, María Estrella; Vicente, Carlos

    1983-01-01

    Arginase (EC 3.5.3.1), l-arginine decarboxylase (EC 4.1.1.19), and agmatine amidinohydrolase (EC 3.5.3.11) activities spontaneously decay in Evernia prunastri thalli incubated on 40 millimolar l-arginine used as inducer of the three enzymes if dithiothreitol is not added to the media. Lichen thalli accumulate both chloroatranorin and evernic acid in parallel to the loss of activity. These substances behave as inactivators of the enzymes at a range of concentrations between 2 and 20 micromolar, whereas several concentrations of dithiothreitol reverse, to some extent, the in vitro inactivation. PMID:16662821

  11. Endogenous Inactivators of Arginase, l-Arginine Decarboxylase, and Agmatine Amidinohydrolase in Evernia prunastri Thallus.

    PubMed

    Legaz, M E; Vicente, C

    1983-02-01

    Arginase (EC 3.5.3.1), l-arginine decarboxylase (EC 4.1.1.19), and agmatine amidinohydrolase (EC 3.5.3.11) activities spontaneously decay in Evernia prunastri thalli incubated on 40 millimolar l-arginine used as inducer of the three enzymes if dithiothreitol is not added to the media. Lichen thalli accumulate both chloroatranorin and evernic acid in parallel to the loss of activity. These substances behave as inactivators of the enzymes at a range of concentrations between 2 and 20 micromolar, whereas several concentrations of dithiothreitol reverse, to some extent, the in vitro inactivation.

  12. Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis.

    PubMed Central

    Diderichsen, B; Wedsted, U; Hedegaard, L; Jensen, B R; Sjøholm, C

    1990-01-01

    A gene for alpha-acetolactate decarboxylase (ALDC) was cloned from Bacillus brevis in Escherichia coli and in Bacillus subtilis. The 1.3-kilobase-pair nucleotide sequence of the gene, aldB, encoding ALDC and its flanking regions was determined. An open reading frame of 285 amino acids included a typical N-terminal signal peptide of 24 or 27 amino acids. A B. subtilis strain harboring the aldB gene on a recombinant plasmid processed and secreted ALDC. In contrast, a similar enzyme from Enterobacter aerogenes is intracellular. Images PMID:2198252

  13. Apraxia in anti-glutamic acid decarboxylase-associated stiff person syndrome: link to corticobasal degeneration?

    PubMed

    Bowen, Lauren N; Subramony, S H; Heilman, Kenneth M

    2015-01-01

    Corticobasal syndrome (CBS) is associated with asymmetrical rigidity as well as asymmetrical limb-kinetic and ideomotor apraxia. Stiff person syndrome (SPS) is characterized by muscle stiffness and gait difficulties. Whereas patients with CBS have several forms of pathology, many patients with SPS have glutamic acid decarboxylase antibodies (GAD-ab), but these 2 disorders have not been reported to coexist. We report 2 patients with GAD-ab-positive SPS who also had signs suggestive of CBS, including asymmetrical limb rigidity associated with both asymmetrical limb-kinetic and ideomotor apraxia. Future studies should evaluate patients with CBS for GAD-ab and people with SPS for signs of CBS.

  14. Expression of Ornithine Decarboxylase Is Transiently Increased by Pollination, 2,4-Dichlorophenoxyacetic Acid, and Gibberellic Acid in Tomato Ovaries1

    PubMed Central

    Alabadí, David; Carbonell, Juan

    1998-01-01

    A cDNA encoding for a functional ornithine decarboxylase has been isolated from a cDNA library of carpels of tomato (Lycopersicon esculentum Mill.). Ornithine decarboxylase in tomato is represented by a single-copy gene that we show to be up-regulated during early fruit growth induced by 2,4-dichlorophenoxyacetic acid and gibberellic acid. PMID:9733552

  15. Amine cations promote concurrent conversion of prohistidine decarboxylase from Lactobacillus 30a to active enzyme and a modified proenzyme.

    PubMed Central

    van Poelje, P D; Snell, E E

    1988-01-01

    Activation of prohistidine decarboxylase (pi 6) from Lactobacillus 30a proceeds by an intramolecular, pH- and monovalent cation-dependent reaction in which its constituent pi chains are cleaved nonhydrolytically between Ser-81 and Ser-82 with loss of NH3 and conversion of Ser-82 to the pyruvoyl residue of active histidine decarboxylase (alpha beta)6. Amines with pKa values more than 7.0 substitute for K+ or NH4+ in the activation of prohistidine decarboxylase, but they also catalyze its inactivation in a competing reaction, pi 6----pi'6. Sequence analysis of the appropriate tryptic peptide from amine-inactivated prohistidine decarboxylase established that inactivation results from conversion of Ser-82 of the pi chain to an aminoacrylate residue. The inactivated proenzyme (pi'6) does not form histidine decarboxylase; this fact eliminates one of two postulated mechanisms of activation and, thus, favors activation by beta-elimination of the acyl group of an intermediate ester formed between Ser-81 and Ser-82. L-Histidine is bound by the proenzyme (Kd = 1.7 x 10(-4) M) and is an effective activator; one binding site is present per pi subunit. K+, NH4+, and Na+ competitively inhibit (Ki values = 2.8-4.4 x 10(-3) M) activation by histidine. The data suggest the presence of two classes of monovalent cation binding sites on prohistidine decarboxylase: one (near Ser-82) is readily saturable and one is unsaturable even by 2.4 M K+. Images PMID:3250558

  16. Local anesthetics inhibit induction of ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate.

    PubMed Central

    Yuspa, S H; Lichti, U; Ben, T

    1980-01-01

    The induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity in mouse epidermal cells in vivo and in vitro occurs rapidly after exposure to the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). This induction has characteristics of a cell surface receptor-mediated process. Local anesthetics modify a variety of cellular responses mediated by membrane receptors. When cultured mouse epidermal cells were exposed to the local anesthetics lidocaine, tetracaine, or procaine (0.1-1 mM), induction of the decarboxylase by TPA was inhibited by more than 90%. In vivo, lidocaine essentially abolishes the decarboxylase response of mouse epidermis when applied shortly after TPA. In contrast, local anesthetics have no effect on the enzyme's activity when added directly to the assay mixture and, in concert with TPA, have only a minimal effect on overall protein synthesis relative to controls. However, lidocaine has no effect on TPA-stimulated DNA synthesis in vitro (12-fold with or without lidocaine). Local anesthetics also markedly inhibit induction of the decarboxylase by ultraviolet light, which is probably not membrane mediated. Furthermore, in culture, lidocaine has only a small inhibitory effect on ornithine decarboxylase when given before TPA but is an effective inhibitor even when given up to 4-5 hr after the promoter, a time when decarboxylase activity has already increased. These findings suggest that local anesthetics, which are tertiary amines, do not act at the site of interaction of TPA and its putative receptor but may be acting specifically on polyamine biosynthesis. These drugs could be useful agents to determine the role of the polyamine pathway in tumor promotion. PMID:6933562

  17. Polyamine formation by arginine decarboxylase as a transducer of hormonal, environmental and stress stimuli in higher plants

    NASA Technical Reports Server (NTRS)

    Galston, A. W.; Flores, H. E.; Kaur-Sawhney, R.

    1982-01-01

    Recent evidence implicates polyamines including putrescine in the regulation of such diverse plant processes as cell division, embryogenesis and senescence. We find that the enzyme arginine decarboxylase, which controls the rate of putrescine formation in some plant systems, is activated by light acting through P(r) phytochrome as a receptor, by the plant hormone gibberellic acid, by osmotic shock and by other stress stimuli. We therefore propose arginine decarboxylase as a possible transducer of the various initially received tropistic stimuli in plants. The putrescine formed could act by affecting cytoskeletal components.

  18. Molecular cloning, characterization, and function analysis of a mevalonate pyrophosphate decarboxylase gene from Ganoderma lucidum.

    PubMed

    Shi, Liang; Qin, Lei; Xu, Yingjie; Ren, Ang; Fang, Xing; Mu, Dashuai; Tan, Qi; Zhao, Mingwen

    2012-05-01

    This study investigated the role of the mevalonate pyrophosphate decarboxylase gene in the triterpene biosynthetic pathway of Ganoderma lucidum. The mevalonate pyrophosphate decarboxylase gene (mvd) was isolated using a degenerate primer-PCR technique. An analysis of the Gl-mvd transcription profile revealed a positive correlation between the expression of the Gl-mvd gene and triterpene content changes in G. lucidum during development. Furthermore, a promoter deletion analysis was conducted in G. lucidum to investigate the promoter activity and the role of methyl jasmonate (MeJA) responsive elements in the mvd promoter under the MeJA elicitor. The overexpression of Gl-mvd increased triterpene accumulation compared with the wild-type strain and increased the expression of several genes involved in the triterpene biosynthetic pathway. The findings of this study suggest that mvd may play an important role in triterpene biosynthesis regulation. Moreover, there may be the interactions among the genes involved in the triterpene biosynthetic pathway in the G. lucidum. Additionally, this study provides an approach for improving triterpene content through the overexpression of a key gene.

  19. Aromatic L-Amino Acid Decarboxylase (AADC) Is Crucial for Brain Development and Motor Functions

    PubMed Central

    Shih, De-Fen; Hsiao, Chung-Der; Min, Ming-Yuan; Lai, Wen-Sung; Yang, Chianne-Wen; Lee, Wang-Tso; Lee, Shyh-Jye

    2013-01-01

    Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare pediatric neuro-metabolic disease in children. Due to the lack of an animal model, its pathogenetic mechanism is poorly understood. To study the role of AADC in brain development, a zebrafish model of AADC deficiency was generated. We identified an aadc gene homolog, dopa decarboxylase (ddc), in the zebrafish genome. Whole-mount in situ hybridization analysis showed that the ddc gene is expressed in the epiphysis, locus caeruleus, diencephalic catecholaminergic clusters, and raphe nuclei of 36-h post-fertilization (hpf) zebrafish embryos. Inhibition of Ddc by AADC inhibitor NSD-1015 or anti-sense morpholino oligonucleotides (MO) reduced brain volume and body length. We observed increased brain cell apoptosis and loss of dipencephalic catecholaminergic cluster neurons in ddc morphants (ddc MO-injected embryos). Seizure-like activity was also detected in ddc morphants in a dose-dependent manner. ddc morphants had less sensitive touch response and impaired swimming activity that could be rescued by injection of ddc plasmids. In addition, eye movement was also significantly impaired in ddc morphants. Collectively, loss of Ddc appears to result in similar phenotypes as that of ADCC deficiency, thus zebrafish could be a good model for investigating pathogenetic mechanisms of AADC deficiency in children. PMID:23940784

  20. Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae

    PubMed Central

    Yoshida, Shiori; Tanaka, Hideki; Hirayama, Makoto; Murata, Kousaku; Kawai, Shigeyuki

    2015-01-01

    Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved. PMID:26588105

  1. Crystal structure of Mycobacterium tuberculosis diaminopimelate decarboxylase, an essential enzyme in bacterial lysine biosynthesis.

    PubMed

    Gokulan, Kuppan; Rupp, Bernhard; Pavelka, Martin S; Jacobs, William R; Sacchettini, James C

    2003-05-16

    The Mycobacterium tuberculosis lysA gene encodes the enzyme meso-diaminopimelate decarboxylase (DAPDC), a pyridoxal-5'-phosphate (PLP)-dependent enzyme. The enzyme catalyzes the final step in the lysine biosynthetic pathway converting meso-diaminopimelic acid (DAP) to l-lysine. The lysA gene of M. tuberculosis H37Rv has been established as essential for bacterial survival in immunocompromised mice, demonstrating that de novo biosynthesis of lysine is essential for in vivo viability. Drugs targeted against DAPDC could be efficient anti-tuberculosis drugs, and the three-dimensional structure of DAPDC from M. tuberculosis complexed with reaction product lysine and the ternary complex with PLP and lysine in the active site has been determined. The first structure of a DAPDC confirms its classification as a fold type III PLP-dependent enzyme. The structure shows a stable 2-fold dimer in head-to-tail arrangement of a triose-phosphate isomerase (TIM) barrel-like alpha/beta domain and a C-terminal beta sheet domain, similar to the ornithine decarboxylase (ODC) fold family. PLP is covalently bound via an internal aldimine, and residues from both domains and both subunits contribute to the binding pocket. Comparison of the structure with eukaryotic ODCs, in particular with a di-fluoromethyl ornithine (DMFO)-bound ODC from Trypanosoma bruceii, indicates that corresponding DAP-analogues might be potential inhibitors for mycobacterial DAPDCs.

  2. Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae.

    PubMed

    Yoshida, Shiori; Tanaka, Hideki; Hirayama, Makoto; Murata, Kousaku; Kawai, Shigeyuki

    2015-01-01

    Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved.

  3. Hypothalamic L-Histidine Decarboxylase Is Up-Regulated During Chronic REM Sleep Deprivation of Rats

    PubMed Central

    Hoffman, Gloria E.; Koban, Michael

    2016-01-01

    A competition of neurobehavioral drives of sleep and wakefulness occurs during sleep deprivation. When enforced chronically, subjects must remain awake. This study examines histaminergic neurons of the tuberomammillary nucleus of the posterior hypothalamus in response to enforced wakefulness in rats. We tested the hypothesis that the rate-limiting enzyme for histamine biosynthesis, L-histidine decarboxylase (HDC), would be up-regulated during chronic rapid eye movement sleep deprivation (REM-SD) because histamine plays a major role in maintaining wakefulness. Archived brain tissues of male Sprague Dawley rats from a previous study were used. Rats had been subjected to REM-SD by the flowerpot paradigm for 5, 10, or 15 days. For immunocytochemistry, rats were transcardially perfused with acrolein-paraformaldehyde for immunodetection of L-HDC; separate controls used carbodiimide-paraformaldehyde for immunodetection of histamine. Immunolocalization of histamine within the tuberomammillary nucleus was validated using carbodiimide. Because HDC antiserum has cross-reactivity with other decarboxylases at high antibody concentrations, titrations localized L-HDC to only tuberomammillary nucleus at a dilution of ≥ 1:300,000. REM-SD increased immunoreactive HDC by day 5 and it remained elevated in both dorsal and ventral aspects of the tuberomammillary complex. Our results suggest that up-regulation of L-HDC within the tuberomammillary complex during chronic REM-SD may be responsible for maintaining wakefulness. PMID:27997552

  4. Rational design of ornithine decarboxylase with high catalytic activity for the production of putrescine.

    PubMed

    Choi, Hyang; Kyeong, Hyun-Ho; Choi, Jung Min; Kim, Hak-Sung

    2014-09-01

    Putrescine finds wide industrial applications in the synthesis of polymers, pharmaceuticals, agrochemicals, and surfactants. Owing to economic and environmental concerns, the microbial production of putrescine has attracted a great deal of attention, and ornithine decarboxylase (ODC) is known to be a key enzyme in the biosynthetic pathway. Herein, we present the design of ODC from Escherichia coli with high catalytic efficiency using a structure-based rational approach. Through a substrate docking into the model structure of the enzyme, we first selected residues that might lead to an increase in catalytic activity. Of the selected residues that are located in the α-helix and the loops constituting the substrate entry site, a mutational analysis of the single mutants identified two key residues, I163 and E165. A combination of two single mutations resulted in a 62.5-fold increase in the catalytic efficiency when compared with the wild-type enzyme. Molecular dynamics simulations of the best mutant revealed that the substrate entry site becomes more flexible through mutations, while stabilizing the formation of the dimeric interface of the enzyme. Our approach can be applied to the design of other decarboxylases with high catalytic efficiency for the production of various chemicals through bio-based processes.

  5. Decarboxylase gene expression and cadaverine and putrescine production by Serratia proteamaculans in vitro and in beef.

    PubMed

    De Filippis, Francesca; Pennacchia, Carmela; Di Pasqua, Rosangela; Fiore, Alberto; Fogliano, Vincenzo; Villani, Francesco; Ercolini, Danilo

    2013-08-01

    Studies of the molecular basis of microbial metabolic activities that are important for the changes in food quality are valuable in order to help in understanding the behavior of spoiling bacteria in food. The growth of a psychrotrophic Serratia proteamaculans strain was monitored in vitro and in artificially inoculated raw beef. Two growth temperatures (25°C and 4°C) were tested in vitro, while growth at 15°C and 4°C was monitored in beef. During growth, the expression of inducible lysine and ornithine-decarboxylase genes was evaluated by quantitative reverse transcription-PCR (qRT-PCR), while the presence of cadaverine and putrescine was quantified by LC-ESI-MS/MS. The expression of the decarboxylase genes, and the consequent production of cadaverine and putrescine were shown to be influenced by the temperature, as well as by the complexity of the growth medium. Generally, the maximum gene expression and amine production took place during the exponential and early stationary phase, respectively. In addition, lower temperatures caused slower growth and gene downregulation. Higher amounts of cadaverine compared to putrescine were found during growth in beef with the highest concentrations corresponding to microbial loads of ca. 9CFU/g. The differences found in gene expression evaluated in vitro and in beef suggested that such activities are more reliably investigated in situ in specific food matrices.

  6. NUTRITIONAL FACTORS STIMULATING THE FORMATION OF LYSINE DECARBOXYLASE IN ESCHERICHIA COLI

    PubMed Central

    Maretzki, Andrew; Mallette, M. F.

    1962-01-01

    Maretzki, Andrew (Pennsylvania State University, University Park) and M. F. Mallette. Nutritional factors stimulating the formation of lysine decarboxylase in Escherichia coli. J. Bacteriol. 83:720–726. 1962 — Inclusion of complex nitrogen sources in the induction medium was shown to be necessary for the synthesis of appreciable amounts of l-lysine decarboxylase by Escherichia coli B. Hy-case, a commercial acid hydrolyzate of casein, was especially effective in enzyme production, which was assayed manometrically after lysis of the bacteria from without by bacteriophage. Partial fractionation of the Hy-case, identification of the free amino acids, and addition of these amino acids to test media revealed stimulatory effects by methionine, threonine, proline, leucine, and tyrosine. A full complement of amino acids did not match the enzyme levels reached in the presence of Hy-case. Certain peptide fractions obtained from this mixture supplemented the effects of the amino acids in such a way as to suggest direct incorporation of peptide rather than transport or protective roles. Added purines, pyrimidines, iron, and water-soluble vitamins were without effect. Neither carbohydrates nor phosphorylated materials could be detected in the stimulatory fractions. PMID:14469751

  7. Crystal Structure and Substrate Specificity of Drosophila 3,4-Dihydroxyphenylalanine Decarboxylase

    SciTech Connect

    Han, Q.; Ding, H; Robinson, H; Christensen, B; Li, J

    2010-01-01

    3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses. In this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine. The crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.

  8. Characterization of Plasmodium phosphatidylserine decarboxylase expressed in yeast and application for inhibitor screening.

    PubMed

    Choi, Jae-Yeon; Kumar, Vidya; Pachikara, Niseema; Garg, Aprajita; Lawres, Lauren; Toh, Justin Y; Voelker, Dennis R; Ben Mamoun, Choukri

    2016-03-01

    Phospholipid biosynthesis is critical for the development, differentiation and pathogenesis of several eukaryotic pathogens. Genetic studies have validated the pathway for phosphatidylethanolamine synthesis from phosphatidylserine catalyzed by phosphatidylserine decarboxylase enzymes (PSD) as a suitable target for development of antimicrobials; however no inhibitors of this class of enzymes have been discovered. We show that the Plasmodium falciparum PSD can restore the essential function of the yeast gene in strains requiring PSD for growth. Genetic, biochemical and metabolic analyses demonstrate that amino acids between positions 40 and 70 of the parasite enzyme are critical for proenzyme processing and decarboxylase activity. We used the essential role of Plasmodium PSD in yeast as a tool for screening a library of anti-malarials. One of these compounds is 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine, an inhibitor with potent activity against P. falciparum, and low toxicity toward mammalian cells. We synthesized an analog of this compound and showed that it inhibits PfPSD activity and eliminates Plasmodium yoelii infection in mice. These results highlight the importance of 4-quinolinamines as a novel class of drugs targeting membrane biogenesis via inhibition of PSD activity.

  9. Transport of phosphatidylserine from the endoplasmic reticulum to the site of phosphatidylserine decarboxylase2 in yeast.

    PubMed

    Kannan, Muthukumar; Riekhof, Wayne R; Voelker, Dennis R

    2015-02-01

    Over the past two decades, most of the genes specifying lipid synthesis and metabolism in yeast have been identified and characterized. Several of these biosynthetic genes and their encoded enzymes have provided valuable tools for the genetic and biochemical dissection of interorganelle lipid transport processes in yeast. One such pathway involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), and its non-vesicular transport to the site of phosphatidylserine decarboxylase2 (Psd2p) in membranes of the Golgi and endosomal sorting system. In this review, we summarize the identification and characterization of the yeast phosphatidylserine decarboxylases, and examine their role in studies of the transport-dependent pathways of de novo synthesis of phosphatidylethanolamine (PtdEtn). The emerging picture of the Psd2p-specific transport pathway is one in which the enzyme and its non-catalytic N-terminal domains act as a hub to nucleate the assembly of a multiprotein complex, which facilitates PtdSer transport at membrane contact sites between the ER and Golgi/endosome membranes. After transport to the catalytic site of Psd2p, PtdSer is decarboxylated to form PtdEtn, which is disseminated throughout the cell to support the structural and functional needs of multiple membranes.

  10. Environmental stress causes oxidative damage to plant mitochondria leading to inhibition of glycine decarboxylase.

    PubMed

    Taylor, Nicolas L; Day, David A; Millar, A Harvey

    2002-11-08

    A cytotoxic product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), rapidly inhibited glycine, malate/pyruvate, and 2-oxoglutarate-dependent O2 consumption by pea leaf mitochondria. Dose- and time-dependence of inhibition showed that glycine oxidation was the most severely affected with a K(0.5) of 30 microm. Several mitochondrial proteins containing lipoic acid moieties differentially lost their reactivity to a lipoic acid antibody following HNE treatment. The most dramatic loss of antigenicity was seen with the 17-kDa glycine decarboxylase complex (GDC) H-protein, which was correlated with the loss of glycine-dependent O2 consumption. Paraquat treatment of pea seedlings induced lipid peroxidation, which resulted in the rapid loss of glycine-dependent respiration and loss of H-protein reactivity with lipoic acid antibodies. Pea plants exposed to chilling and water deficit responded similarly. In contrast, the damage to other lipoic acid-containing mitochondrial enzymes was minor under these conditions. The implication of the acute sensitivity of glycine decarboxylase complex H-protein to lipid peroxidation products is discussed in the context of photorespiration and potential repair mechanisms in plant mitochondria.

  11. Overexpression, purification, crystallization and preliminary structural studies of p-coumaric acid decarboxylase from Lactobacillus plantarum

    PubMed Central

    Rodríguez, Héctor; de las Rivas, Blanca; Muñoz, Rosario; Mancheño, José M.

    2007-01-01

    The substrate-inducible p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum has been overexpressed in Escherichia coli, purified and confirmed to possess decarboxylase activity. The recombinant His6-tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a solution containing 20%(w/v) PEG 4000, 12%(w/v) 2-propanol, 0.2 M sodium acetate, 0.1 M Tris–HCl pH 8.0 with 0.1 M barium chloride as an additive. Diffraction data were collected in-house to 2.04 Å resolution. Crystals belonged to the tetragonal space group P43, with unit-cell parameters a = b = 43.15, c = 231.86 Å. The estimated Matthews coefficient was 2.36 Å3 Da−1, corresponding to 48% solvent content, which is consistent with the presence of two protein molecules in the asymmetric unit. The structure of PDC has been determined by the molecular-replacement method. Currently, the structure of PDC complexed with substrate analogues is in progress, with the aim of elucidating the structural basis of the catalytic mechanism. PMID:17401200

  12. Evolutionary Trails of Plant Group II Pyridoxal Phosphate-Dependent Decarboxylase Genes

    PubMed Central

    Kumar, Rahul

    2016-01-01

    Type II pyridoxal phosphate-dependent decarboxylase (PLP_deC) enzymes play important metabolic roles during nitrogen metabolism. Recent evolutionary profiling of these genes revealed a sharp expansion of histidine decarboxylase genes in the members of Solanaceae family. In spite of the high sequence homology shared by PLP_deC orthologs, these enzymes display remarkable differences in their substrate specificities. Currently, limited information is available on the gene repertoires and substrate specificities of PLP_deCs which renders their precise annotation challenging and offers technical challenges in the immediate identification and biochemical characterization of their full gene complements in plants. Herein, we explored their evolutionary trails in a comprehensive manner by taking advantage of high-throughput data accessibility and computational approaches. We discussed the premise that has enabled an improved reconstruction of their evolutionary lineage and evaluated the factors offering constraints in their rapid functional characterization, till date. We envisage that the synthesized information herein would act as a catalyst for the rapid exploration of their biochemical specificity and physiological roles in more plant species. PMID:27602045

  13. Characterization of Plasmodium phosphatidylserine decarboxylase expressed in yeast and application for inhibitor screening

    PubMed Central

    Choi, Jae-Yeon; Lawres, Lauren; Toh, Justin Y.; Voelker, Dennis R.; Ben Mamoun, Choukri

    2016-01-01

    Summary Phospholipid biosynthesis is critical for the development, differentiation and pathogenesis of several eukaryotic pathogens. Genetic studies have validated the pathway for phosphatidylethanolamine synthesis from phosphatidylserine catalyzed by phosphatidylserine decarboxylase enzymes (PSD) as a suitable target for development of antimicrobials; however no inhibitors of this class of enzymes have been discovered. We show that the Plasmodium falciparum PSD can restore the essential function of the yeast gene in strains requiring PSD for growth. Genetic, biochemical and metabolic analyses demonstrate that amino acids between positions 40 and 70 of the parasite enzyme are critical for proenzyme processing and decarboxylase activity. We used the essential role of Plasmodium PSD in yeast as a tool for screening a library of anti-malarials. One of these compounds is 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine, an inhibitor with potent activity against P. falciparum, and low toxicity toward mammalian cells. We synthesized an analog of this compound and showed that it inhibits PfPSD activity and eliminates Plasmodium yoelii infection in mice. These results highlight the importance of 4-quinolinamines as a novel class of drugs targeting membrane biogenesis via inhibition of PSD activity PMID:26585333

  14. Arginine and Ornithine Decarboxylases, the Polyamine Biosynthetic Enzymes of Mung Bean Seedlings 1

    PubMed Central

    Altman, Arie; Friedman, Ra'Anan; Levin, Nitsa

    1982-01-01

    General properties and relative activities of l-arginine decarboxylase (ADC) (EC 4.1.1.19) and l-ornithine decarboxylase (ODC) (EC 4.1.1.17), two important enzymes in putrescine and polyamine biosynthesis, were investigated in mung bean (Vigna radiata L.) tissues. Both activities increase linearly with increasing concentrations of crude enzyme, but the increase in ADC activity is considerably greater. The decarboxylation reaction is linear for up to 30 to 60 minutes, and both enzymes have a pH optimum of 7.2. α-Difluoromethyl-ornithine inhibits ODC activity of excised roots, while increasing ADC activity. High specific activity of both enzymes is detected in terminal buds and leaves, while root and hypocotyl activity is low. Different ADC-to-ODC activity ratios are found in various tissues of mung bean plants. Substantial increase in the activity of both enzymes is detected in incubated sections as compared with intact plants. A comparison of several plant species indicates a wide range of ADC-to-ODC activity ratio. It is suggested that both ADC and ODC are active in plant tissues and that their relative contribution to putrescine biosynthesis is dependent upon the type of tissue and growth process. PMID:16662312

  15. Hypothalamic L-Histidine Decarboxylase Is Up-Regulated During Chronic REM Sleep Deprivation of Rats.

    PubMed

    Hoffman, Gloria E; Koban, Michael

    2016-01-01

    A competition of neurobehavioral drives of sleep and wakefulness occurs during sleep deprivation. When enforced chronically, subjects must remain awake. This study examines histaminergic neurons of the tuberomammillary nucleus of the posterior hypothalamus in response to enforced wakefulness in rats. We tested the hypothesis that the rate-limiting enzyme for histamine biosynthesis, L-histidine decarboxylase (HDC), would be up-regulated during chronic rapid eye movement sleep deprivation (REM-SD) because histamine plays a major role in maintaining wakefulness. Archived brain tissues of male Sprague Dawley rats from a previous study were used. Rats had been subjected to REM-SD by the flowerpot paradigm for 5, 10, or 15 days. For immunocytochemistry, rats were transcardially perfused with acrolein-paraformaldehyde for immunodetection of L-HDC; separate controls used carbodiimide-paraformaldehyde for immunodetection of histamine. Immunolocalization of histamine within the tuberomammillary nucleus was validated using carbodiimide. Because HDC antiserum has cross-reactivity with other decarboxylases at high antibody concentrations, titrations localized L-HDC to only tuberomammillary nucleus at a dilution of ≥ 1:300,000. REM-SD increased immunoreactive HDC by day 5 and it remained elevated in both dorsal and ventral aspects of the tuberomammillary complex. Our results suggest that up-regulation of L-HDC within the tuberomammillary complex during chronic REM-SD may be responsible for maintaining wakefulness.

  16. Arginine decarboxylase (ADC) and agmatinase (AGMAT): an alternative pathway for synthesis of polyamines in pig conceptuses and uteri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arginine, a precursor for the synthesis of nitric oxide (NO) and polyamines, is critical for implantation and development of the conceptus. We first reported that the arginine decarboxylase (ADC)/agmatinase(AGMAT) pathway as an alternative pathway for synthesis of polyamines in the ovine conceptuses...

  17. Absence of malonyl coenzyme A decarboxylase in mice increases cardiac glucose oxidation and protects the heart from ischemic injury

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acute pharmacological inhibition of cardiac malonyl coenzyme A decarboxylase (MCD) protects the heart from ischemic damage by inhibiting fatty acid oxidation and stimulating glucose oxidation. However, it is unknown whether chronic inhibition of MCD results in altered cardiac function, energy metabo...

  18. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... preparation derived from a recombinant Bacillus subtilis. 173.115 Section 173.115 Food and Drugs FOOD AND DRUG... Bacillus subtilis. The food additive alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation, may be... derived from a modified Bacillus subtilis strain that contains the gene coding for α-ALDC from...

  19. Molecular analysis of a new member of the opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylase gene family.

    PubMed Central

    Maldonado-Mendoza, I E; López-Meyer, M; Galef, J R; Burnett, R J; Nessler, C L

    1996-01-01

    An aromatic amino acid decarboxylase DNA fragment was generated from opium poppy (Papaver somniferum L.) genomic DNA by the PCR using primers designed from conserved amino acid sequences of other aromatic amino acid decarboxylase genes. Using this fragment as a probe, a genomic clone was isolated that encodes a new member of the opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylase gene family (TyDC5). The predicted TyDC5 amino acid sequence shares extensive identity with other opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylases (84%), and when expressed in Escherichia coli, it is active against tyrosine and to a lesser extent against 3,4-dihydroxyphenylalanine. Ribonuclease protection assays indicate that TyDC5 is expressed primarily in the roots of mature poppy plants. A peak of TyDC5 expression was also observed during germination, coincident with the emergence of the radicle from the seed coat. Parallel results were obtained in transgenic tobacco using a TyDC5 promoter fragment (-2060) translationally fused to the beta-glucuronidase reporter gene (GUS). IN TyDC5::GUS tobacco, GUS activity transiently appeared in all parts of the seedling during germination, but was limited to the roots in older plants. These results indicate that TyDC5 expression is transcriptionally regulated and suggest that the TyDC5 enzyme may play an important role in providing precursors for alkaloid synthesis in the roots and germinating seedlings of opium poppy. PMID:8587993

  20. CONFIRMATIONAL IDENTIFICATION OF ESCHERICHIA COLI, A COMPARISON OF GENOTYPIC AND PHENOTYPIC ASSAYS FOR GLUTAMATE DECARBOXYLASE AND B-D-GLUCURONIDASE

    EPA Science Inventory

    Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and B-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E.coli, three major...

  1. In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community

    PubMed Central

    Zargar, K.; Saville, R.; Phelan, R. M.; Tringe, S. G.; Petzold, C. J.; Keasling, J. D.; Beller, H. R.

    2016-01-01

    Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation in complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extracts, (iii) both activities were irreversibly inactivated upon exposure to O2, and (iv) both activities were similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene. PMID:27506494

  2. In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community.

    PubMed

    Zargar, K; Saville, R; Phelan, R M; Tringe, S G; Petzold, C J; Keasling, J D; Beller, H R

    2016-08-10

    Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation in complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extracts, (iii) both activities were irreversibly inactivated upon exposure to O2, and (iv) both activities were similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene.

  3. Control by Ethylene of Arginine Decarboxylase Activity in Pea Seedlings and Its Implication for Hormonal Regulation of Plant Growth 1

    PubMed Central

    Apelbaum, Akiva; Goldlust, Arie; Icekson, Isaac

    1985-01-01

    Activity of arginine decarboxylase in etiolated pea seedlings appears 24 hours after seed imbibition, reaches its highest level on the 4th day, and levels off until the 7th day. This activity was found in the apical and subapical tissue of the roots and shoots where intensive DNA synthesis occurs. Exposure of the seedlings to ethylene greatly reduced the specific activity of this enzyme. The inhibition was observed within 30 min of the hormone application, and maximal effect—90% inhibition—after 18 hours. Ethylene at physiological concentrations affected the enzyme activity; 50% inhibitory rate was recorded at 0.12 microliters per liter ethylene and maximal response at 1.2 microliters per liter. Ethylene provoked a 5-fold increase in the Kmapp of arginine decarboxylase for its substrate and reduced the Vmaxapp by 10-fold. However, the enzyme recovered from the inhibition and regained control activity 7 hours after transferral of the seedlings to ethylene-free atmosphere. Reducing the endogenous level of ethylene in the tissue by hypobaric pressure, or by exposure to light, as well as interfering with ethylene action by treatment with silver thiosulfate or 2,5-norbornadiene, caused a gradual increase in the specific activity of arginine decarboxylase in the apical tissue of the etiolated seedlings. On the basis of these findings, the possible control of arginine decarboxylase activity by endogenous ethylene, and its implication for the hormone effect on plant growth, are discussed. PMID:16664464

  4. Identification and measurement of acid (specific) histidine decarboxylase activity in rabbit gastric mucosa: ending an old controversy?

    PubMed

    Neugebauer, E; Lorenz, W

    1985-04-01

    One of the main obstacles in assigning any distinct function to histamine in health and disease was the longlasting controversy on the existence of any physiological, endogenous histamine formation in man and most of the other mammals except the rat. Using a modification of Schayer's isotope dilution method, a renewed attempt was made to identify the very low activities of an acid (specific) histidine decarboxylase in rabbit gastric mucosa capable of producing endogenous histamine in physiological conditions, to develop tests for its identification in crude enzyme extracts and to demonstrate the specificity of the enzymatic assay by excluding any relevant Dopa decarboxylase activity and also nonenzymatic decarboxylation interfering with the determination of acid (specific) histidine decarboxylase. To achieve this aim five tests were developed: In the pH profile (test 1), a pH optimum was found at 7.0 in the presence of a low substrate concentration (1.6 X 10(-6)M L-[ring-2-14C]-histidine). The apparent Michaelis concentration at the pH optimum (test 2) was 1.8 X 10(-4)M, the maximum rate 12.5pmol [14C]histamine formed X min-1. To increase the specificity of inhibition experiments with alpha-methylhistidine and alpha-methyl-L-Dopa a pH profile was determined in the presence of these two enzymatic inhibitors (test 3 and 4). alpha-Methylhistidine was used for a reliable diagnostic confirmation test, alpha-methyl-L-Dopa for a reliable exclusion test. Benzene showed no influence on either blanks or recovery rates, but inhibited the enzymic activity at pH 7.0, not however that of unspecific histidine decarboxylase and hence was very valuable as an additional diagnostic exclusion test (test 5). Although these new tests identifying acid (specific) histidine decarboxylase and demonstrating the specificity of its determination were tedious, despite the use of the modified isotope dilution method, they excluded the presence of any Dopa decarboxylase activity in mixtures with

  5. Expression and stereochemical and isotope effect studies of active 4-oxalocrotonate decarboxylase.

    PubMed

    Stanley, T M; Johnson, W H; Burks, E A; Whitman, C P; Hwang, C C; Cook, P F

    2000-02-01

    4-Oxalocrotonate decarboxylase (4-OD) and vinylpyruvate hydratase (VPH) from Pseudomonas putida mt-2 form a complex that converts 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate in the catechol meta fission pathway. To facilitate mechanistic and structural studies of the complex, the two enzymes have been coexpressed and the complex has been purified to homogeneity. In addition, Glu-106, a potential catalytic residue in VPH, has been changed to glutamine, and the resulting E106QVPH mutant has been coexpressed with 4-OD and purified to homogeneity. The 4-OD/E106QVPH complex retains full decarboxylase activity, with comparable kinetic parameters to those observed for 4-OD in the wild-type complex, but is devoid of any detectable hydratase activity. Decarboxylation of (5S)-2-oxo-3-[5-D]hexenedioate by either the 4-OD/VPH complex or the mutant complex generates 2-hydroxy-2,4E-[5-D]pentadienoate in D(2)O. Ketonization of 2-hydroxy-2,4-pentadienoate by the wild-type complex is highly stereoselective and results in the formation of 2-oxo-(3S)-[3-D]-4-pentenoate, while the mutant complex generates a racemic mixture. These results indicate that 2-hydroxy-2, 4-pentadienoate is the product of 4-OD and that 2-oxo-4-pentenoate results from a VPH-catalyzed process. On this basis, the previously proposed hypothesis for the conversion of 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate has been revised [Lian, H., and Whitman, C. P. (1994) J. Am. Chem. Soc. 116, 10403-10411]. Finally, the observed (13)C kinetic isotope effect on the decarboxylation of 2-oxo-3-hexenedioate by the 4-OD/VPH complex suggests that the decarboxylation step is nearly rate-limiting. Because the value is not sensitive to either magnesium or manganese, it is likely that the transition state for carbon-carbon bond cleavage is late and that the metal positions the substrate and polarizes the carbonyl group, analogous to its role in oxalacetate decarboxylase.

  6. Structural determinants for the inhibitory ligands of orotidine-5′-monophosphate decarboxylase

    SciTech Connect

    Meza-Avina, Maria Elena; Wei, Lianhu; Liu, Yan; Poduch, Ewa; Bello, Angelica M.; Mishra, Ram K.; Pai, Emil F.; Kotra, Lakshmi P.

    2010-06-14

    In recent years, orotidine-5{prime}-monophosphate decarboxylase (ODCase) has gained renewed attention as a drug target. As a part of continuing efforts to design novel inhibitors of ODCase, we undertook a comprehensive study of potent, structurally diverse ligands of ODCase and analyzed their structural interactions in the active site of ODCase. These ligands comprise of pyrazole or pyrimidine nucleotides including the mononucleotide derivatives of pyrazofurin, barbiturate ribonucleoside, and 5-cyanouridine, as well as, in a computational approach, 1,4-dihydropyridine-based non-nucleoside inhibitors such as nifedipine and nimodipine. All these ligands bind in the active site of ODCase exhibiting distinct interactions paving the way to design novel inhibitors against this interesting enzyme. We propose an empirical model for the ligand structure for rational modifications in new drug design and potentially new lead structures.

  7. Maternal immune activation alters glutamic acid decarboxylase-67 expression in the brains of adult rat offspring

    PubMed Central

    Cassella, Sarah N.; Hemmerle, Ann M.; Lundgren, Kerstin H.; Kyser, Tara L.; Ahlbrand, Rebecca; Bronson, Stefanie L.; Richtand, Neil M.; Seroogy, Kim B.

    2016-01-01

    Activation of the maternal innate immune system, termed “maternal immune activation” (MIA), represents a common environmental risk factor for schizophrenia. Whereas evidence suggests dysregulation of GABA systems may underlie the pathophysiology of schizophrenia, a role for MIA in alteration of GABAergic systems is less clear. Here, pregnant rats received either the viral mimetic polyriboinosinic-polyribocytidilic acid or vehicle injection on gestational day 14. Glutamic acid decarboxylase-67 (GAD67) mRNA expression was examined in male offspring at postnatal day (P)14, P30 and P60. At P60, GAD67 mRNA was elevated in hippocampus and thalamus and decreased in prefrontal cortex of MIA offspring. MIA-induced alterations in GAD expression could contribute to the pathophysiology of schizophrenia. PMID:26830319

  8. Crystallization and preliminary X-ray analysis of the inducible lysine decarboxylase from Escherichia coli

    SciTech Connect

    Alexopoulos, E.; Kanjee, U.; Snider, J.; Houry, W.A.; Pai, E.F.

    2010-02-11

    The decameric inducible lysine decarboxylase (LdcI) from Escherichia coli has been crystallized in space groups C2 and C222{sub 1}; the Ta{sub 6}Br{sub 12}{sup 2+} cluster was used to derivatize the C2 crystals. The method of single isomorphous replacement with anomalous scattering (SIRAS) as implemented in SHELXD was used to solve the Ta{sub 6}Br{sub 12}{sup 2+}-derivatized structure to 5 {angstrom} resolution. Many of the Ta{sub 6}Br{sub 12}{sup 2+}-binding sites had twofold and fivefold noncrystallographic symmetry. Taking advantage of this feature, phase modification was performed in DM. The electron-density map of LdcI displays many features in agreement with the low-resolution negative-stain electron-density map [Snider et al. (2006), J. Biol. Chem. 281, 1532-1546].

  9. Histidine decarboxylase deficiency causes Tourette syndrome: parallel findings in humans and mice

    PubMed Central

    Baldan, Lissandra Castellan; Rapanelli, Maximiliano; Crowley, Michael; Anderson, George M.; Loring, Erin; Gorczyca, Roxanne; Billingslea, Eileen; Wasylink, Suzanne; Panza, Kaitlyn E.; Ercan-Sencicek, A. Gulhan; Krusong, Kuakarun; Leventhal, Bennett L.; Ohtsu, Hiroshi; Bloch, Michael H.; Hughes, Zoë A.; Krystal, John H.; Mayes, Linda; de Araujo, Ivan; Ding, Yu-Shin; State, Matthew W.; Pittenger, Christopher

    2013-01-01

    Tourette syndrome (TS) is characterized by tics, sensorimotor gating deficiencies, and abnormalities of cortico-basal ganglia circuits. A mutation in histidine decarboxylase (Hdc), the key enzyme for the biosynthesis of histamine (HA), has been implicated as a rare genetic cause. Hdc knockout mice exhibited potentiated tic-like stereotypies, recapitulating core phenomenology of TS; these were mitigated by the dopamine D2 antagonist haloperidol, a proven pharmacotherapy, and by HA infusion into the brain. Prepulse inhibition was impaired in both mice and humans carrying Hdc mutations. HA infusion reduced striatal dopamine (DA) levels; in Hdc knockout mice, striatal DA was increased and the DA-regulated immediate early gene Fos was upregulated. Dopamine D2/D3 receptor binding was altered both in mice and in humans carrying the Hdc mutation. These data confirm HDC deficiency as a rare cause of TS and identify histamine-dopamine interactions in the basal ganglia as an important locus of pathology. PMID:24411733

  10. Extralimbic autoimmune encephalitis associated with glutamic acid decarboxylase antibodies: an underdiagnosed entity?

    PubMed

    Najjar, Souhel; Pearlman, Daniel; Najjar, Amanda; Ghiasian, Vahid; Zagzag, David; Devinsky, Orrin

    2011-07-01

    Nonparaneoplastic glutamic acid decarboxylase antibody (GADAb)-related autoimmune encephalitis is a syndrome characterized by refractory seizures, progressive cognitive deficits, and psychiatric manifestations. The limbic subtype is well described, has characteristic affective and memory disturbances, and typical mesial temporal MRI abnormalities. We found only one single case report of the extralimbic subtype. We report clinical, radiological, and pathological findings of two additional cases with contrast-enhancing lesions. One of our cases presented as vasculitis, and the other imitated a tumor. Pathological evidence of both vasculitis and encephalitis has never been previously reported in any inflammatory condition affecting the brain. Our cases confirm prior reports that immune therapy can better control seizures associated with GADAb autoimmune encephalitis, and support the rationale for assaying for GADAb titers in patients with etiologically unclear extralimbic lesions and refractory epilepsy, independent of seizure types.

  11. Identification and molecular cloning of glutamate decarboxylase gene from Lactobacillus casei

    PubMed Central

    Tavakoli, Yasaman; Esmaeili, Abolghasem; Rabbani, Mohammad

    2015-01-01

    Gamma-amino butyric acid (GABA) possesses several physiological functions such as neurotransmission, induction of hypotension, diuretic and tranquilizer effects. Production of GABA-enriched products by lactic acid bacteria has been a focus of different researches in recent years because of their safety and health-promoting specifities. In this study, glutamate decarboxylase (gad) gene of a local strains Lactobacillus casei was identified and cloned. In order to clone the gad gene from this strain, the PCR was carried out using primers designed based on conserved regions. The PCR product was purified and ligated into PGEM-T vector. Comparison of obtained sequences shows that this fragment codes the pyridoxal 5′-phosphate binding region. This strain could possibly be used for the industrial GABA production and also for development of functional fermented foods. Gad gene manipulation can also either decrease or increase the activity of enzyme in bacteria. PMID:27844008

  12. Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase.

    PubMed

    Versées, Wim; Spaepen, Stijn; Wood, Martin D H; Leeper, Finian J; Vanderleyden, Jos; Steyaert, Jan

    2007-11-30

    Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

  13. Some Aspects of Yeast Anaerobic Metabolism Examined by the Inhibition of Pyruvate Decarboxylase

    NASA Astrophysics Data System (ADS)

    Martin, Earl V.

    1998-10-01

    Incubation of yeast cells with various sugars in aqueous alkaline phosphate solutions under anaerobic conditions results in the accumulation of pyruvate in the cell medium after short periods of up to 15 minutes. This accumulation of pyruvate as the end product of glycolysis results from the inhibition of pyruvate decarboxylase under the conditions. This pyruvate production can be readily measured in the cell-free medium by a spectrophotometric assay using lactic dehydrogenase and NADH. The production of pyruvate can be directly related to the ability of the yeast cells to metabolize particular carbon sources provided. Comparison of pyruvate production by yeast from a variety of common sugars, for example, provides students with a means to assess what sugars are readily utilized by this organism. An additional advantage for student laboratory studies is the availability of Sacchromyces cerevisiae at minimal cost as dry granules which are easily weighed and quickly activated.

  14. Improvement of the Process Stability of Arylmalonate Decarboxylase by Immobilization for Biocatalytic Profen Synthesis

    PubMed Central

    Aßmann, Miriam; Mügge, Carolin; Gaßmeyer, Sarah Katharina; Enoki, Junichi; Hilterhaus, Lutz; Kourist, Robert; Liese, Andreas; Kara, Selin

    2017-01-01

    The enzyme arylmalonate decarboxylase (AMDase) enables the selective synthesis of enantiopure (S)-arylpropinates in a simple single-step decarboxylation of dicarboxylic acid precursors. However, the poor enzyme stability with a half-life time of about 1.2 h under process conditions is a serious limitation of the productivity, which results in a need for high catalyst loads. By immobilization on an amino C2 acrylate carrier the operational stability of the (S)-selective AMDase variant G74C/M159L/C188G/V43I/A125P/V156L was increased to a half-life of about 8.6 days, which represents a 158-fold improvement. Further optimization was achieved by simple immobilization of the cell lysate to eliminate the cost- and time intensive enzyme purification step. PMID:28360905

  15. Opsoclonus-myoclonus-ataxia syndrome with autoantibodies to glutamic acid decarboxylase.

    PubMed

    Markakis, Ioannis; Alexiou, Eleni; Xifaras, Michael; Gekas, Georgios; Rombos, Antonios

    2008-06-01

    Opsoclonus-myoclonus-ataxia syndrome (OMS) is a rare neurological disorder of probably autoimmune origin. Most cases are associated with a remote neoplasm or a viral infection; however in some instances no underlying aetiology can be demonstrated. We report the presence of anti-glutamic acid decarboxylase antibodies (anti-GAD Abs) in the serum and CSF of a patient with idiopathic OMS. Treatment with intravenous immunoglobulin led to a remarkable clinical improvement with parallel reduction of anti-GAD titers. Anti-GAD Abs have been associated with several neurological syndromes. They could also be responsible for the clinical triad of OMS, by impairing GABAergic transmission in specific brainstem and cerebellar circuits. We propose that testing for anti-GAD Abs should be performed in OMS, especially when no other aetiological association can be demonstrated.

  16. The importance of SERINE DECARBOXYLASE1 (SDC1) and ethanolamine biosynthesis during embryogenesis of Arabidopsis thaliana.

    PubMed

    Yunus, Ian Sofian; Liu, Yu-Chi; Nakamura, Yuki

    2016-11-01

    In plants, ethanolamine is considered a precursor for the synthesis of choline, which is an essential dietary nutrient for animals. An enzyme serine decarboxylase (SDC) has been identified and characterized in Arabidopsis, which directly converts serine to ethanolamine, a precursor to phosphorylethanolamine and its subsequent metabolites in plants. However, the importance of SDC and ethanolamine production in plant growth and development remains unclear. Here, we show that SDC is required for ethanolamine biosynthesis in vivo and essential in plant embryogenesis in Arabidopsis. The knockout of SDC1 caused an embryonic lethal defect due to the developmental arrest of the embryos at the heart stage. During embryo development, the expression was observed at the later stages, at which developmental defect occurred in the knockout mutant. Overexpression of SDC1 in planta increased levels of ethanolamine, phosphatidylethanolamine, and phosphatidylcholine both in leaves and siliques. These results suggest that SDC1 plays an essential role in ethanolamine biosynthesis during the embryogenesis in Arabidopsis.

  17. Regioselective Enzymatic β-Carboxylation of para-Hydroxy- styrene Derivatives Catalyzed by Phenolic Acid Decarboxylases

    PubMed Central

    Wuensch, Christiane; Pavkov-Keller, Tea; Steinkellner, Georg; Gross, Johannes; Fuchs, Michael; Hromic, Altijana; Lyskowski, Andrzej; Fauland, Kerstin; Gruber, Karl; Glueck, Silvia M; Faber, Kurt

    2015-01-01

    We report on a ‘green’ method for the utilization of carbon dioxide as C1 unit for the regioselective synthesis of (E)-cinnamic acids via regioselective enzymatic carboxylation of para-hydroxystyrenes. Phenolic acid decarboxylases from bacterial sources catalyzed the β-carboxylation of para-hydroxystyrene derivatives with excellent regio- and (E/Z)-stereoselectivity by exclusively acting at the β-carbon atom of the C=C side chain to furnish the corresponding (E)-cinnamic acid derivatives in up to 40% conversion at the expense of bicarbonate as carbon dioxide source. Studies on the substrate scope of this strategy are presented and a catalytic mechanism is proposed based on molecular modelling studies supported by mutagenesis of amino acid residues in the active site. PMID:26190963

  18. Genetic Confirmation of the Role of Sulfopyruvate Decarboxylase in Coenzyme M Biosynthesis in Methanococcus maripaludis

    DOE PAGES

    Sarmiento, Felipe; Ellison, Courtney K.; Whitman, William B.

    2013-01-01

    Coenzyme M is an essential coenzyme for methanogenesis. The proposed biosynthetic pathway consists of five steps, of which the fourth step is catalyzed by sulfopyruvate decarboxylase (ComDE). Disruption of the gene comE by transposon mutagenesis resulted in a partial coenzyme M auxotroph, which grew poorly in the absence of coenzyme M and retained less than 3% of the wild type level of coenzyme M biosynthesis. Upon coenzyme M addition, normal growth of the mutant was restored. Moreover, complementation of the mutation with the wild type comE gene in trans restored full growth in the absence of coenzyme M. Thesemore » results confirm that ComE plays an important role in coenzyme M biosynthesis. The inability to yield a complete CoM auxotroph suggests that either the transposon insertion failed to completely inactivate the gene or M. maripaludis possesses a promiscuous activity that partially complemented the mutation.« less

  19. Hydroxydibenzoylmethane induces apoptosis through repressing ornithine decarboxylase in human promyelocytic leukemia HL-60 cells

    PubMed Central

    Wang, Ming-Fu; Liao, Ya-Fan; Hung, Ying-Cheng; Lin, Chih-Li; Hour, Tzyh-Chyuan; Lue, Ko-Huang; Hung, Hui-Chih

    2011-01-01

    Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Δψm), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway. PMID:21372632

  20. Glutamate alteration of glutamic acid decarboxylase (GAD) in GABAergic neurons: the role of cysteine proteases.

    PubMed

    Monnerie, Hubert; Le Roux, Peter D

    2008-09-01

    Brain cell vulnerability to neurologic insults varies greatly, depending on their neuronal subpopulation. Among cells that survive a pathological insult such as ischemia or brain trauma, some may undergo morphological and/or biochemical changes that could compromise brain function. We previously reported that surviving cortical GABAergic neurons exposed to glutamate in vitro displayed an NMDA receptor (NMDAR)-mediated alteration in the levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) [Monnerie, H., Le Roux, P., 2007. Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro. Exp. Neurol. 205, 367-382]. In this study, we examined the mechanisms by which glutamate excitotoxicity caused a change in cortical GABAergic neurons' GAD protein levels. Removing extracellular calcium prevented the NMDAR-mediated decrease in GAD protein levels, measured using Western blot techniques, whereas inhibiting calcium entry through voltage-gated calcium channels had no effect. Glutamate's effect on GAD protein isoforms was significantly attenuated by preincubation with the cysteine protease inhibitor N-Acetyl-L-Leucyl-L-Leucyl-L-norleucinal (ALLN). Using class-specific protease inhibitors, we observed that ALLN's effect resulted from the blockade of calpain and cathepsin protease activities. Cell-free proteolysis assay confirmed that both proteases were involved in glutamate-induced alteration in GAD protein levels. Together these results suggest that glutamate-induced excitotoxic stimulation of NMDAR in cultured cortical neurons leads to altered GAD protein levels from GABAergic neurons through intracellular calcium increase and protease activation including calpain and cathepsin. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation

  1. [Cloning, prokaryotic expression and characterization of lysine decarboxylase gene from Huperzia serrata].

    PubMed

    Di, Ci; Li, Jing; Tang, Yuntao; Peng, Qingzhong

    2014-08-01

    Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD 19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 μg/mL IPTG at 30 degrees C, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 degrees C and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures.

  2. Evaluation of the Substrate Scope of Benzoic Acid (De)carboxylases According to Chemical and Biochemical Parameters.

    PubMed

    Pesci, Lorenzo; Kara, Selin; Liese, Andreas

    2016-10-04

    The enzymatic carboxylation of phenolic compounds has been attracting increasing interest in recent years, owing to its regioselectivity and technical potential as a biocatalytic equivalent for the Kolbe-Schmitt reaction. Mechanistically the reaction was demonstrated to occur through electrophilic aromatic substitution/water elimination with bicarbonate as a cosubstrate. The effects of the substituents on the phenolic ring have not yet been elucidated in detail, but this would give detailed insight into the substrate-activity relationship and would provide predictability for the acceptance of future substrates. In this report we show how the kinetic and (apparent) thermodynamic behavior can be explained through the evaluation of linear free energy relationships based on electronic, steric, and geometric parameters and through the consideration of enzyme-ligand interactions. Moreover, the similarity between the benzoic acid decarboxylases and the amidohydrolases superfamily is investigated, and promiscuous hydrolytic activity of the decarboxylase in the context of the hydrolysis of an activated ester bond has been established.

  3. Threonine 57 is required for the post-translational activation of Escherichia coli aspartate α-decarboxylase

    PubMed Central

    Webb, Michael E.; Yorke, Briony A.; Kershaw, Tom; Lovelock, Sarah; Lobley, Carina M. C.; Kilkenny, Mairi L.; Smith, Alison G.; Blundell, Tom L.; Pearson, Arwen R.; Abell, Chris

    2014-01-01

    Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formed via the intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation. PMID:24699660

  4. Threonine 57 is required for the post-translational activation of Escherichia coli aspartate α-decarboxylase.

    PubMed

    Webb, Michael E; Yorke, Briony A; Kershaw, Tom; Lovelock, Sarah; Lobley, Carina M C; Kilkenny, Mairi L; Smith, Alison G; Blundell, Tom L; Pearson, Arwen R; Abell, Chris

    2014-04-01

    Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formed via the intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

  5. Computational, structural, and kinetic evidence that Vibrio vulnificus FrsA is not a cofactor-independent pyruvate decarboxylase.

    PubMed

    Kellett, Whitney F; Brunk, Elizabeth; Desai, Bijoy J; Fedorov, Alexander A; Almo, Steven C; Gerlt, John A; Rothlisberger, Ursula; Richards, Nigel G J

    2013-03-19

    The fermentation-respiration switch (FrsA) protein in Vibrio vulnificus was recently reported to catalyze the cofactor-independent decarboxylation of pyruvate. We now report quantum mechanical/molecular mechenical calculations that examine the energetics of C-C bond cleavage for a pyruvate molecule bound within the putative active site of FrsA. These calculations suggest that the barrier to C-C bond cleavage in the bound substrate is 28 kcal/mol, which is similar to that estimated for the uncatalyzed decarboxylation of pyruvate in water at 25 °C. In agreement with the theoretical predictions, no pyruvate decarboxylase activity was detected for recombinant FrsA protein that could be crystallized and structurally characterized. These results suggest that the functional annotation of FrsA as a cofactor-independent pyruvate decarboxylase is incorrect.

  6. Computational, Structural and Kinetic Evidence that Vibrio vulnificus FrsA is not a Cofactor-Independent Pyruvate Decarboxylase

    PubMed Central

    Kellett, Whitney F.; Brunk, Elizabeth; Desai, Bijoy J.; Fedorov, Alexander A.; Almo, Steven C.; Gerlt, John A.; Rothlisberger, Ursula; Richards, Nigel G. J.

    2013-01-01

    The fermentation-respiration switch (FrsA) protein in Vibrio vulnificus was recently reported to catalyze the cofactor-independent decarboxylation of pyruvate. We now report QM/MM calculations that examine the energetics of C-C bond cleavage for a pyruvate molecule bound within the putative active site of FrsA. These calculations suggest that the barrier to C-C bond cleavage in the bound substrate is 28 kcal/mol, which is similar to that estimated for the uncatalyzed decarboxylation of pyruvate in water at 25 °C. In agreement with the theoretical predictions, no pyruvate decarboxylase activity was detected for recombinant FrsA protein that could be crystallized and structurally characterized. These results suggest that the functional annotation of FrsA as a cofactor-independent pyruvate decarboxylase is incorrect. PMID:23452154

  7. /sup 3/H-DFMA metabolism in tobacco: non-specific, arginase mediated inhibition of ornithine decarboxylase activity

    SciTech Connect

    Slocum, R.D.; Feirer, R.L.

    1987-04-01

    ..cap alpha..-Difluoromethylarginine (DFMA) is a specific, enzyme-activated, irreversible inhibit of arginine decarboxylase (ADC) in vitro. ADC catalyzes the first step leading to putrescine biosynthesis and the activity of this enzyme is closely linked to overall polyamine (PA) biosynthesis in non-meristematic plant tissues. Consequently, ADC represents an important target enzyme for inhibitors of PA metabolism. DFMA has been shown to inhibit ADC activity in a variety of tissues in vivo but its specificity in tobacco was questioned since ornithine decarboxylase (ODC) activity was also inhibited. Further studies have shown that (/sup 3/H)-DFMA is efficiently hydrolyzed in tobacco to (/sup 3/H)-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. Tobacco and bovine arginases also catalyze the hydrolysis of DFMA in vitro, suggesting a role for this enzyme in mediating the non-specific inhibition of ODC by DFMA in tobacco flowers.

  8. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

    PubMed Central

    Dalton, Heidi L.; Blomstedt, Cecilia K.; Neale, Alan D.; Gleadow, Ros; DeBoer, Kathleen D.; Hamill, John D.

    2016-01-01

    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana. PMID

  9. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

    PubMed

    Dalton, Heidi L; Blomstedt, Cecilia K; Neale, Alan D; Gleadow, Ros; DeBoer, Kathleen D; Hamill, John D

    2016-05-01

    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana.

  10. Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola.

    PubMed Central

    Brandl, M T; Lindow, S E

    1996-01-01

    Erwinia herbicola 299R synthesizes indole-3-acetic acid (IAA) primarily by the indole-3-pyruvic acid pathway. A gene involved in the biosynthesis of IAA was cloned from strain 299R. This gene (ipdC) conferred the synthesis of indole-3-acetaldehyde and tryptophol upon Escherichia coli DH5 alpha in cultures supplemented with L-tryptophan. The deduced amino acid sequence of the gene product has high similarity to that of the indolepyruvate decarboxylase of Enterobacter cloacae. Regions within pyruvate decarboxylases of various fungal and plant species also exhibited considerable homology to portions of this gene. This gene therefore presumably encodes an indolepyruvate decarboxylase (IpdC) which catalyzes the conversion of indole-3-pyruvic acid to indole-3-acetaldehyde. Insertions of Tn3-spice within ipdC abolished the ability of strain 299R to synthesize indole-3-acetaldehyde and tryptophol and reduced its IAA production in tryptophan-supplemented minimal medium by approximately 10-fold, thus providing genetic evidence for the role of the indolepyruvate pathway in IAA synthesis in this strain. An ipdC probe hybridized strongly with the genomic DNA of all E. herbicola strains tested in Southern hybridization studies, suggesting that the indolepyruvate pathway is common in this species. Maximum parsimony analysis revealed that the ipdC gene is highly conserved within this group and that strains of diverse geographic origin were very similar with respect to ipdC. PMID:8900003

  11. Lysine Decarboxylase Catalyzes the First Step of Quinolizidine Alkaloid Biosynthesis and Coevolved with Alkaloid Production in Leguminosae[W][OA

    PubMed Central

    Bunsupa, Somnuk; Katayama, Kae; Ikeura, Emi; Oikawa, Akira; Toyooka, Kiminori; Saito, Kazuki; Yamazaki, Mami

    2012-01-01

    Lysine decarboxylase (LDC) catalyzes the first-step in the biosynthetic pathway of quinolizidine alkaloids (QAs), which form a distinct, large family of plant alkaloids. A cDNA of lysine/ornithine decarboxylase (L/ODC) was isolated by differential transcript screening in QA-producing and nonproducing cultivars of Lupinus angustifolius. We also obtained L/ODC cDNAs from four other QA-producing plants, Sophora flavescens, Echinosophora koreensis, Thermopsis chinensis, and Baptisia australis. These L/ODCs form a phylogenetically distinct subclade in the family of plant ornithine decarboxylases. Recombinant L/ODCs from QA-producing plants preferentially or equally catalyzed the decarboxylation of l-lysine and l-ornithine. L. angustifolius L/ODC (La-L/ODC) was found to be localized in chloroplasts, as suggested by the transient expression of a fusion protein of La-L/ODC fused to the N terminus of green fluorescent protein in Arabidopsis thaliana. Transgenic tobacco (Nicotiana tabacum) suspension cells and hairy roots produced enhanced levels of cadaverine-derived alkaloids, and transgenic Arabidopsis plants expressing (La-L/ODC) produced enhanced levels of cadaverine, indicating the involvement of this enzyme in lysine decarboxylation to form cadaverine. Site-directed mutagenesis and protein modeling studies revealed a structural basis for preferential LDC activity, suggesting an evolutionary implication of L/ODC in the QA-producing plants. PMID:22415272

  12. Lysine decarboxylase catalyzes the first step of quinolizidine alkaloid biosynthesis and coevolved with alkaloid production in leguminosae.

    PubMed

    Bunsupa, Somnuk; Katayama, Kae; Ikeura, Emi; Oikawa, Akira; Toyooka, Kiminori; Saito, Kazuki; Yamazaki, Mami

    2012-03-01

    Lysine decarboxylase (LDC) catalyzes the first-step in the biosynthetic pathway of quinolizidine alkaloids (QAs), which form a distinct, large family of plant alkaloids. A cDNA of lysine/ornithine decarboxylase (L/ODC) was isolated by differential transcript screening in QA-producing and nonproducing cultivars of Lupinus angustifolius. We also obtained L/ODC cDNAs from four other QA-producing plants, Sophora flavescens, Echinosophora koreensis, Thermopsis chinensis, and Baptisia australis. These L/ODCs form a phylogenetically distinct subclade in the family of plant ornithine decarboxylases. Recombinant L/ODCs from QA-producing plants preferentially or equally catalyzed the decarboxylation of L-lysine and L-ornithine. L. angustifolius L/ODC (La-L/ODC) was found to be localized in chloroplasts, as suggested by the transient expression of a fusion protein of La-L/ODC fused to the N terminus of green fluorescent protein in Arabidopsis thaliana. Transgenic tobacco (Nicotiana tabacum) suspension cells and hairy roots produced enhanced levels of cadaverine-derived alkaloids, and transgenic Arabidopsis plants expressing (La-L/ODC) produced enhanced levels of cadaverine, indicating the involvement of this enzyme in lysine decarboxylation to form cadaverine. Site-directed mutagenesis and protein modeling studies revealed a structural basis for preferential LDC activity, suggesting an evolutionary implication of L/ODC in the QA-producing plants.

  13. Herbacetin is a novel allosteric inhibitor of ornithine decarboxylase with antitumor activity

    PubMed Central

    Lee, Mee-Hyun; Oi, Naomi; Lim, Do Young; Kim, Myoung Ok; Cho, Young-Yeon; Pugliese, Angelo; Shim, Jung-Hyun; Chen, Hanyong; Cho, Eun Jin; Kim, Jong-Eun; Kang, Sun Chul; Paul, Souren; Kang, Hee Eun; Jung, Ji Won; Lee, Sung-Young; Kim, Sung-Hyun; Reddy, Kanamata; Yeom, Young Il; Bode, Ann M; Dong, Zigang

    2015-01-01

    Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the first step of polyamine biosynthesis that is associated with cell growth and tumor formation. Existing catalytic inhibitors of ODC have lacked efficacy in clinical testing or displayed unacceptable toxicity. In this study, we report the identification of an effective and nontoxic allosteric inhibitor of ODC. Using computer docking simulation and an in vitro ODC enzyme assay, we identified herbacetin, a natural compound found in flax and other plants, as a novel ODC inhibitor. Mechanistic investigations defined aspartate 44 in ODC as critical for binding. Herbacetin exhibited potent anticancer activity in colon cancer cell lines expressing high levels of ODC. Intraperitoneal or oral administration of herbacetin effectively suppressed HCT116 xenograft tumor growth and also reduced the number and size of polyps in a mouse model of APC-driven colon cancer (ApcMin/+). Unlike the well established ODC inhibitor DFMO, herbacetin treatment was not associated with hearing loss. Taken together, our findings defined the natural product herbacetin as an allosteric inhibitor of ODC with chemopreventive and antitumor activity in preclinical models of colon cancer, prompting its further investigation in clinical trials. PMID:26676750

  14. Structural requirements for novel coenzyme-substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation.

    PubMed

    Wu, Fang; Gehring, Heinz

    2009-02-01

    Creating transition-state mimics has proven to be a powerful strategy in developing inhibitors to treat malignant diseases in several cases. In the present study, structurally diverse coenzyme-substrate derivatives mimicking this type for pyridoxal 5'-phosphate-dependent human ornithine decarboxylase (hODC), a potential anticancer target, were designed, synthesized, and tested to elucidate the structural requirements for optimal inhibition of intracellular ODC as well as of tumor cell proliferation. Of 23 conjugates, phosphopyridoxyl- and pyridoxyl-L-tryptophan methyl ester (pPTME, PTME) proved significantly more potent in suppression proliferation (IC(50) up to 25 microM) of glioma cells (LN229) than alpha-DL-difluoromethylornithine (DFMO), a medically used irreversible inhibitor of ODC. In agreement with molecular modeling predictions, the inhibitory action of pPTME and PTME toward intracellular ODC of LN229 cells exceeded that of the previous designed lead compound POB. The inhibitory active compounds feature hydrophobic side chain fragments and a kind of polyamine motif (-NH-(CH(X))(4)-NH-). In addition, they induce, as polyamine analogs often do, the activity of the polyamine catabolic enzymes polyamine oxidase and spermine/spermidine N(1)-acetyltransferase up to 250 and 780%, respectively. The dual-action mode of these compounds in LN229 cells affects the intracellular polyamine metabolism and might underlie the more favorable cell proliferation inhibition in comparison with DFMO.

  15. Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme.

    PubMed

    Torrens-Spence, Michael P; Gillaspy, Glenda; Zhao, Bingyu; Harich, Kim; White, Robert H; Li, Jianyong

    2012-02-10

    Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46mM) and dopa (km 1.40mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine.

  16. Enhanced histamine production through the induction of histidine decarboxylase expression by phorbol ester in Jurkat cells.

    PubMed

    Nagashima, Yusuke; Kako, Koichiro; Kim, Jun-Dal; Fukamizu, Akiyoshi

    2012-11-01

    Histamine (HA), a mediator of inflammation, type I allergic responses and neurotransmission, is synthesized from L-histidine, the reaction of which is catalyzed by histidine decarboxylase (HDC). HDC has been reported to be induced by various stimuli, not only in mast cells and basophils, but also in T lymphocytes and macrophages. Although its mRNA has been shown to be increased in Jurkat cells when treated with phorbol 12-myristate 13-acetate (TPA), little is known concerning the induced production of HA by HDC. The present study quantified the trace amounts of intracellular HA using ultra-high liquid chromatography in combination with the 6-aminoquinoline carbamate-derivatization technique. To test whether the cellular level of HA is elevated by the induction of HDC in Jurkat cells treated with TPA, the peak corresponding to authentic HA in the cell lysate was fractioned and its molecular weight determined by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry. The results of this study show that the HA level is increased by the induction of HDC expression by TPA in Jurkat cells. Therefore, this method is useful in elucidating the physiological significance of HA production.

  17. Multiple promoter elements govern expression of the human ornithine decarboxylase gene in colon carcinoma cells.

    PubMed Central

    Moshier, J A; Osborne, D L; Skunca, M; Dosescu, J; Gilbert, J D; Fitzgerald, M C; Polidori, G; Wagner, R L; Friezner Degen, S J; Luk, G D

    1992-01-01

    Overexpression of the ornithine decarboxylase (ODC) gene may be important to the development and maintenance of colonic neoplasms, as well as tumors in general. In this study, we examined the promoter elements governing constitutive expression of the human ODC gene in HCT 116 human colon carcinoma cells and, for comparison, K562 human erythro-leukemia cells. It was determined by functional analysis that the promoter elements responsible reside within the 378 bp immediately upstream from the transcription start site. Within this sequence, there are at least three regions that modulate the efficiency of the ODC promoter cooperatively. Both DNA bandshift and footprint assays demonstrated all three regions to be rich in sites that bind to nuclear proteins isolated from HCT 116 and K562 cells; the protein binding pattern of non-transformed, diploid fibroblasts was found to be much less complex. Several of the protein binding sequences have little or no homology to common regulatory elements. We suggest that the constitutive activity of the ODC gene in HCT 116 colon carcinoma cells, and perhaps transformed cells in general, involves a complex interaction of multiple regulatory sequences and their associated nuclear proteins. Finally, the saturation of the promoter in these transformed cell lines suggests that high levels of protein binding in the ODC promoter may contribute to elevated constitutive expression of this gene. Images PMID:1598217

  18. Characterization of the activity and expression of arginine decarboxylase in human and animal Chlamydia pathogens.

    PubMed

    Bliven, Kimberly A; Fisher, Derek J; Maurelli, Anthony T

    2012-12-01

    Chlamydia pneumoniae encodes a functional arginine decarboxylase (ArgDC), AaxB, that activates upon self-cleavage and converts l-arginine to agmatine. In contrast, most Chlamydia trachomatis serovars carry a missense or nonsense mutation in aaxB abrogating activity. The G115R missense mutation was not predicted to impact AaxB functionality, making it unclear whether AaxB variations in other Chlamydia species also result in enzyme inactivation. To address the impact of gene polymorphism on functionality, we investigated the activity and production of the Chlamydia AaxB variants. Because ArgDC plays a critical role in the Escherichia coli acid stress response, we studied the ability of these Chlamydia variants to complement an E. coli ArgDC mutant in an acid shock assay. Active AaxB was detected in four additional species: Chlamydia caviae, Chlamydia pecorum, Chlamydia psittaci, and Chlamydia muridarum. Of the C. trachomatis serovars, only E appears to encode active enzyme. To determine when functional enzyme is present during the chlamydial developmental cycle, we utilized an anti-AaxB antibody to detect both uncleaved and cleaved enzyme throughout infection. Uncleaved enzyme production peaked around 20 h postinfection, with optimal cleavage around 44 h. While the role ArgDC plays in Chlamydia survival or virulence is unclear, our data suggest a niche-specific function.

  19. Aspartate Decarboxylase is Required for a Normal Pupa Pigmentation Pattern in the Silkworm, Bombyx mori

    PubMed Central

    Dai, Fangyin; Qiao, Liang; Cao, Cun; Liu, Xiaofan; Tong, Xiaoling; He, Songzhen; Hu, Hai; Zhang, Li; Wu, Songyuan; Tan, Duan; Xiang, Zhonghuai; Lu, Cheng

    2015-01-01

    The pigmentation pattern of Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of β-alanine and N-β-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of β-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera. PMID:26077025

  20. Ornithine decarboxylase or gamma-glutamylcysteine synthetase overexpression protects Leishmania (Vianna) guyanensis against antimony.

    PubMed

    Fonseca, Maisa S; Comini, Marcelo A; Resende, Bethânia V; Santi, Ana Maria M; Zoboli, Antônio P; Moreira, Douglas S; Murta, Silvane M F

    2017-04-01

    Trypanosomatids present a unique mechanism for detoxification of peroxides that is dependent on trypanothione (bisglutathionylspermidine). Ornithine decarboxylase (ODC) and γ-glutamylcysteine synthetase (GSH1) produce molecules that are direct precursors of trypanothione. In this study, Leishmania guyanensis odc and gsh1 overexpressor cell lines were generated to investigate the contribution of these genes to the trivalent antimony (Sb(III))-resistance phenotype. The ODC- or GSH1-overexpressors parasites presented an increase of two and four-fold in Sb(III)-resistance index, respectively, when compared with the wild-type line. Pharmacological inhibition of ODC and GSH1 with the specific inhibitors α-difluoromethylornithine (DFMO) and buthionine sulfoximine (BSO), respectively, increased the antileishmanial effect of Sb(III) in all cell lines. However, the ODC- and GSH1-overexpressor were still more resistant to Sb(III) than the parental cell line. Together, our data shows that modulation of ODC and GSH1 levels and activity is sufficient to affect L. guyanensis susceptibility to Sb(III), and confirms a role of these genes in the Sb(III)-resistance phenotype.

  1. Effect of cocaine, ethanol or nicotine on ornithine decarboxylase activity in early chick embryo brain.

    PubMed

    Beeker, K; Smith, C; Pennington, S

    1992-09-18

    Fetal drug exposure causes multiple deficits in the developing child. For both humans and animal models, the single most common drug-related problem is fetal growth suppression. This defect is associated with significant perinatal morbidity and mortality and may also be related to significant behavioral problems appearing later in life. Studies focussed on the molecular mechanism of fetal drug effects in placental models are complicated by multiple interactions of the drug with mother, placenta and fetus. Using early (76-168 h) chick embryos as a non-placental model, and three common drugs of abuse (nicotine, ethanol and cocaine) it was found that each drug suppressed the peak in fetal brain ornithine decarboxylase (ODC) activity that normally occurs at 120 h of development. For each drug, the decrease in ODC activity at 120 h was followed by a small but significant increase in ODC. Thus, although the drug-treated embryos were smaller in size, they appeared to be undergoing compensatory growth and, in fact, became equal in weight to the vehicle-treated animals, if allowed to hatch.

  2. Substrate shuttling between active sites of uroporphyrinogen decarboxylase is not required to generate coproporphyrinogen

    PubMed Central

    Phillips, John D.; Warby, Christy A.; Whitby, Frank G.; Kushner, James P.; Hill, Christopher P.

    2009-01-01

    Summary Uroporphyrinogen Decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of the four acetate side chains on the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer with the active site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single chain protein (scURO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposible with wild-type activity and have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of scURO-D resulted in approximately half of wild-type activity. The distribution of reaction intermediates was the same for mutant and wild-type sequences, and was unaltered in a competition experiment using the I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function, and suggest that the dimeric structure of URO-D is required to achieve conformational stability and create a large active site cleft. PMID:19362562

  3. Coexpression of pyruvate decarboxylase and alcohol dehydrogenase genes in Lactobacillus brevis.

    PubMed

    Liu, Siqing; Dien, Bruce S; Nichols, Nancy N; Bischoff, Kenneth M; Hughes, Stephen R; Cotta, Michael A

    2007-09-01

    Lactobacillus brevis ATCC367 was engineered to express pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) genes in order to increase ethanol fermentation from biomass-derived residues. First, a Gram-positive Sarcina ventriculi PDC gene (Svpdc) was introduced into L. brevis ATCC 367 to obtain L. brevis bbc03. The SvPDC was detected by immunoblot using an SvPDC oligo peptide antiserum, but no increased ethanol was detected in L. brevis bbc03. Then, an ADH gene from L. brevis (Bradh) was cloned behind the Svpdc gene that generated a pdc/adh-coupled ethanol cassette pBBC04. The pBBC04 restored anaerobic growth and conferred ethanol production of Escheirichia coli NZN111 (a fermentative defective strain incapable of growing anaerobically). Approximately 58 kDa (SvPDC) and 28 kDa (BrADH) recombinant proteins were observed in L. brevis bbc04. These results indicated that the Gram-positive ethanol production genes can be expressed in L. brevis using a Gram-positive promoter and pTRKH2 shuttle vector. This work provides evidence that expressing Gram-positive ethanol genes in pentose utilizing L. brevis will further aid manipulation of this microbe toward biomass to ethanol production.

  4. Tyrosine decarboxylase activity of Lactobacillus brevis IOEB 9809 isolated from wine and L. brevis ATCC 367.

    PubMed

    Moreno-Arribas, V; Lonvaud-Funel, A

    1999-11-01

    Tyramine, a frequent amine in wines, is produced from tyrosine by the tyrosine decarboxylase (TDC) activity of bacteria. The tyramine-producing strain Lactobacillus brevis IOEB 9809 isolated from wine and the reference strain L. brevis ATCC 367 were studied. At the optimum pH, 5.0, K(m) values of IOEB 9809 and ATCC 367 crude extracts for L-tyrosine were 0.58 mM and 0.67 mM, and V(max) was higher for the wine strain (115 U) than the ATCC 367 (66 U). TDC exhibited a preference for L-tyrosine over L-DOPA as substrate. Enzyme activity was pyridoxal-5'-phosphate (PLP)-dependent and it was stabilized by the substrate and coenzyme. In contrast, glycerol and beta-mercaptoethanol strongly inhibited TDC. Tyramine competitively inhibited TDC for both strains. Citric acid, lactic acid and ethanol had an inhibitory effect on cells and crude extracts, but none could inhibit TDC at the usual concentrations in wines.

  5. Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

    SciTech Connect

    Phillips, J.; Warby, C; Whitby, F; Kushner, J; Hill, C

    2009-01-01

    Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.

  6. Functional and conformational transitions of mevalonate diphosphate decarboxylase from Bacopa monniera.

    PubMed

    Abbassi, Shakeel; Patel, Krunal; Khan, Bashir; Bhosale, Siddharth; Gaikwad, Sushama

    2016-02-01

    Functional and conformational transitions of mevalonate diphosphate decarboxylase (MDD), a key enzyme of mevalonate pathway in isoprenoid biosynthesis, from Bacopa monniera (BmMDD), cloned and overexpressed in Escherichia coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and Circular dichroism spectroscopy. Native BmMDD is a helix dominant structure with 45% helix and 11% sheets and possesses seven tryptophan residues with two residues exposed on surface, three residues partially exposed and two situated in the interior of the protein. Thermal denaturation of BmMDD causes rapid structural transitions at and above 40°C and transient exposure of hydrophobic residues at 50°C, leading to aggregation of the protein. An acid induced molten globule like structure was observed at pH 4, exhibiting altered but compact secondary structure, distorted tertiary structure and exposed hydrophobic residues. The molten globule displayed different response at higher temperature and similar response to chemical denaturation as compared to the native protein. The surface tryptophans have predominantly positively charged amino acids around them, as indicated by higher KSV for KI as compared to that for CsCl. The native enzyme displayed two different lifetimes, τ1 (1.203±0.036 ns) and τ2 (3.473±0.12 ns) indicating two populations of tryptophan.

  7. Partial purification and characterization of a novel histidine decarboxylase from Enterobacter aerogenes DL-1.

    PubMed

    Zou, Yu; Hu, Wenzhong; Jiang, Aili; Tian, Mixia

    2015-08-18

    Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca(2+) and Mn(2+) showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver-Burk plot showed that K m and V max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.

  8. Induction of histidine decarboxylase in macrophages inhibited by the novel NF-{kappa}B inhibitor (-)-DHMEQ

    SciTech Connect

    Suzuki, Eriko Ninomiya, Yoko; Umezawa, Kazuo

    2009-02-06

    Histamine often causes inflammation, and this amine is produced by histidine decarboxylase (HDC). We found that (-)-DHMEQ, an NF-{kappa}B inhibitor, inhibited lipopolysaccharide (LPS)-induced histamine production and HDC induction in mouse macrophage cell line RAW264.7. However, as there is no {kappa}B site in the HDC promoter, we studied the mechanism of inhibition. Knockdown of the transcription factor C/EBP{beta} reduced the HDC expression in LPS-treated cells. (-)-DHMEQ inhibited the C/EBP{beta} transcriptional activity in a reporter assay and in an electrophoresis mobility shift assay. But it did not inhibit the in vitro binding of C/EBP{beta} to DNA. It also did not lower the nuclear amount of C/EBP{beta}. On the other hand, the addition of recombinant p65, a component of NF-{kappa}B, enhanced the activity of C/EBP{beta} acting as a cofactor in vitro. Then, we found that (-)-DHMEQ lowered the nuclear amount of p65. Thus, inhibition of the C/EBP{beta} activity by (-)-DHMEQ would be due to a reduction in the amount of nuclear p65, which has a co-activator activity for C/EBP{beta} that is essential for the HDC induction. (-)-DHMEQ may be useful as an anti-inflammatory agent by lowering the histamine production in the body.

  9. Renal ornithine decarboxylase activity, polyamines, and compensatory renal hypertrophy in the rat

    SciTech Connect

    Humphreys, M.H.; Etheredge, S.B.; Lin, Shanyan; Ribstein, J.; Marton, L.J. Univ. of California, San Francisco )

    1988-08-01

    The authors determined the role of ornithine decarboxylase (ODC) in compensatory renal hypertrophy (CRH) by relating renal ODC activity and polyamine content to kidney size, expressed as a percent of body weight, 1 wk after unilateral nephrectomy (UN). In normal rats, renal ODC activity increased after UN; 1 wk later the remaining kidney weight had increased. Renal concentration of putrescine, the product of ODC's decarboxylation of ornithine, was increased 3, 8, and 48 h after UN, but concentrations of polyamines synthesized later in the pathway, spermidine and spermine, were not appreciably affected. Pretreatment with difluoromethylornithine (DFMO), an irreversible inhibitor of ODC inhibited both base-line renal ODC activity and putrescine concentration as well as increases stimulated by UN, although concentrations of spermidine and spermine were not decreased. In hypophysectomized rats, both increased renal ODC activity and CRH occurred as well, indicating that these two consequences of UN do not require intact pituitary function. Thus stimulation of renal ODC activity and putrescine content do not appear critical to the process of CRH after UN.

  10. Aspartate beta-decarboxylase from Alcaligenes faecalis: carbon-13 kinetic isotope effect and deuterium exchange experiments

    SciTech Connect

    Rosenberg, R.M.; O'Leary, M.H.

    1985-03-26

    The authors have measured the /sup 13/C kinetic isotope effect at pH 4.0, 5.0, 6.0, and 6.5 and in D/sub 2/O at pH 5.0 and the rate of D-H exchange of the alpha and beta protons of aspartic acid in D/sub 2/O at pH 5.0 for the reaction catalyzed by the enzyme aspartate beta-decarboxylase from Alcaligenes faecalis. The /sup 13/C kinetic isotope effect, with a value of 1.0099 +/- 0.0002 at pH 5.0, is less than the intrinsic isotope effect for the decarboxylation step, indicating that the decarboxylation step is not entirely rate limiting. The authors have been able to estimate probable values of the relative free energies of the transition states of the enzymatic reaction up to and including the decarboxylation step from the /sup 13/C kinetic isotope effect and the rate of D-H exchange of alpha-H. The pH dependence of the kinetic isotope effect reflects the pKa of the pyridine nitrogen of the coenzyme pyridoxal 5'-phosphate but not that of the imine nitrogen. A mechanism is proposed for the exchange of aspartate beta-H that is consistent with the stereochemistry suggested earlier.

  11. Identification and characterization of barley mutants lacking glycine decarboxylase and carboxyl esterase activities

    SciTech Connect

    Blackwell, R.; Lewis, K.; Lea, P. )

    1990-05-01

    A barley mutant has been isolated, from a selection of fifty air-sensitive seed-lines, using a standard gel stain technique which lacks carboxyl esterase activity, but has normal levels of carbonic anhydrase. In addition, two barley mutants lacking the ability to convert glycine to serine in the mitochondria, have been characterized. Both plants accumulate glycine in air and are unable to metabolize ({sup 14}C)glycine in the short-term. When ({sup 14}C)glycine was supplied over 2h LaPr 85/55 metabolized 90%, whereas the second mutant (LaPr 87/30) metabolized 10%. Results indicate that the mutation in LaPr 85/55 is almost certainly in the glycine transporter into the mitochondrion. The mutation in LaPr 87/30 has been shown, using western blotting, to be in both the P and H proteins, two of four proteins which comprise glycine decarboxylase (P, H, T and L).

  12. Induction of an oxalate decarboxylase in the filamentous fungus Trametes versicolor by addition of inorganic acids.

    PubMed

    Zhu, Cui Xia; Hong, Feng

    2010-01-01

    In order to improve yields and to reduce the cost of oxalate decarboxylase (OxDC, EC 4.1.1.2), the induction of OxDC in the white-rot fungus Trametes versicolor was studied in this work. OxDC was induced by addition of inorganic acids including hydrochloric acid, sulfuric acid, and phosphoric acid to culture media. The results showed that all the acids could enhance OxDC expression. The activity of the acid-induced OxDC rose continuously. All of the OxDC volumetric activities induced by the inorganic acids were higher than 20.0 U/L and were two times higher than that obtained with oxalic acid. OxDC productivity was around 4.0 U*L(-1)*day(-1). The highest specific activity against total protein was 3.2 U/mg protein at day 8 after induction of sulfuric acid, and the specific activity against mycelial dry weight was 10.6 U/g at day 9 after induction of hydrochloric acid. The growth of mycelia was inhibited slightly when the pH values in culture media was around 2.5-3.0, while the growth was inhibited heavily when the pH was lower than 2.5.

  13. A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase.

    PubMed

    Yu, Kai; Hu, Sheng; Huang, Jun; Mei, Le-He

    2011-08-10

    A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of L-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pK(a) to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.

  14. Simultaneous Silencing of Two Arginine Decarboxylase Genes Alters Development in Arabidopsis

    PubMed Central

    Sánchez-Rangel, Diana; Chávez-Martínez, Ana I.; Rodríguez-Hernández, Aída A.; Maruri-López, Israel; Urano, Kaoru; Shinozaki, Kazuo; Jiménez-Bremont, Juan F.

    2016-01-01

    Polyamines (PAs) are small aliphatic polycations that are found ubiquitously in all organisms. In plants, PAs are involved in diverse biological processes such as growth, development, and stress responses. In Arabidopsis thaliana, the arginine decarboxylase enzymes (ADC1 and 2) catalyze the first step of PA biosynthesis. For a better understanding of PA biological functions, mutants in PA biosynthesis have been generated; however, the double adc1/adc2 mutant is not viable in A. thaliana. In this study, we generated non-lethal A. thaliana lines through an artificial microRNA that simultaneously silenced the two ADC genes (amiR:ADC). The generated transgenic lines (amiR:ADC-L1 and -L2) showed reduced AtADC1 and AtADC2 transcript levels. For further analyses the amiR:ADC-L2 line was selected. We found that the amiR:ADC-L2 line showed a significant decrease of their PA levels. The co-silencing revealed a stunted growth in A. thaliana seedlings, plantlets and delay in its flowering rate; these phenotypes were reverted with PA treatment. In addition, amiR:ADC-L2 plants displayed two seed phenotypes, such as yellow and brownish seeds. The yellow mutant seeds were smaller than adc1, adc2 mutants and wild type seeds; however, the brownish were the smallest seeds with arrested embryos at the torpedo stage. These data reinforce the importance of PA homeostasis in the plant development processes. PMID:27014322

  15. Increasing thermal stability and catalytic activity of glutamate decarboxylase in E. coli: An in silico study.

    PubMed

    Tavakoli, Yasaman; Esmaeili, Abolghasem; Saber, Hossein

    2016-10-01

    Glutamate decarboxylase (GAD) is an enzyme that converts l-glutamate to gamma amino butyric acid (GABA) that is a widely used drug to treat mental disorders like Alzheimer's disease. In this study for the first time point mutation was performed virtually in the active site of the E. coli GAD in order to increase thermal stability and catalytic activity of the enzyme. Energy minimization and addition of water box were performed using GROMACS 5.4.6 package. PoPMuSiC 2.1 web server was used to predict potential spots for point mutation and Modeller software was used to perform point mutation on three dimensional model. Molegro virtual docker software was used for cavity detection and stimulated docking study. Results indicate that performing mutation separately at positions 164, 302, 304, 393, 396, 398 and 410 increase binding affinity to substrate. The enzyme is predicted to be more thermo- stable in all 7 mutants based on ΔΔG value.

  16. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    PubMed

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe

    2016-01-01

    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity.

  17. Quantitative expression analysis and prognostic significance of L-DOPA decarboxylase in colorectal adenocarcinoma

    PubMed Central

    Kontos, C K; Papadopoulos, I N; Fragoulis, E G; Scorilas, A

    2010-01-01

    Background: L-DOPA decarboxylase (DDC) is an enzyme that catalyses, mainly, the decarboxylation of L-DOPA to dopamine and was found to be involved in many malignancies. The aim of this study was to investigate the mRNA expression levels of the DDC gene and to evaluate its clinical utility in tissues with colorectal adenocarcinoma. Methods: Total RNA was isolated from colorectal adenocarcinoma tissues of 95 patients. After having tested RNA quality, we prepared cDNA by reverse transcription. Highly sensitive quantitative real-time PCR method for DDC mRNA quantification was developed using the SYBR Green chemistry. GAPDH served as a housekeeping gene. Relative quantification analysis was performed using the comparative CT method (2−ΔΔCT). Results: DDC mRNA expression varied remarkably among colorectal tumours examined in this study. High DDC mRNA expression levels were found in well-differentiated and Dukes' stage A and B tumours. Kaplan–Meier survival curves showed that patients with DDC-positive tumours have significantly longer disease-free survival (P=0.009) and overall survival (P=0.027). In Cox regression analysis of the entire cohort of patients, negative DDC proved to be a significant predictor of reduced disease-free (P=0.021) and overall survival (P=0.047). Conclusions: The results of the study suggest that DDC mRNA expression may be regarded as a novel potential tissue biomarker in colorectal adenocarcinoma. PMID:20424616

  18. Aspartate Decarboxylase is Required for a Normal Pupa Pigmentation Pattern in the Silkworm, Bombyx mori.

    PubMed

    Dai, Fangyin; Qiao, Liang; Cao, Cun; Liu, Xiaofan; Tong, Xiaoling; He, Songzhen; Hu, Hai; Zhang, Li; Wu, Songyuan; Tan, Duan; Xiang, Zhonghuai; Lu, Cheng

    2015-06-16

    The pigmentation pattern of Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of β-alanine and N-β-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of β-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera.

  19. Elevated production of melatonin in transgenic rice seeds expressing rice tryptophan decarboxylase.

    PubMed

    Byeon, Yeong; Park, Sangkyu; Lee, Hyoung Yool; Kim, Young-Soon; Back, Kyoungwhan

    2014-04-01

    A major goal of plant biotechnology is to improve the nutritional qualities of crop plants through metabolic engineering. Melatonin is a well-known bioactive molecule with an array of health-promoting properties, including potent antioxidant capability. To generate melatonin-rich rice plants, we first independently overexpressed three tryptophan decarboxylase isogenes in the rice genome. Melatonin levels were altered in the transgenic lines through overexpression of TDC1, TDC2, and TDC3; TDC3 transgenic seed (TDC3-1) had melatonin concentrations 31-fold higher than those of wild-type seeds. In TDC3 transgenic seedlings, however, only a doubling of melatonin content occurred over wild-type levels. Thus, a seed-specific accumulation of melatonin appears to occur in TDC3 transgenic lines. In addition to increased melatonin content, TDC3 transgenic lines also had enhanced levels of melatonin intermediates including 5-hydroxytryptophan, tryptamine, serotonin, and N-acetylserotonin. In contrast, expression levels of melatonin biosynthetic mRNA did not increase in TDC3 transgenic lines, indicating that increases in melatonin and its intermediates in these lines are attributable exclusively to overexpression of the TDC3 gene.

  20. S-adenosyl-L-methionine decarboxylase activity in the rat epididymis: ontogeny and androgenic control.

    PubMed

    de las Heras, M A; Calandra, R S

    1991-01-01

    The authors describe the occurrence of high levels of S-adenosyl-L-methionine decarboxylase (SAMDC) activity in the rat epididymis, and its ontogeny and androgenic control. As early as 15 days of age, SAMDC activity exists, although a peak of activity is observed at 25 days. Bilateral orchidectomy resulted in a decline of epididymal SAMDC activity. However, an androgen-independent fraction, accounting for 34% of total activity, appears to exist in the epididymis. In 45-day-old orchidectomized rats, SAMDC activity was stimulated by testosterone treatment in a dose-dependent manner. However, treatment of 45-day-old intact animals with a high dose of the androgen failed to modify SAMDC activity, indicating that, at this age, the enzyme is maximally stimulated by endogenous androgens. The observed effect of testosterone on castrated rats was completely abolished by concomitant treatment with the antiandrogen flutamide. This compound was ineffective on the androgen-insensitive fraction. To assess the contribution of circulating and luminal androgens to the maintenance of epididymal SAMDC, rats were unilaterally orchidectomized and activity was determined in both epididymides after 7 days. The SAMDC activity was identical in epididymides from both sides, suggesting circulating androgens suffice to maintain normal levels of activity. It was concluded that androgens regulate epididymal SAMDC activity, although an androgen-independent fraction appears to exist.

  1. Acute effect of prolactin on ornithine decarboxylase activity in the rat testis.

    PubMed

    de Las Heras, M A; Calandra, R S

    1992-01-01

    A study was conducted to evaluate the effect of the acute treatment with prolactin (PRL) on ornithine decarboxylase (ODC) activity in the rat testis. Injection of a single SC dose of ovine PRL to puberal rats resulted in the activation of ODC from whole testis. This effect was maximal at 4 h after injection, and statistically significant at the dose of 500 micrograms. The effect of PRL was confined to the interstitial space; no change was observed in seminiferous tubules. PRL was unable to further increase testicular ODC activity when injected together with a stimulatory dose of human chorionic gonadotropin (hCG). The effect of PRL was mimicked by injection of a single dose of the dopamine antagonist sulpiride, which provoked a ninefold increase in serum PRL levels. In contrast, PRL did not stimulate testicular ODC activity in hypophysectomized rats, either under basal conditions or during treatment with PRL-hCG, indicating the requirement of a functional hypophysis for the expression of PRL action. These results suggest that the stimulation of testicular ODC activity by PRL is a marker of the trophic response of the testis to this hormone, different from the stimulation of steroidogenesis. This activity could be useful for the study of PRL action on the testis as well as of the interaction between PRL and LH at the testicular level.

  2. The bifunctional pyruvate decarboxylase/pyruvate ferredoxin oxidoreductase from Thermococcus guaymasensis.

    PubMed

    Eram, Mohammad S; Oduaran, Erica; Ma, Kesen

    2014-01-01

    The hyperthermophilic archaeon Thermococcus guaymasensis produces ethanol as a metabolic end product, and an alcohol dehydrogenase (ADH) catalyzing the reduction of acetaldehyde to ethanol has been purified and characterized. However, the enzyme catalyzing the formation of acetaldehyde has not been identified. In this study an enzyme catalyzing the production of acetaldehyde from pyruvate was purified and characterized from T. guaymasensis under strictly anaerobic conditions. The enzyme had both pyruvate decarboxylase (PDC) and pyruvate ferredoxin oxidoreductase (POR) activities. It was oxygen sensitive, and the optimal temperatures were 85°C and >95°C for the PDC and POR activities, respectively. The purified enzyme had activities of 3.8 ± 0.22 U mg(-1) and 20.2 ± 1.8 U mg(-1), with optimal pH-values of 9.5 and 8.4 for each activity, respectively. Coenzyme A was essential for both activities, although it did not serve as a substrate for the former. Enzyme kinetic parameters were determined separately for each activity. The purified enzyme was a heterotetramer. The sequences of the genes encoding the subunits of the bifunctional PDC/POR were determined. It is predicted that all hyperthermophilic β -keto acids ferredoxin oxidoreductases are bifunctional, catalyzing the activities of nonoxidative and oxidative decarboxylation of the corresponding β -keto acids.

  3. Differential induction of pyruvate decarboxylase subunits and transcripts in anoxic rice seedlings.

    PubMed Central

    Rivoal, J; Thind, S; Pradet, A; Ricard, B

    1997-01-01

    In 2-d-old rice (Oryza sativa L.) seedlings subjected to anoxic stress, pyruvate decarboxylase (PDC) activity increased 9-fold during a 168-h period. A polyclonal PDC antiserum that recognized alpha- and beta-subunits was used to quantify PDC protein by an enzyme-linked immunosorbant assay and showed a 5.6-fold increase, suggesting that the anoxically induced enzyme has a higher specific activity than the PDC isoform present under normoxia. Immunoblot analysis showed that levels of both PDC subunits were induced by anoxia. Immunoprecipitation of proteins labeled in vivo during anoxic treatment demonstrated that the alpha-subunit was preferentially synthesized at the onset of anoxia. Two partial cDNAs, including a novel sequence, were cloned from a cDNA library made from seedlings subjected to anoxia for 6 h. Gene-specific probes used to quantify northern blots showed that two or three PDC mRNAs are differentially induced by anoxia in rice seedlings. Immunoprecipitation of in vitro translation products of mRNAs isolated a different times of anoxic treatment confirmed this findings Our results suggest that anoxic induction of rice PDC involves transcriptional and posttranscriptional regulation of gene expression as well as differences in enzyme characteristics. PMID:9232881

  4. Partial purification and characterization of arginine decarboxylase from avocado fruit, a thermostable enzyme.

    PubMed

    Winer, L; Vinkler, C; Apelbaum, A

    1984-09-01

    A partially purified preparation of arginine decarboxylase (EC 4.1.1.19), a key enzyme in polyamine metabolism in plants, was isolated from avocado (Persea americana Mill. cv Fuerte) fruit. The preparation obtained from the crude extract after ammonium sulfate precipitation, dialysis, and heat treatment, had maximal activity between pH 8.0 and 9.0 at 60 degrees C, in the presence of 1.2 millimolar MnCl(2), 2 millimolar dithiothreitol, and 0.06 millimolar pyridoxal phosphate. The K(m), of arginine for the decarboxylation reaction was determined for enzymes prepared from the seed coat of both 4-week-old avocado fruitlet and fully developed fruit, and was found to have a value of 1.85 and 2.84 millimolar, respectively. The value of V(app) (max) of these enzymes was 1613 and 68 nanomoles of CO(2) produced per milligram of protein per hour for the fruitlet and the fully developed fruit, respectively. Spermine, an end product of polyamine metabolism, caused less than 5% inhibition of the enzyme from fully developed fruit and 65% inhibition of the enzyme from the seed coat of 4-week-old fruitlets at 1 millimolar under similar conditions. The effect of different inhibitors on the enzyme and the change in the nature of the enzyme during fruit development are discussed.

  5. Biochemical and Genetic Characterization of the Enterococcus faecalis Oxaloacetate Decarboxylase Complex

    PubMed Central

    Repizo, Guillermo D.; Blancato, Víctor S.; Mortera, Pablo; Lolkema, Juke S.

    2013-01-01

    Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef-A), membrane pump Ef-B, biotin acceptor protein Ef-D, and the novel subunit Ef-H. Our results show that in E. faecalis, subunits Ef-A, Ef-D, and Ef-H form a cytoplasmic soluble complex (termed Ef-AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef-H subunit, coexpression of oad genes was performed in Escherichia coli, showing that this subunit is vital for Ef-A and Ef-D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef-AHD complex is functional in E. faecalis. These results suggest that the Ef-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef-B subunit to achieve its maximal alkalinization capacity during citrate fermentation. PMID:23435880

  6. pH shift enhancement of Candida utilis pyruvate decarboxylase production.

    PubMed

    Chen, Allen Kuan-Liang; Breuer, Michael; Hauer, Bernhard; Rogers, Peter L; Rosche, Bettina

    2005-10-20

    Pyruvate decarboxylase (PDC) catalyses the synthesis of asymmetric carbinols, e.g., chiral precursors for pharmaceuticals such as ephedrine and pseudoephedrine. The production of PDC by Candida utilis in a minimal medium was improved by manipulating the pH during fermentation in a 5 L bioreactor. At an aeration rate of 0.1 vvm with a stirrer speed of 300 rpm at constant pH 6, a specific PDC activity of 141 U/g dry cell weight (DCW) was achieved (average of two fermentations +/-13%). By allowing the yeast to acidify the growth medium from pH 6 to 2.9, the final specific PDC activity increased by a factor of 2.7 to 385 U/g DCW (average from 4 fermentations +/-16%). The effect of this pH drift on PDC production was confirmed by another experiment with a manual shift of pH from 6 to 3 by addition of 5 M sulfuric acid. The final PDC activity was 392 U/g DCW (average from two fermentations +/-5%). However, experiments with constant pH of 6, 5, 4, or 3 resulted in average specific activities of only 102 to 141 U/g DCW, suggesting that a transitional pH change rather than the absolute pH value was responsible for the increased specific PDC activity.

  7. Effect of undernutrition on the regional development of transmitter enzymes: glutamate decarboxylase and choline acetyltransferase.

    PubMed

    Patel, A J; del Vecchio, M; Atkinson, D J

    1978-01-01

    The effect of undernutrition on the activity of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAc) (markers for the GABA-ergic and the cholinergic transmitter system, respectively) was studied in various parts of the rat brain at the age of 10, 15 and 21 days, and at day 54 following 33 days of rehabilitation. The brain regions investigated were the olfactory bulbs, cerebellum, pons-medulla, hypothalamus, colliculi, cerebral cortex hippocampus and the residual brain. Undernutrition resulted in a marked retardation of the developmental rise of the activities of both enzymes, expressed in terms of either total brain part or unit weight or protein. The effect diminished with age even during the period of nutritional deprivation. In most brain regions the enzyme activities were restored to normal after rehabilitation. In the cerebral cortex the total activity of both enzymes was persistently reduced, although the concentration of GAD exceeded the control levels. A negative correlation was manifested between the activities of GAD and ChAc in the different brain parts (except the cerebellum) during development. The correlation became significant by day 21 in the controls, but only after postweaning rehabilitation of the undernourished rats. The results showed therefore that undernutrition caused a reversible retardation in the development of these two transmitter enzymes, and they suggested that even the balance of the GABA-ergic and cholinergic systems throughout the brain can be restored to normal by rehabilitation.

  8. Polyamine metabolism and osmotic stress. II. Improvement of oat protoplasts by an inhibitor of arginine decarboxylase

    NASA Technical Reports Server (NTRS)

    Tiburcio, A. F.; Kaur-Sawhney, R.; Galston, A. W.

    1986-01-01

    We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with DL-alpha-difluoromethylarginine (DFMA), a specific 'suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.

  9. Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells

    SciTech Connect

    Kucharzewska, Paulina; Welch, Johanna E.; Svensson, Katrin J.; Belting, Mattias

    2010-10-01

    The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by {alpha}-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo.

  10. Expanding the active pH range of Escherichia coli glutamate decarboxylase by breaking the cooperativeness.

    PubMed

    Thu Ho, Ngoc Anh; Hou, Chen Yuan; Kim, Woo Hyun; Kang, Taek Jin

    2013-02-01

    Bacterial glutamate decarboxylase (GAD) transforms glutamate into γ-aminobutyric acid (GABA) with the consumption of a proton. The enzyme is active under acidic environments only and sharply loses its activity as pH approaches neutrality with concomitant structural deformation. In an attempt to understand better the role of this cooperative loss of activity upon pH shifts, we prepared and studied a series of GAD site-specific mutants. In this report, we show that the cooperativeness was kept intact by at least two residues, Glu89 and His465, of which Glu89 is newly identified to be involved in the cooperativity system of GAD. Double mutation on these residues not only broke the cooperativity in the activity change but also yielded a mutant GAD that retained the activity at neutral pH. The resulting mutant GAD that was active at neutral pH inhibited the cell growth in a glycerol medium by converting intracellular Glu into GABA in an uncontrolled manner, which explains in part why the cooperativeness of GAD has to be kept by several layers of safety keepers. This unexpected result might be utilized to convert a low-valued by-product of biodiesel production, glycerol, into value-added product, GABA.

  11. Hepatoerythropoietic Porphyria Caused by a Novel Homoallelic Mutation in Uroporphyrinogen Decarboxylase Gene in Egyptian Patients.

    PubMed

    Farrag, M S; Mikula, I; Richard, E; Saudek, V; De Verneuil, H; Martásek, P

    2015-01-01

    Porphyrias are metabolic disorders resulting from mutations in haem biosynthetic pathway genes. Hepatoerythropoietic porphyria (HEP) is a rare type of porphyria caused by the deficiency of the fifth enzyme (uroporphyrinogen decarboxylase, UROD) in this pathway. The defect in the enzymatic activity is due to biallelic mutations in the UROD gene. Currently, 109 UROD mutations are known. The human disease has an early onset, manifesting in infancy or early childhood with red urine, skin photosensitivity in sun-exposed areas, and hypertrichosis. Similar defects and links to photosensitivity and hepatopathy exist in several animal models, including zebrafish and mice. In the present study, we report a new mutation in the UROD gene in Egyptian patients with HEP. We show that the homozygous c.T163A missense mutation leads to a substitution of a conserved phenylalanine (amino acid 55) for isoleucine in the enzyme active site, causing a dramatic decrease in the enzyme activity (19 % of activity of wild-type enzyme). Inspection of the UROD crystal structure shows that Phe-55 contacts the substrate and is located in the loop that connects helices 2 and 3. Phe-55 is strictly conserved in both prokaryotic and eukaryotic UROD. The F55I substitution likely interferes with the enzyme-substrate interaction.

  12. Dynamic expression of a glutamate decarboxylase gene in multiple non-neural tissues during mouse development

    PubMed Central

    Maddox, Dennis M; Condie, Brian G

    2001-01-01

    Background Glutamate decarboxylase (GAD) is the biosynthetic enzyme for the neurotransmitter γ-aminobutyric acid (GABA). Mouse embryos lacking the 67-kDa isoform of GAD (encoded by the Gad1 gene) develop a complete cleft of the secondary palate. This phenotype suggests that this gene may be involved in the normal development of tissues outside of the CNS. Although Gad1 expression in adult non-CNS tissues has been noted previously, no systematic analysis of its embryonic expression outside of the nervous system has been performed. The objective of this study was to define additional structures outside of the central nervous system that express Gad1, indicating those structures that may require its function for normal development. Results Our analysis detected the localized expression of Gad1 transcripts in several developing tissues in the mouse embryo from E9.0-E14.5. Tissues expressing Gad1 included the tail bud mesenchyme, the pharyngeal pouches and arches, the ectodermal placodes of the developing vibrissae, and the apical ectodermal ridge (AER), mesenchyme and ectoderm of the limb buds. Conclusions Some of the sites of Gad1 expression are tissues that emit signals required for patterning and differentiation (AER, vibrissal placodes). Other sites correspond to proliferating stem cell populations that give rise to multiple differentiated tissues (tail bud mesenchyme, pharyngeal endoderm and mesenchyme). The dynamic expression of Gad1 in such tissues suggests a wider role for GABA signaling in development than was previously appreciated. PMID:11178105

  13. Novel interactions of fluorinated nucleotide derivatives targeting orotidine-5′-monophosphate decarboxylase

    PubMed Central

    Lewis, Melissa; Avina, Maria Elena Meza; Wei, Lianhu; Crandall, Ian E.; Bello, Angelica Mara; Poduch, Ewa; Liu, Yan; Paige, Christopher J.; Kain, Kevin C.; Pai, Emil F.; Kotra, Lakshmi P.

    2011-01-01

    Fluorinated nucleosides and nucleotides are of considerable interest to medicinal chemists due to their antiviral, anticancer, and other biological activities. However, their direct interactions at target binding sites are not well understood. A new class of 2′-deoxy-2′-fluoro-C6-substituted uridine and UMP derivatives were synthesized and evaluated as inhibitors of orotidine-5′-monophosphate decarboxylase (ODCase). These compounds were synthesized from the key intermediate, fully-protected 2′-deoxy-2′-fluorouridine. Among the synthesized compounds, 2′-deoxy-2′-fluoro-6-iodo-UMP covalently inhibited human ODCase with a second-order rate constant of 0.62 ± 0.02 M−1sec−1. Interestingly, the 6-cyano-2′-fluoro derivative covalently interacted with ODCase defying the conventional thinking, where its ribosyl derivative undergoes transformation into BMP by ODCase. This confirms that the 2′-fluoro moiety influences the chemistry at the C6 position of the nucleotides, thus interactions in the active site of ODCase. Molecular interactions of the 2′-fluorinated nucleotides are compared to those with the 3′-fluorinated nucleotides bound to the corresponding target enzyme, and the carbohydrate moieties were shown to bind in different conformations. PMID:21417464

  14. Gene cloning, expression, and characterization of phenolic acid decarboxylase from Lactobacillus brevis RM84.

    PubMed

    Landete, José María; Rodríguez, Héctor; Curiel, José Antonio; de las Rivas, Blanca; Mancheño, José Miguel; Muñoz, Rosario

    2010-06-01

    Phenolic acid decarboxylase (PAD) catalyzes the synthesis of vinyl phenols from hydroxycinnamic acids. The gene encoding PAD from Lactobacillus brevis was cloned and expressed as a fusion protein in Escherichia coli. The recombinant PAD enzyme is a heat-labile enzyme that functions optimally at 22 degrees C and pH 6.0. The purified enzyme did not show thermostability at temperatures above 22 degrees C. L. brevis PAD is able to decarboxylate exclusively the hydroxycinnamic acids, such as p-coumaric, caffeic, and ferulic acids, with K (m) values of 0.98, 0.96, and 0.78 mM, respectively. The substrate specificity exhibited by L. brevis PAD is similar to the PAD isolated from Bacillus subtilis and B. pumilus, but different from that of L. plantarum and Pediococcus pentosaceus. As the C-terminal region may be involved in determining PAD substrate specificity and catalytic capacity, amino acid differences among these proteins could explain the differences observed. The substrate specificity shown by L. brevis PAD shows promise for the synthesis of high-added value products from plant wastes.

  15. Glycine decarboxylase deficiency causes neural tube defects and features of non-ketotic hyperglycinemia in mice

    PubMed Central

    Pai, Yun Jin; Leung, Kit-Yi; Savery, Dawn; Hutchin, Tim; Prunty, Helen; Heales, Simon; Brosnan, Margaret E.; Brosnan, John T.; Copp, Andrew J.; Greene, Nicholas D.E.

    2015-01-01

    Glycine decarboxylase (GLDC) acts in the glycine cleavage system to decarboxylate glycine and transfer a one-carbon unit into folate one-carbon metabolism. GLDC mutations cause a rare recessive disease non-ketotic hyperglycinemia (NKH). Mutations have also been identified in patients with neural tube defects (NTDs); however, the relationship between NKH and NTDs is unclear. We show that reduced expression of Gldc in mice suppresses glycine cleavage system activity and causes two distinct disease phenotypes. Mutant embryos develop partially penetrant NTDs while surviving mice exhibit post-natal features of NKH including glycine accumulation, early lethality and hydrocephalus. In addition to elevated glycine, Gldc disruption also results in abnormal tissue folate profiles, with depletion of one-carbon-carrying folates, as well as growth retardation and reduced cellular proliferation. Formate treatment normalizes the folate profile, restores embryonic growth and prevents NTDs, suggesting that Gldc deficiency causes NTDs through limiting supply of one-carbon units from mitochondrial folate metabolism. PMID:25736695

  16. 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

  17. Polyamines directly promote antizyme-mediated degradation of ornithine decarboxylase by the proteasome

    PubMed Central

    Beenukumar, R. R.; Gödderz, Daniela; Palanimurugan, R.; Dohmen, R. J.

    2015-01-01

    Ornithine decarboxylase (ODC), a ubiquitin-independent substrate of the proteasome, is a homodimeric protein with a rate-limiting function in polyamine biosynthesis. Polyamines regulate ODC levels by a feedback mechanism mediated by ODC antizyme (OAZ). Higher cellular polyamine levels trigger the synthesis of OAZ and also inhibit its ubiquitin-dependent proteasomal degradation. OAZ binds ODC monomers and targets them to the proteasome. Here, we report that polyamines, aside from their role in the control of OAZ synthesis and stability, directly enhance OAZ-mediated ODC degradation by the proteasome. Using a stable mutant of OAZ, we show that polyamines promote ODC degradation in Saccharomyces cerevisiae cells even when OAZ levels are not changed. Furthermore, polyamines stimulated the in vitro degradation of ODC by the proteasome in a reconstituted system using purified components. In these assays, spermine shows a greater effect than spermidine. By contrast, polyamines do not have any stimulatory effect on the degradation of ubiquitin-dependent substrates. PMID:28357293

  18. Characterization of an avian histidine decarboxylase and localization of histaminergic neurons in the chicken brain.

    PubMed

    Bessho, Yuki; Iwakoshi-Ukena, Eiko; Tachibana, Tetsuya; Maejima, Sho; Taniuchi, Shusuke; Masuda, Keiko; Shikano, Kenshiro; Kondo, Kunihiro; Furumitsu, Megumi; Ukena, Kazuyoshi

    2014-08-22

    In mammals, it is established that histamine is a neurotransmitter and/or neuromodulator in the central nervous system. It is produced by the enzyme histidine decarboxylase (HDC) in the tuberomammillary nucleus of the posterior hypothalamus. However, HDC as well as histaminergic neurons have not yet been characterized in the avian brain. We have cloned the cDNA for HDC from the chicken hypothalamus and demonstrated that the chicken HDC sequence is highly homologous to the mammalian counterpart, and that the expressed protein shows high enzymatic activity. The expression of HDC mRNA at various sites in the brain was investigated using quantitative RT-PCR. The results showed that the HDC mRNA was highly expressed in the hypothalamic infundibulum. In situ hybridization analyses revealed that the cells containing HDC mRNA were localized in the medial mammillary nucleus of the hypothalamic infundibulum. Intracerebroventricular injection of histamine in chicks resulted in inhibition of feeding behavior. This is the first report of the characterization of histaminergic neurons in the avian brain, and our findings indicate that neuronal histamine exerts anorexigenic effects in chicks.

  19. Tryptamine-induced resistance in tryptophan decarboxylase transgenic poplar and tobacco plants against their specific herbivores.

    PubMed

    Gill, Rishi I S; Ellis, Brian E; Isman, Murray B

    2003-04-01

    The presence of amines and their derivatives in plant tissues is known to influence insect feeding and reproduction. The enzyme tryptophan decarboxylase (TDC) catalyzes the decarboxylation of tryptophan to tryptamine, which is both a bioactive amine and a precursor of other indole derivatives. Transgenic poplar and tobacco plants ectopically expressing TDC1 accumulated elevated levels of tryptamine without affecting plant growth and development. This accumulation was consistently associated with adverse effects on feeding behavior and physiology of Malacosoma disstria Hub. (forest tent caterpillar, FTC) and Manduca sexta L. (tobacco hornworm, THW). Behavior studies with FTC and THW larvae showed that acceptability of the leaf tissue to larvae was inversely related to foliar tryptamine levels. Physiological studies with FTC and THW larvae showed that consumption of leaf tissue from the transgenic lines is deleterious to larvae growth, apparently due to a postingestive mechanism. Thus, ectopic expression of TDC1 can allow sufficient tryptamine to accumulate in poplar and tobacco leaf tissue to suppress significantly the growth of insect pests that normally feed on these plants.

  20. Catecholamines are required for testosterone induction of ornithine decarboxylase in the mouse kidney.

    PubMed

    Cremades, A; Tovar, A; Peñafiel, R

    1992-08-01

    This report presents a study on the influence of the adrenergic system on androgen-regulated ornithine decarboxylase activity (ODC) in the mouse kidney. We have found that the existing levels of renal ODC in male mice and the increase induced by testosterone in male, female, castrated male and hypophysectomized mice were dramatically impaired by catecholamine depletion produced by treatment with either alpha-methyl-p-tyrosine or reserpine. Blockade of beta-receptors by treatment of the animals with the antagonists propranolol or bisoprolol was unable to prevent the effect of testosterone on the renal enzyme. However, alpha i-blockade obtained by treatment with prazosin or phenoxybenzamine was partially effective to produce the decrease of basal ODC in control male or to diminish ODC induction produced by androgens. All these findings demonstrate that catecholamines and alpha 1-adrenergic receptors are implicated in androgen-regulated ODC activity in the mouse kidney and suggest a plausible role of sympathetic renal innervation in enzyme induction by steroid hormones in this organ.

  1. Differential roles of pyruvate decarboxylase in aerial and embedded mycelia of the ascomycete Gibberella zeae.

    PubMed

    Son, Hokyoung; Min, Kyunghun; Lee, Jungkwan; Choi, Gyung Ja; Kim, Jin-Cheol; Lee, Yin-Won

    2012-04-01

    The pyruvate-acetaldehyde-acetate (PAA) pathway has diverse roles in eukaryotes. Our previous study on acetyl-coenzyme A synthetase 1 (ACS1) in Gibberella zeae suggested that the PAA pathway is important for lipid production, which is required for perithecia maturation. In this study, we deleted all three pyruvate decarboxylase (PDC) genes, which encode enzymes that function upstream of ACS1 in the PAA pathway. Results suggest PDC1 is required for lipid accumulation in the aerial mycelia, and deletion of PDC1 resulted in highly wettable mycelia. However, the total amount of lipids in the PDC1 deletion mutants was similar to that of the wild-type strain, likely due to compensatory lipid production processes in the embedded mycelia. PDC1 was expressed both in the aerial and embedded mycelia, whereas ACS1 was observed only in the aerial mycelia in a PDC1-dependent manner. PDC1 is also involved in vegetative growth of embedded mycelia in G. zeae, possibly through initiating the ethanol fermentation pathway. Thus, PDC1 may function as a key metabolic enzyme crucial for lipid production in the aerial mycelia, but play a different role in the embedded mycelia, where it might be involved in energy generation by ethanol fermentation.

  2. Crystal Structure and Pyridoxal 5-Phosphate Binding Property of Lysine Decarboxylase from Selenomonas ruminantium

    PubMed Central

    Sagong, Hye-Young; Son, Hyeoncheol Francis; Kim, Sunghwan; Kim, Yong-Hwan; Kim, Il-Kwon; Kim, Kyung-Jin

    2016-01-01

    Lysine decarboxylase (LDC) is a crucial enzyme for acid stress resistance and is also utilized for the biosynthesis of cadaverine, a promising building block for bio-based polyamides. We determined the crystal structure of LDC from Selenomonas ruminantium (SrLDC). SrLDC functions as a dimer and each monomer consists of two distinct domains; a PLP-binding barrel domain and a sheet domain. We also determined the structure of SrLDC in complex with PLP and cadaverine and elucidated the binding mode of cofactor and substrate. Interestingly, compared with the apo-form of SrLDC, the SrLDC in complex with PLP and cadaverine showed a remarkable structural change at the PLP binding site. The PLP binding site of SrLDC contains the highly flexible loops with high b-factors and showed an open-closed conformational change upon the binding of PLP. In fact, SrLDC showed no LDC activity without PLP supplement, and we suggest that highly flexible PLP binding site results in low PLP affinity of SrLDC. In addition, other structurally homologous enzymes also contain the flexible PLP binding site, which indicates that high flexibility at the PLP binding site and low PLP affinity seems to be a common feature of these enzyme family. PMID:27861532

  3. Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10

    SciTech Connect

    Karlsen, A.E.; Hagopian, W.A.; Grubin, C.E.; Dube, S.; Disteche, C.M.; Adler, D.A.; Baermeier, H.; Lernmark, A. ); Mathewes, S.; Grant, F.J.; Foster, D. )

    1991-10-01

    Glutamic acid decarboxylase which catalyzes formation of {gamma}-aminobutyric acid from L-glutamic acid, is detectable in different isoforms with distinct electrophoretic and kinetic characteristics. GAD has also been implicated as an autoantigen in the vastly differing autoimmune disease stiff-man syndrome and insulin-dependent diabetes mellitus. Despite the differing GAD isoforms, only one type of GAD cDNA (GAD-1), localized to a syntenic region of chromosome 2, has been isolated from rat, mouse, and cat. Using sequence information from GAD-1 to screen a human pancreatic islet cDNA library, the authors describe the isolation of an additional GAD cDNA (GAD-2), which was mapped to the short arm of human chromosome 10. Genomic Southern blotting with GAD-2 demonstrated a hybridization pattern different form that detected by GAD-1. GAD-2 recognizes a 5.6-kilobase transcript in both islets and brain, in contrast to GAD-1, which detects a 3.7-kilobase transcript in brain only. The deduced 585-amino acid sequence coded for by GAD-2 shows < 65% identify to previously published, highly conserved GAD-1 brain sequences, which show > 96% deduced amino acid sequence homology among the three species.

  4. Overexpression of Actinidia deliciosa pyruvate decarboxylase 1 gene enhances waterlogging stress in transgenic Arabidopsis thaliana.

    PubMed

    Zhang, Ji-Yu; Huang, Sheng-Nan; Wang, Gang; Xuan, Ji-Ping; Guo, Zhong-Ren

    2016-09-01

    Ethanolic fermentation is classically associated with waterlogging tolerance when plant cells switch from respiration to anaerobic fermentation. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we cloned a full-length PDC cDNA sequence from kiwifruit, named AdPDC1. We determined the expression of the AdPDC1 gene in kiwifruit under different environmental stresses using qRT-PCR, and the results showed that the increase of AdPDC1 expression during waterlogging stress was much higher than that during salt, cold, heat and drought stresses. Overexpression of kiwifruit AdPDC1 in transgenic Arabidopsis enhanced the resistance to waterlogging stress but could not enhance resistance to cold stress at five weeks old seedlings. Overexpression of kiwifruit AdPDC1 in transgenic Arabidopsis could not enhance resistance to NaCl and mannitol stresses at the stage of seed germination and in early seedlings. These results suggested that the kiwifruit AdPDC1 gene is required during waterlogging but might not be required during other environmental stresses. Expression of the AdPDC1 gene was down-regulated by abscisic acid (ABA) in kiwifruit, and overexpression of the AdPDC1 gene in Arabidopsis inhibited seed germination and root length under ABA treatment, indicating that ABA might negatively regulate the AdPDC1 gene under waterlogging stress.

  5. Cloning and sequence analysis of ornithine decarboxylase gene fragments from the Ascomycota.

    PubMed

    Jiménez-Bremont, Juan Francisco; Rodríguez-Kessler, Margarita; Rodríguez-Guerra, Raul; Cortes-Penagos, Carlos; Torres-Guzman, Juan Carlos; Williamson, June Simpson

    2006-06-01

    Ornithine decarboxylase (ODC; EC 4.1.1.17) catalyzes the initial step in the biosynthesis of polyamines, the conversion of ornithine to putrescine. Based on the most conserved regions of fungal ODCs, we designed and synthesized oligonucleotides to amplify homologous fragments of three important plant pathogenic Pyrenomycete fungi (Ascomycota), Magnaporthe grisea, Colletotrichum lindemuthianum and Fusarium solani, and one insect pathogenic fungus Metarhizium anisopliae. Cloning and sequencing of the amplified fragments revealed homologies of between 37 to 88% with other fungal ODCs. The predicted peptide sequences were compared by Clustal analysis and conserved sequences corresponding to the substrate and cofactor binding sites were identified. Comparative analyses of the ODC fragments isolated in this study, revealed high homology between them (68.3-81.1%) and also with other Pyrenomycetes such as Neurospora crassa (order Sordariales; 68.6-72.9%) and Fusarium graminearum (order Hypocreales; 70.8-88.1%). Data obtained in this work revealed that these fungi constitute a compact group separated from other eukaryotic ODCs.

  6. Immobilization and characterization of benzoylformate decarboxylase from Pseudomonas putida on spherical silica carrier.

    PubMed

    Peper, Stephanie; Kara, Selin; Long, Wei Sing; Liese, Andreas; Niemeyer, Bernd

    2011-08-01

    If an adequate biocatalyst is identified for a specific reaction, immobilization is one possibility to further improve its properties. The immobilization allows easy recycling, improves the enzyme performance, and it often enhances the stability of the enzyme. In this work, the immobilization of the benzoylformate decarboxylase (BFD) variant, BFD A460I-F464I, from Pseudomonas putida was accomplished on spherical silica. Silicagel is characterized by its high mechanical stability, which allows its application in different reactor types without restrictions. The covalently bound enzyme was characterized in terms of its activity, stability, and kinetics for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde. Moreover, temperature as well as pressure dependency of immobilized BFD A460I-F464I activity and enantioselectivity were analyzed. The used wide-pore silicagel shows a good accessibility of the immobilized enzyme. The activity of the immobilized BFD A460I-F464I variant was determined to be 70% related to the activity of the free enzyme. Thereby, the enantioselectivity of the enzyme was not influenced by the immobilization. In addition, a pressure-induced change in stereoselectivity was found both for the free and for the immobilized enzyme. With increasing pressure, the enantiomeric excess (ee) of (R)-2-HPP can be increased from 44% (0.1 MPa) to 76% (200 MPa) for the free enzyme and from 43% (0.1 MPa) to 66% (200 MPa) for the immobilized enzyme.

  7. Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme

    SciTech Connect

    Matte, Allan; Grosse, Stephan; Bergeron, Hélène; Abokitse, Kofi; Lau, Peter C.K.

    2012-04-30

    The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavoring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a -barrel structure and two -helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally related proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the -barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site.

  8. A heterozygous deletion in the glutamate decarboxylase 67 gene enhances maternal and fetal stress vulnerability.

    PubMed

    Uchida, Taku; Oki, Yutaka; Yanagawa, Yuchio; Fukuda, Atsuo

    2011-04-01

    Both down-regulation of glutamate decarboxylase 67 (GAD67) and maternal exposure to severe stress during pregnancy can increase the risk of schizophrenia and related psychotic disorders in the offspring. To investigate a gene-environment interaction, we performed the restraint-and-light stress to pregnant GAD67-GFP knock-in (GAD67(+/GFP)) and wild-type (GAD67(+/+)) mice three times a day for 45 min per session during gestational day (G) 15.0-17.5. The stress hormone (corticosterone) level of pregnant GAD67(+/GFP) mice (the overall GABA content is reduced because of the destruction of one allele of the endogenous GAD67 gene) was higher than that of GAD67(+/+), even without stress. The fetal body weights (GAD67(+/+)) in the GAD67(+/GFP) mothers were lower than those in the GAD67(+/+) mothers. GAD67(+/GFP) fetuses exhibited higher corticosterone (CORT) levels than GAD67(+/+) fetuses, even in non-stressed GAD67(+/+) mothers. Fetal body weight-decreases and CORT-increases by maternal stress (GAD67(+/+) mother) were significantly more in the GAD67(+/GFP) fetuses than the GAD67(+/+) fetuses. These results indicate that a GAD67 heterozygous deletion itself enhances vulnerability by many aspects, e.g., maternal stress, maternity, and being in utero. Thus, an abnormality in GAD67 could interact with environmental risk factors of psychiatric disorders, including schizophrenia.

  9. Pathogenic Roles of Glutamic Acid Decarboxylase 65 Autoantibodies in Cerebellar Ataxias

    PubMed Central

    Hampe, Christiane S.

    2017-01-01

    Reports suggesting a pathogenic role of autoantibodies directed against glutamic acid decarboxylase 65 (GAD65Abs) in cerebellar ataxias (CAs) are reviewed, and debatable issues such as internalization of antibodies by neurons and roles of epitopes are discussed. GAD65 is one of two enzymes that catalyze the conversion of glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). A pathogenic role of GAD65Ab in CAs is suggested by in vivo and in vitro studies. (1) Intracerebellar administration of cerebrospinal fluid (CSF) immunoglobulins (IgGs) obtained from GAD65Ab-positive CA patients impairs cerebellar modulation of motor control in rats. (2) CSF IgGs act on terminals of GABAergic neurons and decrease the release of GABA in cerebellar slices from rats and mice. (3) Absorption of GAD65Ab by recombinant GAD65 diminishes the above effects, and monoclonal human GAD65Ab (b78) mimic the effects of CSF IgGs in vivo and in vitro. Studies using GAD65-KO mice confirm that the target molecule is GAD65. (4) Notably, the effects of GAD65Ab depend on the epitope specificity of the monoclonal GAD65Ab. Taken together, these results indicate that epitope-specific GAD65Ab-induced impairment of GABA release is involved in the pathogenesis of GAD65Ab-positive CA and support the early detection of GAD65Ab-associated CA to initiate immunotherapy before irreversible neuronal death in the cerebellum. PMID:28386570

  10. Characterization of the p-coumaric acid decarboxylase from Lactobacillus plantarum CECT 748(T).

    PubMed

    Rodríguez, Héctor; Landete, José María; Curiel, José Antonio; de Las Rivas, Blanca; Mancheño, José Miguel; Muñoz, Rosario

    2008-05-14

    It was previously reported that cell cultures from Lactobacillus plantarum CECT 748 (T) were able to decarboxylate phenolic acids, such as p-coumaric, m-coumaric, caffeic, ferulic, gallic, and protocatechuic acid. The p-coumaric acid decarboxylase (PDC) from this strain has been overexpressed and purified. This PDC differs at its C-terminal end when compared to the previously reported PDC from L. plantarum LPCHL2. Because the C-terminal region of PDC is involved in enzymatic activity, especially in substrate activity, it was decided to biochemically characterize the PDC from L. plantarum CECT 748 (T). Contrarily to L. plantarum LPCHL2 PDC, the recombinant PDC from L. plantarum CECT 748 (T) is a heat-labile enzyme, showing optimal activity at 22 degrees C. This PDC is able to decarboxylate exclusively the hydroxycinnamic acids p-coumaric, caffeic, and ferulic acids. Kinetic analysis showed that the enzyme has a 14-fold higher K(M) value for p-coumaric and caffeic acids than for ferulic acid. PDC catalyzes the formation of the corresponding 4-vinyl derivatives (vinylphenol and vinylguaiacol) from p-coumaric and ferulic acids, respectively, which are valuable food additives that have been approved as flavoring agents. The biochemical characteristics showed by L. plantarum PDC should be taken into account for its potential use in the food-processing industry.

  11. Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1

    PubMed Central

    Pal Chowdhury, Piyali; Basu, Soumik; Dutta, Arindam

    2016-01-01

    ABSTRACT The gene encoding a nonoxidative decarboxylase capable of catalyzing the transformation of 2-hydroxy-1-naphthoic acid (2H1NA) to 2-naphthol was identified, recombinantly expressed, and purified to homogeneity. The putative gene sequence of the decarboxylase (hndA) encodes a 316-amino-acid protein (HndA) with a predicted molecular mass of 34 kDa. HndA exhibited high identity with uncharacterized amidohydrolase 2 proteins of various Burkholderia species, whereas it showed a modest 27% identity with γ-resorcylate decarboxylase, a well-characterized nonoxidative decarboxylase belonging to the amidohydrolase superfamily. Biochemically characterized HndA demonstrated strict substrate specificity toward 2H1NA, whereas inhibition studies with HndA indicated the presence of zinc as the transition metal center, as confirmed by atomic absorption spectroscopy. A three-dimensional structural model of HndA, followed by docking analysis, identified the conserved metal-coordinating and substrate-binding residues, while their importance in catalysis was validated by site-directed mutagenesis. IMPORTANCE Microbial nonoxidative decarboxylases play a crucial role in the metabolism of a large array of carboxy aromatic chemicals released into the environment from a variety of natural and anthropogenic sources. Among these, hydroxynaphthoic acids are usually encountered as pathway intermediates in the bacterial degradation of polycyclic aromatic hydrocarbons. The present study reveals biochemical and molecular characterization of a 2-hydroxy-1-naphthoic acid nonoxidative decarboxylase involved in an alternative metabolic pathway which can be classified as a member of the small repertoire of nonoxidative decarboxylases belonging to the amidohydrolase 2 family of proteins. The strict substrate specificity and sequence uniqueness make it a novel member of the metallo-dependent hydrolase superfamily. PMID:27068590

  12. Boron (B) deprivation increases plasma homocysteine and decreases liver S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The diverse effects of B deprivation suggest that B affects a biomolecule involved in a variety of biochemical reactions. An experiment was conducted to determine whether dietary B affects the liver concentration of SAM, a frequently used enzyme substrate, especially for methylation reactions that y...

  13. Treatment of idiopathic parkinsonism with L-dopa in the absence and presence of decarboxylase inhibitors: effects on plasma levels of L-dopa, dopa decarboxylase, catecholamines and 3-O-methyl-dopa.

    PubMed

    Boomsma, F; Meerwaldt, J D; Man in't Veld, A J; Hovestadt, A; Schalekamp, M A

    1989-05-01

    The effect of levodopa (L-dopa), alone or in combination with a peripheral decarboxylase inhibitor (PDI), on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD, = dopa decarboxylase), L-dopa, 3-O-methyl-dopa (3-OMD), dopamine (DA), noradrenaline, adrenaline and dopamine beta-hydroxylase has been studied. In healthy subjects and in patients with parkinsonism plasma ALAAD level fell after administration of L-dopa + benserazide, but returned to previous levels within 90 min. In a cross-sectional study blood was obtained, 2 h after dosing, from 104 patients with idiopathic parkinsonism, divided into four groups: no L-dopa treatment (group 1), L-dopa alone (group 2), L-dopa + benserazide (Madopar) (group 3) and L-dopa + carbidopa (Sinemet) (group 4). Plasma ALAAD, which was normal in groups 1 and 2, was increased 3-fold in groups 3 and 4, indicating that there was induction of ALAAD by the co-administration of PDI. Despite this induction of ALAAD, in groups 3 and 4, with half the daily L-dopa dose compared with group 2, plasma L-dopa and 3-OMD levels were 5 times higher, while plasma DA levels were not different. The DA/L-dopa ratio was decreased 5-fold in group 2 and 16-fold in groups 3 and 4 as compared with group 1. Neither 3-OMD levels nor 3-OMD/L-dopa ratios correlated with the occurrence of on-off fluctuations. In a longitudinal study of three patients started on Madopar treatment the induction of plasma ALAAD was found to occur gradually over 3-4 weeks. Further detailed pharmacokinetic studies in plasma and cerebrospinal fluid are required in order to elucidate whether the ALAAD induction by PDI may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena.

  14. Mesomere-derived glutamate decarboxylase-expressing blastocoelar mesenchyme cells of sea urchin larvae

    PubMed Central

    Katow, Hideki; Katow, Tomoko; Abe, Kouki; Ooka, Shioh; Kiyomoto, Masato; Hamanaka, Gen

    2014-01-01

    Summary The ontogenetic origin of blastocoelar glutamate decarboxylase (GAD)-expressing cells (GADCs) in larvae of the sea urchin Hemicentrotus pulcherrimus was elucidated. Whole-mount in situ hybridisation (WISH) detected transcription of the gene that encodes GAD in H. pulcherrimus (Hp-gad) in unfertilised eggs and all blastomeres in morulae. However, at and after the swimming blastula stage, the transcript accumulation was particularly prominent in clumps of ectodermal cells throughout the embryonic surface. During the gastrula stage, the transcripts also accumulated in the endomesoderm and certain blastocoelar cells. Consistent with the increasing number of Hp-gad transcribing cells, immunoblot analysis indicated that the relative abundance of Hp-Gad increased considerably from the early gastrula stage until the prism stage. The expression pattern of GADCs determined by immunohistochemistry was identical to the pattern of Hp-gad transcript accumulation determined using WISH. In early gastrulae, GADCs formed blastocoelar cell aggregates around the blastopore with primary mesenchyme cells. The increase in the number of blastocoelar GADCs was inversely proportional to the number of ectodermal GADCs ranging from a few percent of total GADCs in early gastrulae to 80% in late prism larvae; this depended on ingression of ectodermal GADCs into the blastocoel. Some of the blastocoelar GADCs were fluorescein-positive in the larvae that developed from the 16-cell stage chimeric embryos; these comprised fluorescein-labeled mesomeres and unlabelled macromeres and micromeres. Our finding indicates that some of the blastocoelar GADCs are derived from the mesomeres and thus they are the new group of mesenchyme cells, the tertiary mesenchyme cells. PMID:24357228

  15. Sequestered end products and enzyme regulation: the case of ornithine decarboxylase.

    PubMed Central

    Davis, R H; Morris, D R; Coffino, P

    1992-01-01

    The polyamines (putrescine, spermidine, and spermine) are synthesized by almost all organisms and are universally required for normal growth. Ornithine decarboxylase (ODC), an initial enzyme of polyamine synthesis, is one of the most highly regulated enzymes of eucaryotic organisms. Unusual mechanisms have evolved to control ODC, including rapid, polyamine-mediated turnover of the enzyme and control of the synthetic rate of the protein without change of its mRNA level. The high amplitude of regulation and the rapid variation in the level of the protein led biochemists to infer that polyamines had special cellular roles and that cells maintained polyamine concentrations within narrow limits. This view was sustained in part because of our continuing uncertainty about the actual biochemical roles of polyamines. In this article, we challenge the view that ODC regulation is related to precise adjustment of polyamine levels. In no organism does ODC display allosteric feedback inhibition, and in three types of organism, bacteria, fungi, and mammals, the size of polyamine pools may vary radically without having a profound effect on growth. We suggest that the apparent stability of polyamine pools in unstressed cells is due to their being largely bound to cellular polyanions. We further speculate that allosteric feedback inhibition, if it existed, would be inappropriately responsive to changes in the small, freely diffusible polyamine pool. Instead, mechanisms that control the amount of the ODC protein have appeared in most organisms, and even these are triggered inappropriately by variation of the binding of polyamines to ionic binding sites. In fact, feedback inhibition of ODC might be maladaptive during hypoosmotic stress or at the onset of growth, when organisms appear to require rapid increases in the size of their cellular polyamine pools. PMID:1620066

  16. Role of modulation on the effect of microwaves on ornithine decarboxylase activity in L929 cells

    SciTech Connect

    Penafiel, L.M.; Litovitz, T.; Krause, D.; Desta, A.; Mullins, J.M.

    1997-05-01

    The effect of 835 MHz microwaves on the activity of ornithine decarboxylase (ODC) in L929 murine cells was investigated at an SAR of {approximately}2.5 W/kg. The results depended upon the type of modulation employed. AM frequencies of 16 Hz and 60 Hz produced a transient increase in ODC activity that reached a peak at 8 h of exposure and returned to control levels after 24 h of exposure. In this case, ODC was increased by a maximum of 90% relative to control levels. A 40% increase in ODC activity was also observed after 8 h of exposure with a typical signal from a TDMA digital cellular telephone operating in the middle of its transmission frequency range. This signal was burst modulated at 50 Hz, with approximately 30% duty cycle. By contrast, 8 h exposure with 835 MHz microwaves amplitude modulated with speech produced no significant change in ODC activity. Further investigations, with 8 h of exposure to AM microwaves, as a function of modulation frequency, revealed that the response is frequency dependent, decreasing sharply at 6 Hz and 600 Hz. Exposure with 835 MHz microwaves, frequency modulated with a 60 Hz sinusoid, yielded no significant enhancement in ODC activity for exposure times ranging between 2 and 24 h. Similarly, exposure with a typical signal from an AMPS analog cellular telephone, which uses a form of frequency modulation, produced no significant enhancement in ODC activity. Exposure with 835 MHz continuous wave microwaves produced no effects for exposure times between 2 and 24 h, except for a small but statistically significant enhancement in ODC activity after 6 h of exposure.

  17. Substrate Distortion and the Catalytic Reaction Mechanism of 5-Carboxyvanillate Decarboxylase

    PubMed Central

    2015-01-01

    5-Carboxyvanillate decarboxylase (LigW) catalyzes the conversion of 5-carboxyvanillate to vanillate in the biochemical pathway for the degradation of lignin. This enzyme was shown to require Mn2+ for catalytic activity and the kinetic constants for the decarboxylation of 5-carboxyvanillate by the enzymes from Sphingomonas paucimobilis SYK-6 (kcat = 2.2 s–1 and kcat/Km = 4.0 × 104 M–1 s–1) and Novosphingobium aromaticivorans (kcat = 27 s–1 and kcat/Km = 1.1 × 105 M–1 s–1) were determined. The three-dimensional structures of both enzymes were determined in the presence and absence of ligands bound in the active site. The structure of LigW from N. aromaticivorans, bound with the substrate analogue, 5-nitrovanillate (Kd = 5.0 nM), was determined to a resolution of 1.07 Å. The structure of this complex shows a remarkable enzyme-induced distortion of the nitro-substituent out of the plane of the phenyl ring by approximately 23°. A chemical reaction mechanism for the decarboxylation of 5-carboxyvanillate by LigW was proposed on the basis of the high resolution X-ray structures determined in the presence ligands bound in the active site, mutation of active site residues, and the magnitude of the product isotope effect determined in a mixture of H2O and D2O. In the proposed reaction mechanism the enzyme facilitates the transfer of a proton to C5 of the substrate prior to the decarboxylation step. PMID:26714575

  18. Molecular cloning and expression analysis of an arginine decarboxylase gene from peach (Prunus persica).

    PubMed

    Liu, Ji Hong; Ban, Yusuke; Wen, Xiao-Peng; Nakajima, Ikuko; Moriguchi, Takaya

    2009-01-15

    Arginine decarboxylase (ADC), one of the enzymes responsible for putrescine (Put) biosynthesis, has been shown to be implicated in stress response. In the current paper attempts were made to clone and characterize a gene encoding ADC from peach (Prunus persica (L.) Batsch, 'Akatsuki'). Rapid amplification of cDNA ends (RACE) gave rise to a full-length ADC cDNA (PpADC) with a complete open reading frame of 2178 bp, encoding a 725 amino acid polypeptide. Homology search and sequence multi-alignment demonstrated that the deduced PpADC protein sequence shared a high identity with ADCs from other plants, including several highly conservative motifs and amino acids. Southern blotting indicated that PpADC existed in peach genome as a single gene. Expression levels of PpADC in different tissues of peach (P. persica 'Akatsuki') were spatially and developmentally regulated. Treatment of peach shoots from 'Mochizuki' with exogenous 5 mM Put, an indirect product of ADC, remarkably induced accumulation of PpADC mRNA. Transcripts of PpADC in peach leaves from 'Mochizuki' were quickly induced, either transiently or continuously, in response to dehydration, high salinity (200 mM NaCl), low temperature (4 degrees C) and heavy metal (150 microM CdCl(2)), but repressed by high temperature 37 degrees C) during a 2-day treatment, which changed in an opposite direction when the stresses were otherwise removed with the exception of CdCl(2) treatment. In addition, steady-state of PpADC mRNA could be also transiently up-regulated by abscisic acid (ABA) in 'Mochizuki' leaves. All of these, taken together, suggest that PpADC is a stress-responsive gene and can be considered as a potential target that is genetically manipulated so as to create novel germplasms with enhanced stress tolerance in the future.

  19. Effects of immunization with natural and recombinant lysine decarboxylase on canine gingivitis development.

    PubMed

    Peters, Jennifer L; DeMars, Paul L; Collins, Lindsay M; Stoner, Julie A; Matsumoto, Hiroyuki; Komori, Naoka; Singh, Anil; Feasley, Christa L; Haddock, James A; Levine, Martin

    2012-10-19

    Periodontal disease, gingival inflammation (gingivitis) and periodontal attachment loss (periodontitis), causes tooth loss and susceptibility to chronic inflammation. Professionally scaling and cleaning the teeth regularly controls the disease, but is expensive in companion animals. Eikenella corrodens is common in canine oral cavities where it is a source of lysine decarboxylase (LDC). In human dental biofilms (plaques), LDC converts lysine to cadaverine and impairs the gingival epithelial barrier to bacteria. LDC vaccination may therefore retard gingivitis development. Year-old beagle dogs provided blood samples, and had weight and clinical measurements (biofilm and gingivitis) recorded. After scaling and cleaning, two dogs were immunized subcutaneously with 0.2mg native LDC from E. corrodens and 2 sets of four dogs with 0.2mg recombinant LDC purified from Escherichia coli. A third set of 4 dogs was immunized intranasally. Rehydragel(®), Emulsigen(®), Polygen™ or Carbigen™ were used as adjuvant. Four additional pairs of dogs were sham-immunized with each adjuvant alone (controls). Immunizations were repeated twice, 3 weeks apart, and clinical measurements were obtained after another 2 weeks, when the teeth were scaled and cleaned again. Tooth brushing was then stopped and the diet was changed from hard to soft chow. Clinical measurements were repeated after 1, 2, 3, 4, 6 and 8 weeks. Compared with sham-immunized dogs, gingivitis was reduced over all 8 weeks of soft diet after subcutaneous immunization with native LDC, or after intranasal immunization with recombinant LDC in Carbigen™, but for only 6 of the 8 weeks after subcutaneous immunization with recombinant LDC in Emulsigen(®) (repeated measures ANOVA). Subcutaneous vaccination induced a strong serum IgG antibody response that decreased during the soft diet period, whereas intranasal immunization induced a weak serum IgA antibody response that did not decrease. Immunization with recombinant LDC may

  20. Glutamate Decarboxylase 1 Overexpression as a Poor Prognostic Factor in Patients with Nasopharyngeal Carcinoma

    PubMed Central

    Lee, Yi-Ying; Chao, Tung-Bo; Sheu, Ming-Jen; Tian, Yu-Feng; Chen, Tzu-Ju; Lee, Sung-Wei; He, Hong-Lin; Chang, I-Wei; Hsing, Chung-Hsi; Lin, Ching-Yih; Li, Chien-Feng

    2016-01-01

    Background: Glutamate decarboxylase 1 (GAD1) which serves as a rate-limiting enzyme involving in the production of γ-aminobutyric acid (GABA), exists in the GABAergic neurons in the central nervous system (CNS). Little is known about the relevance of GAD1 to nasopharyngeal carcinoma (NPC). Through data mining on a data set derived from a published transcriptome database, this study first identified GAD1 as a differentially upregulated gene in NPC. We aimed to evaluate GAD1 expression and its prognostic effect on patients with early and locoregionally advanced NPC. Methods: We evaluated GAD1 immunohistochemistry and performed an H-score analysis on biopsy specimens from 124 patients with nonmetastasized NPC receiving treatment. GAD1 overexpression was defined as an H score higher than the median value. The findings of such an analysis are correlated with clinicopathological behaviors and survival rates, namely disease-specific survival (DSS), distant-metastasis-free survival (DMeFS), and local recurrence-free survival (LRFS) rates. Results: GAD1 overexpression was significantly associated with an increase in the primary tumor status (p < 0.001) and American Joint Committee on Cancer (AJCC) stages III-IV (p = 0.002) and was a univariate predictor of adverse outcomes of DSS (p = 0.002), DMeFS (p < 0.0001), and LRFS (p = 0.001). In the multivariate comparison, in addition to advanced AJCC stages III-IV, GAD1 overexpression remained an independent prognosticator of short DSS (p = 0.004, hazard ratio = 2.234), DMeFS (p < 0.001, hazard ratio = 4.218), and LRFS (p = 0.013, hazard ratio = 2.441) rates. Conclusions: Our data reveal that GAD1 overexpression was correlated with advanced disease status and may thus be a critical prognostic indicator of poor outcomes in NPC and a potential therapeutic target to facilitate the development of effective treatment modalities. PMID:27698909

  1. Role of glutamate decarboxylase-like protein 1 (GADL1) in taurine biosynthesis.

    PubMed

    Liu, Pingyang; Ge, Xiaomei; Ding, Haizhen; Jiang, Honglin; Christensen, Bruce M; Li, Jianyong

    2012-11-30

    This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to β-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no β-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although β-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in β-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).

  2. Role of Arginine decarboxylase (ADC) in Arabidopsis thaliana defence against the pathogenic bacterium Pseudomonas viridiflava.

    PubMed

    Rossi, F R; Marina, M; Pieckenstain, F L

    2015-07-01

    Polyamine biosynthesis starts with putrescine production through the decarboxylation of arginine or ornithine. In Arabidopsis thaliana, putrescine is synthesised exclusively by arginine decarboxylase (ADC), which exists as two isoforms (ADC1 and 2) that are differentially regulated by abiotic stimuli, but their role in defence against pathogens has not been studied in depth. This work analysed the participation of ADC in Arabidopsis defence against Pseudomonas viridiflava. ADC activity and expression, polyamine levels and bacterial resistance were analysed in null mutants of each ADC isoform. In non-infected wild-type (WT) plants, ADC2 expression was much higher than ADC1. Analysis of adc mutants demonstrated that ADC2 contributes to a much higher extent than ADC1 to basal ADC activity and putrescine biosynthesis. In addition, adc2 mutants showed increased basal expression of salicylic acid- and jasmonic acid-dependent PR genes. Bacterial infection induced putrescine accumulation and ADC1 expression in WT plants, but pathogen-induced putrescine accumulation was blocked in adc1 mutants. Results suggest a specific participation of ADC1 in defence, although basal resistance was not decreased by dysfunction of either of the two ADC genes. In addition, and as opposed to WT plants, bacterial infection increased ADC2 expression and ADC activity in adc1 mutants, which could counterbalance the lack of ADC1. Results demonstrate a major contribution of ADC2 to total ADC activity and the specific induction of ADC1 in response to infection. A certain degree of functional redundancy between the two isoforms in relation to their contribution to basal resistance is also evident.

  3. Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

    SciTech Connect

    Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V.

    2012-09-17

    Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

  4. Functional Roles of the Dimer-Interface Residues in Human Ornithine Decarboxylase

    PubMed Central

    Lee, Chien-Yun; Liu, Yi-Liang; Lin, Chih-Li; Liu, Guang-Yaw; Hung, Hui-Chih

    2014-01-01

    Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to putrescine and is the rate-limiting enzyme in the polyamine biosynthesis pathway. ODC is a dimeric enzyme, and the active sites of this enzyme reside at the dimer interface. Once the enzyme dissociates, the enzyme activity is lost. In this paper, we investigated the roles of amino acid residues at the dimer interface regarding the dimerization, protein stability and/or enzyme activity of ODC. A multiple sequence alignment of ODC and its homologous protein antizyme inhibitor revealed that 5 of 9 residues (residues 165, 277, 331, 332 and 389) are divergent, whereas 4 (134, 169, 294 and 322) are conserved. Analytical ultracentrifugation analysis suggested that some dimer-interface amino acid residues contribute to formation of the dimer of ODC and that this dimerization results from the cooperativity of these interface residues. The quaternary structure of the sextuple mutant Y331S/Y389D/R277S/D332E/V322D/D134A was changed to a monomer rather than a dimer, and the Kd value of the mutant was 52.8 µM, which is over 500-fold greater than that of the wild-type ODC (ODC_WT). In addition, most interface mutants showed low but detectable or negligible enzyme activity. Therefore, the protein stability of these interface mutants was measured by differential scanning calorimetry. These results indicate that these dimer-interface residues are important for dimer formation and, as a consequence, are critical for enzyme catalysis. PMID:25140796

  5. Ornithine decarboxylase activity as a marker of androgen and antiandrogen action in the rat epididymis.

    PubMed

    de las Heras, M A; Suescun, M O; Calandra, R S

    1988-05-01

    After castration, there was a marked decrease in serum androgen concentration at 6 h, and a dramatic inhibition of ornithine decarboxylase (ODC) at 12 h. Administration of testosterone propionate to castrated rats at a dose of 0.05 mg/animal restored ODC activity to the normal value. However, no change was observed when intact rats were treated with testosterone even at a 40-fold higher dose, indicating that endogenous androgens present in intact rats are far in excess for maintenance of maximal levels of activity. Administration of the antiandrogen flutamide to intact rats caused a moderate decrease in epididymal weight, whereas this effect was more pronounced in castrated, androgen-treated rats. In the latter, the effect of flutamide was significant at the lowest dose used (0.5 mg/day). ODC activity was significantly decreased by flutamide treatment of intact rats, but even at the highest dose used (10 mg/day) only a 39% inhibition was observed. In flutamide-treated rats, LH concentrations were markedly increased, as were serum and epididymal androgens. In androgen-treated castrated rats, flutamide caused epididymal ODC to fall to undetectable values. These results show that: (1) androgens are essential for the maintenance of ODC activity in the epididymis; (2) epididymal ODC activity is maximally stimulated by endogenous androgens, at least in the pubertal rat; (3) the apparent potency of flutamide is substantially lowered by an increase in epididymal androgens. We suggest that ODC is a sensitive marker of the action of androgens and antiandrogens in the epididymis.

  6. Snapshot of a Reaction Intermediate: Analysis of Benzoylformate Decarboxylase in Complex with a Benzoylphosphonate Inhibitor

    SciTech Connect

    Brandt, Gabriel S.; Kneen, Malea M.; Chakraborty, Sumit; Baykal, Ahmet T.; Nemeria, Natalia; Yep, Alejandra; Ruby, David I.; Petsko, Gregory A.; Kenyon, George L.; McLeish, Michael J.; Jordan, Frank; Ringe, Dagmar

    2009-04-22

    Benzoylformate decarboxylase (BFDC) is a thiamin diphosphate- (ThDP-) dependent enzyme acting on aromatic substrates. In addition to its metabolic role in the mandelate pathway, BFDC shows broad substrate specificity coupled with tight stereo control in the carbon-carbon bond-forming reverse reaction, making it a useful biocatalyst for the production of chiral-hydroxy ketones. The reaction of methyl benzoylphosphonate (MBP), an analogue of the natural substrate benzoylformate, with BFDC results in the formation of a stable analogue (C2{alpha}-phosphonomandelyl-ThDP) of the covalent ThDP-substrate adduct C2{alpha}-mandelyl-ThDP. Formation of the stable adduct is confirmed both by formation of a circular dichroism band characteristic of the 1',4'-iminopyrimidine tautomeric form of ThDP (commonly observed when ThDP forms tetrahedral complexes with its substrates) and by high-resolution mass spectrometry of the reaction mixture. In addition, the structure of BFDC with the MBP inhibitor was solved by X-ray crystallography to a spatial resolution of 1.37 {angstrom} (PDB ID 3FSJ). The electron density clearly shows formation of a tetrahedral adduct between the C2 atom of ThDP and the carbonyl carbon atom of the MBP. This adduct resembles the intermediate from the penultimate step of the carboligation reaction between benzaldehyde and acetaldehyde. The combination of real-time kinetic information via stopped-flow circular dichroism with steady-state data from equilibrium circular dichroism measurements and X-ray crystallography reveals details of the first step of the reaction catalyzed by BFDC. The MBP-ThDP adduct on BFDC is compared to the recently solved structure of the same adduct on benzaldehyde lyase, another ThDP-dependent enzyme capable of catalyzing aldehyde condensation with high stereospecificity.

  7. Role of modulation on the effect of microwaves on ornithine decarboxylase activity in L929 cells.

    PubMed

    Penafiel, L M; Litovitz, T; Krause, D; Desta, A; Mullins, J M

    1997-01-01

    The effect of 835 MHz microwaves on the activity of ornithine decarboxylase (ODC) in L929 murine cell was investigated at an SAR of approximately 2.5 W/kg. The results depended upon the type of modulation employed. AM frequencies of 16 Hz and 60 Hz produced a transient increase in ODC activity that reached a peak at 8 h of exposure and returned to control levels after 24 h of exposure. In this case, ODC was increased by a maximum of 90% relative to control levels. A 40% increase in ODC activity was also observed after 8 h of exposure with a typical signal from a TDMA digital cellular telephone operating in the middle of its transmission frequency range (approximately 840 MHz). This signal was burst modulated at 50 Hz, with approximately 30% duty cycle. By contrast, 8 h exposure with 835 MHz microwaves amplitude modulated with speech produced no significant change in ODC activity. Further investigations, with 8 h of exposure to AM microwaves, as a function of modulation frequency, revealed that the response is frequency dependent, decreasing sharply at 6 Hz an 600 Hz. Exposure with 835 MHz microwaves, frequency modulated with a 60 Hz sinusoid, yielded no significant enhancement in ODC activity for exposure times ranging between 2 and 24 h. Similarly, exposure with a typical signal from an AMPS analog cellular telephone, which uses a form of frequency modulation, produced no significant enhancement in ODC activity. Exposure with 835 MHz continuous wave microwaves produced no effects for exposure times between 2 and 24 h, except for a small but statistically significant enhancement in ODC activity after 6 h of exposure. Comparison of these results suggests that effects are much more robust when the modulation causes low-frequency periodic changes in the amplitude of the microwave carrier.

  8. Uroporphyrinogen decarboxylase: Complete human gene sequence and molecular study of three families with hepatoerythropoietic porphyria

    SciTech Connect

    Moran-Jimenez, M.J.; Ged, C.; Verneuil, H. de

    1996-04-01

    A deficiency in uroporphyrinogen decarboxylase (UROD) enzyme activity, the fifth enzyme of the heme biosynthetic pathway, is found in patients with sporadic porphyria cutanea tarda (s-PCT), familial porphyria cutanea tarda (f-PCT), and hepatoerythropoietic porphyria (HEP). Subnormal UROD activity is due to mutations of the UROD gene in both f-PCT and HEP, but no mutations have been found in s-PCT. Genetic analysis has determined that f-PCT is transmitted as an autosomal dominant trait. In contrast, HEP, a severe form of cutaneous porphyria, is transmitted as an autosomal recessive trait. HEP is characterized by a profound deficiency of UROD activity, and the disease is usually manifest in childhood. In this study, a strategy was designed to identify alleles responsible for the HEP phenotype in three unrelated families. Mutations of UROD were identified by direct sequencing of four amplified fragments that contained the entire coding sequence of the UROD gene. Two new missense mutations were observed at the homoallelic state: P62L (proline-to-leucine substitution at codon 62) in a Portuguese family and Y311C (tyrosine-to-cysteine substitution at codon 311) in an Italian family. A third mutation, G281E, was observed in a Spanish family. This mutation has been previously described in three families from Spain and one from Tunisia. In the Spanish family described in this report, a paternal uncle of the proband developed clinically overt PCT as an adult and proved to be heterozygous for the G281E mutation. Mutant cDNAs corresponding to the P62L and Y311C changes detected in these families were created by site-directed mutagenesis. Recombinant proteins proved to have subnormal enzyme activity, and the Y311C mutant was thermolabile. 24 refs., 7 figs., 4 tabs.

  9. Epilepsy and hippocampal neurodegeneration induced by glutamate decarboxylase inhibitors in awake rats.

    PubMed

    Salazar, Patricia; Tapia, Ricardo

    2015-10-01

    Glutamic acid decarboxylase (GAD), the enzyme responsible for GABA synthesis, requires pyridoxal phosphate (PLP) as a cofactor. Thiosemicarbazide (TSC) and γ-glutamyl-hydrazone (PLPGH) inhibit the free PLP-dependent isoform (GAD65) activity after systemic administration, leading to epilepsy in mice and in young, but not in adult rats. However, the competitive GAD inhibitor 3-mercaptopropionic acid (MPA) induces convulsions in both immature and adult rats. In the present study we tested comparatively the epileptogenic and neurotoxic effects of PLPGH, TSC and MPA, administered by microdialysis in the hippocampus of adult awake rats. Cortical EEG and motor behavior were analyzed during the next 2h, and aspartate, glutamate and GABA were measured by HPLC in the microdialysis-collected fractions. Twenty-four hours after drug administration rats were fixed for histological analysis of the hippocampus. PLPGH or TSC did not affect the motor behavior, EEG or cellular morphology, although the extracellular concentration of GABA was decreased. In contrast, MPA produced intense wet-dog shakes, EEG epileptiform discharges, a >75% reduction of extracellular GABA levels and remarkable neurodegeneration of the CA1 region, with >80% neuronal loss. The systemic administration of the NMDA glutamate receptor antagonist MK-801 30 min before MPA did not prevent the MPA-induced epilepsy but significantly protected against its neurotoxic effect, reducing neuronal loss to <30%. We conclude that in adult awake rats, drugs acting on PLP availability have only a weak effect on GABA neurotransmission, whereas direct GAD inhibition produced by MPA induces hyperexcitation leading to epilepsy and hippocampal neurodegeneration. Because this degeneration was prevented by the blockade of NMDA receptors, we conclude that it is due to glutamate-mediated excitotoxicity consequent to disinhibition of the hippocampal excitatory circuits.

  10. Arginine Decarboxylase expression, polyamines biosynthesis and reactive oxygen species during organogenic nodule formation in hop.

    PubMed

    Fortes, Ana M; Costa, Joana; Santos, Filipa; Seguí-Simarro, José M; Palme, Klaus; Altabella, Teresa; Tiburcio, Antonio F; Pais, Maria S

    2011-02-01

    Hop (Humulus lupulus L.) is an economically important plant species used in beer production and as a health-promoting medicine. Hop internodes develop upon stress treatments organogenic nodules which can be used for genetic transformation and micropropagation. Polyamines are involved in plant development and stress responses. Arginine decarboxylase (ADC; EC 4·1.1·19) is a key enzyme involved in the biosynthesis of putrescine in plants. Here we show that ADC protein was increasingly expressed at early stages of hop internode culture (12h). Protein continued accumulating until organogenic nodule formation after 28 days, decreasing thereafter. The same profile was observed for ADC transcript suggesting transcriptional regulation of ADC gene expression during morphogenesis. The highest transcript and protein levels observed after 28 days of culture were accompanied by a peak in putrescine levels. Reactive oxygen species accumulate in nodular tissues probably due to stress inherent to in vitro conditions and enhanced polyamine catabolism. Conjugated polyamines increased during plantlet regeneration from nodules suggesting their involvement in plantlet formation and/or in the control of free polyamine levels. Immunogold labeling revealed that ADC is located in plastids, nucleus and cytoplasm of nodular cells. In vacuolated cells, ADC immunolabelling in plastids doubled the signal of proplastids in meristematic cells. Location of ADC in different subcellular compartments may indicate its role in metabolic pathways taking place in these compartments. Altogether these data suggest that polyamines play an important role in organogenic nodule formation and represent a progress towards understanding the role played by these growth regulators in plant morphogenesis.

  11. Developmental PCB Exposure Increases Audiogenic Seizures and Decreases Glutamic Acid Decarboxylase in the Inferior Colliculus

    PubMed Central

    Bandara, Suren B.; Eubig, Paul A.; Sadowski, Renee N.; Schantz, Susan L.

    2016-01-01

    Previously, we observed that developmental polychlorinated biphenyl (PCB) exposure resulted in an increase in audiogenic seizures (AGSs) in rats. However, the rats were exposed to loud noise in adulthood, and were not tested for AGS until after 1 year of age, either of which could have interacted with early PCB exposure to increase AGS susceptibility. This study assessed susceptibility to AGS in young adult rats following developmental PCB exposure alone (without loud noise exposure) and investigated whether there was a decrease in GABA inhibitory neurotransmission in the inferior colliculus (IC) that could potentially explain this effect. Female Long-Evans rats were dosed orally with 0 or 6 mg/kg/day of an environmentally relevant PCB mixture from 28 days prior to breeding until the pups were weaned at postnatal day 21. One male-female pair from each litter was retained for the AGS study whilst another was retained for Western blot analysis of glutamic acid decarboxylase (GAD) and GABAAα1 receptor in the IC, the site in the auditory midbrain where AGS are initiated. There was a significant increase in the number and severity of AGSs in the PCB groups, with females somewhat more affected than males. GAD65 was decreased but there was no change in GAD67 or GABAAα1 in the IC indicating decreased inhibitory regulation in the PCB group. These results confirm that developmental PCB exposure alone is sufficient to increase susceptibility to AGS, and provide the first evidence for a possible mechanism of action at the level of the IC. PMID:26543103

  12. Hippocampal interneurons expressing glutamic acid decarboxylase and calcium-binding proteins decrease with aging in Fischer 344 rats.

    PubMed

    Shetty, A K; Turner, D A

    1998-05-04

    Aging leads to alterations in the function and plasticity of hippocampal circuitry in addition to behavioral changes. To identify critical alterations in the substrate for inhibitory circuitry as a function of aging, we evaluated the numbers of hippocampal interneurons that were positive for glutamic acid decarboxylase and those that expressed calcium-binding proteins (parvalbumin, calbindin, and calretinin) in young adult (4-5 months old) and aged (23-25 months old) male Fischer 344 rats. Both the overall interneuron population and specific subpopulations of interneurons demonstrated a commensurate decline in numbers throughout the hippocampus with aging. Interneurons positive for glutamic acid decarboxylase were significantly depleted in the stratum radiatum of CA1, the strata oriens, radiatum and pyramidale of CA3, the dentate molecular layer, and the dentate hilus. Parvalbumin interneurons showed significant reductions in the strata oriens and pyramidale of CA1, the stratum pyramidale of CA3, and the dentate hilus. The reductions in calbindin interneurons were more pronounced than other calcium-binding protein-positive interneurons and were highly significant in the strata oriens and radiatum of both CA1 and CA3 subfields and in the dentate hilus. Calretinin interneurons were decreased significantly in the strata oriens and radiatum of CA3, in the dentate granule cell and molecular layers, and in the dentate hilus. However, the relative ratio of parvalbumin-, calbindin-, and calretinin-positive interneurons compared with glutamic acid decarboxylase-positive interneurons remained constant with aging, suggesting actual loss of interneurons expressing calcium-binding proteins with age. This loss contrasts with the reported preservation of pyramidal neurons with aging in the hippocampus. Functional decreases in inhibitory drive throughout the hippocampus may occur due to this loss, particularly alterations in the processing of feed-forward information through the

  13. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    NASA Astrophysics Data System (ADS)

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

    1992-09-01

    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  14. Anti glutamate-decarboxylase antibodies: a liaison between localisation related epilepsy, stiff-person syndrome and type-1 diabetes mellitus.

    PubMed

    Szűcs, Anna; Barcs, Gábor; Winkler, Gábor; Soós, Zsuzsanna; Folyovich, András; Kelemen, Anna; Várallyay, Péter; Kamondi, Anita

    2014-07-30

    We present two patients with partial epilepsy, type-1 diabetes and stiff person syndrome associated with high serum auto-antibody levels to glutamate-decarboxylase (anti-GAD). Both patients were or have suffered from additional autoimmune conditions. The presence of stiff person syndrome and elevated anti-GAD levels have to make clinicians look for additional autoimmune conditions including type-1 diabetes. On the other hand, the co-morbidity of partial epilepsy with autoimmune conditions in patients with elevated serum anti-GAD suggests an autoimmune mechanism of partial epilepsy in these cases.

  15. Oxalate decarboxylase and oxalate oxidase activities can be interchanged with a specificity switch of up to 282,000 by mutating an active site lid.

    PubMed

    Burrell, Matthew R; Just, Victoria J; Bowater, Laura; Fairhurst, Shirley A; Requena, Laura; Lawson, David M; Bornemann, Stephen

    2007-10-30

    Oxalate decarboxylases and oxalate oxidases are members of the cupin superfamily of proteins that have many common features: a manganese ion with a common ligand set, the substrate oxalate, and dioxygen (as either a unique cofactor or a substrate). We have hypothesized that these enzymes share common catalytic steps that diverge when a carboxylate radical intermediate becomes protonated. The Bacillus subtilis decarboxylase has two manganese binding sites, and we proposed that Glu162 on a flexible lid is the site 1 general acid. We now demonstrate that a decarboxylase can be converted into an oxidase by mutating amino acids of the lid that include Glu162 with specificity switches of 282,000 (SEN161-3DAS), 275,000 (SENS161-4DSSN), and 225,000 (SENS161-4DASN). The structure of the SENS161-4DSSN mutant showed that site 2 was not affected. The requirement for substitutions other than of Glu162 was, at least in part, due to the need to decrease the Km for dioxygen for the oxidase reaction. Reversion of decarboxylase activity could be achieved by reintroducing Glu162 to the SENS161-4DASN mutant to give a relative specificity switch of 25,600. This provides compelling evidence for the crucial role of Glu162 in the decarboxylase reaction consistent with it being the general acid, for the role of the lid in controlling the Km for dioxygen, and for site 1 being the sole catalytically active site. We also report the trapping of carboxylate radicals produced during turnover of the mutant with the highest oxidase activity. Such radicals were also observed with the wild-type decarboxylase.

  16. Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells

    PubMed Central

    Baydoun, Anwar R; Morgan, David M L

    1998-01-01

    We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by α-difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages.DFMO potentiated LPS-stimulated nitrite production in both a concentration- and time-dependent manner, increasing nitrite levels by 48±5% at 10 mM. This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production.Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10  mM) to potentiate LPS-induced NO synthesis.Metabolism of L-[3H]arginine to L-[3H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1–10 mM) or by putrescine (0.001–1 mM), spermidine (0.001–1 mM) or spermine (0.001–1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70±0.07%) by L-NMMA (100 μM).Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (∼2 fold) iNOS protein expression.These results show that DFMO potentiates LPS-induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines. PMID:9884080

  17. Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5'-Monophosphate Decarboxylase.

    PubMed

    Goryanova, Bogdana; Goldman, Lawrence M; Ming, Shonoi; Amyes, Tina L; Gerlt, John A; Richard, John P

    2015-07-28

    The caged complex between orotidine 5'-monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5'-monophosphate (FOMP) undergoes decarboxylation ∼300 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5'-monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5'-monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on kcat/Km and kcat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in kcat and a decrease in kcat/Km, due to a weakening of the protein-phosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in kcat/Km for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that kcat = 8.2 s(-1) for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the

  18. Determinants of the Differential Antizyme-Binding Affinity of Ornithine Decarboxylase

    PubMed Central

    Liu, Yen-Chin; Hsu, Den-Hua; Huang, Chi-Liang; Liu, Yi-Liang; Liu, Guang-Yaw; Hung, Hui-Chih

    2011-01-01

    Ornithine decarboxylase (ODC) is a ubiquitous enzyme that is conserved in all species from bacteria to humans. Mammalian ODC is degraded by the proteasome in a ubiquitin-independent manner by direct binding to the antizyme (AZ). In contrast, Trypanosoma brucei ODC has a low binding affinity toward AZ. In this study, we identified key amino acid residues that govern the differential AZ binding affinity of human and Trypanosoma brucei ODC. Multiple sequence alignments of the ODC putative AZ-binding site highlights several key amino acid residues that are different between the human and Trypanosoma brucei ODC protein sequences, including residue 119, 124,125, 129, 136, 137 and 140 (the numbers is for human ODC). We generated a septuple human ODC mutant protein where these seven bases were mutated to match the Trypanosoma brucei ODC protein sequence. The septuple mutant protein was much less sensitive to AZ inhibition compared to the WT protein, suggesting that these amino acid residues play a role in human ODC-AZ binding. Additional experiments with sextuple mutants suggest that residue 137 plays a direct role in AZ binding, and residues 119 and 140 play secondary roles in AZ binding. The dissociation constants were also calculated to quantify the affinity of the ODC-AZ binding interaction. The Kd value for the wild type ODC protein-AZ heterodimer ([ODC_WT]-AZ) is approximately 0.22 μM, while the Kd value for the septuple mutant-AZ heterodimer ([ODC_7M]-AZ) is approximately 12.4 μM. The greater than 50-fold increase in [ODC_7M]-AZ binding affinity shows that the ODC-7M enzyme has a much lower binding affinity toward AZ. For the mutant proteins ODC_7M(-Q119H) and ODC_7M(-V137D), the Kd was 1.4 and 1.2 μM, respectively. These affinities are 6-fold higher than the WT_ODC Kd, which suggests that residues 119 and 137 play a role in AZ binding. PMID:22073206

  19. Homology-based molecular modelling of PLP-dependent histidine decarboxylase from Mmorganella morganii.

    PubMed

    Tahanejad, F S; Naderi-Manesh, H; Habibinejad, B; Mahmoudian, M

    2000-06-01

    The 3-D structural information is a prerequisite for a rational ligand design. In the absence of experimental data, model building on the basis of a known 3-D structure of a homologous protein is at present the only reliable method to obtain structural information. A homology model building study of the pyridoxal 5'-phosphate (PLP)-dependent histidine decarboxylase from Morganella morganii (HDC-MM) has been carried out based on the crystal structure of the aspartate aminotransferase from Escherichia coli (AAT-EC). The primary sequences of AAT-EC and HDC-MM were aligned by automated alignment procedure. A 3-D model of HDC-MM was constructed by copying the coordinates of the residues from the crystal structure of AAT-EC into the corresponding residues in HDC-MM. After energy-minimization of the resulting 3-D model of HDC-MM, possible active site residues were identified by fitting the substrate (l-histidine) into the proposed active-site. In our model, several residues, which have an important role in the AAT-EC active-site, are located in positions spatially identical to those in AAT-EC structure. The back-bone of the modelled active site pocket is constructed by residues; Gly-92, Gly-93, Thr-93, Ser-115, Asp-200, Ala-202, Ser-229 and Lys-232 together with residues Asn-8, His-119, Thr-171, His-198, Leu-203, His-231, Ser-236 and Ile-238. In the ligand binding site, it appears that the HDC-MM model will position l-histidine (substrate) in the area consisting of the residues; Glu-29, Ser-30, Leu-38, His-231 and Lys-232. The nitrogen atom of the imidazole ring (N2) of the substrate is predicted to interact with the carboxylate group of Ser-30. The alpha-carboxylate of histidine points toward the Lys-232 to have electrostatic interaction with its side chain nitrogen atom (N(Z)). In conclusion, this combination of sequence and 3-D structural homology between AAT-EC and HDC-MM model could provide insight in assigning the probable active site residues.

  20. Substrate Binding Mode and Molecular Basis of a Specificity Switch in Oxalate Decarboxylase

    PubMed Central

    2016-01-01

    Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into formate and carbon dioxide in a remarkable reaction that requires manganese and dioxygen. Previous studies have shown that replacing an active-site loop segment Ser161-Glu162-Asn163-Ser164 in the N-terminal domain of OxDC with the cognate residues Asp161-Ala162-Ser-163-Asn164 of an evolutionarily related, Mn-dependent oxalate oxidase gives a chimeric variant (DASN) that exhibits significantly increased oxidase activity. The mechanistic basis for this change in activity has now been investigated using membrane inlet mass spectrometry (MIMS) and isotope effect (IE) measurements. Quantitative analysis of the reaction stoichiometry as a function of oxalate concentration, as determined by MIMS, suggests that the increased oxidase activity of the DASN OxDC variant is associated with only a small fraction of the enzyme molecules in solution. In addition, IE measurements show that C–C bond cleavage in the DASN OxDC variant proceeds via the same mechanism as in the wild-type enzyme, even though the Glu162 side chain is absent. Thus, replacement of the loop residues does not modulate the chemistry of the enzyme-bound Mn(II) ion. Taken together, these results raise the possibility that the observed oxidase activity of the DASN OxDC variant arises from an increased level of access of the solvent to the active site during catalysis, implying that the functional role of Glu162 is to control loop conformation. A 2.6 Å resolution X-ray crystal structure of a complex between oxalate and the Co(II)-substituted ΔE162 OxDC variant, in which Glu162 has been deleted from the active site loop, reveals the likely mode by which the substrate coordinates the catalytically active Mn ion prior to C–C bond cleavage. The “end-on” conformation of oxalate observed in the structure is consistent with the previously published V/K IE data and provides an empty coordination site for the dioxygen ligand that is thought to

  1. Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization

    SciTech Connect

    Chan, K.; Wood, M; Fedorov, A; Fedorov, E; Imker, H; Amyes, T; Richard, J; Almo, S; Gerlt, J

    2009-01-01

    The reaction catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) involves a stabilized anionic intermediate, although the structural basis for the rate acceleration (kcat/knon, 7.1 x 1016) and proficiency (kcat/KM)/knon, 4.8 x 1022 M-1 is uncertain. That the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC) catalyze the exchange of H6 of the UMP product with solvent deuterium allows an estimate of a lower limit on the rate acceleration associated with stabilization of the intermediate and its flanking transition states (=1010). The origin of the 'missing' contribution, =107 (1017 total - =1010), is of interest. Based on structures of liganded complexes, unfavorable electrostatic interactions between the substrate carboxylate group and a proximal Asp (Asp 70 in MtOMPDC and Asp 91 in ScOMPDC) have been proposed to contribute to the catalytic efficiency. We investigated that hypothesis by structural and functional characterization of the D70N and D70G mutants of MtOMPDC and the D91N mutant of ScOMPDC. The substitutions for Asp 70 in MtOMPDC significantly decrease the value of kcat for decarboxylation of FOMP (a more reactive substrate analogue) but have little effect on the value of kex for exchange of H6 of FUMP with solvent deuterium; the structures of wild-type MtOMPDC and its mutants are superimposable when complexed with 6-azaUMP. In contrast, the D91N mutant of ScOMPDC does not catalyze exchange of H6 of FUMP; the structures of wild-type ScOMPDC and its D91N mutant are not superimposable when complexed with 6-azaUMP, with differences in both the conformation of the active site loop and the orientation of the ligand vis vis the active site residues. We propose that the differential effects of substitutions for Asp 70 of MtOMPDC on decarboxylation and exchange provide additional evidence for a carbanionic intermediate as well as the involvement of Asp 70 in substrate destabilization.

  2. Cholera Toxin B Subunit Linked to Glutamic Acid Decarboxylase Suppresses Dendritic Cell Maturation and Function

    PubMed Central

    Odumosu, Oludare; Nicholas, Dequina; Payne, Kimberly; Langridge, William

    2012-01-01

    Dendritic cells are the largest population of antigen presenting cells in the body. One of their main functions is to regulate the delicate balance between immunity and tolerance responsible for maintenance of immunological homeostasis. Disruption of this delicate balance often results in chronic inflammation responsible for initiation of organ specific autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and type I diabetes. The cholera toxin B subunit (CTB) is a weak mucosal adjuvant known for its ability to stimulate immunity to antigenic proteins. However, conjugation of CTB to many autoantigens can induce immunological tolerance resulting in suppression of autoimmunity. In this study, we examined whether linkage of CTB to a 5 kDa C-terminal protein fragment of the major diabetes autoantigen glutamic acid decarboxylase (GAD35), can block dendritic cell (DC) functions such as biosynthesis of co-stimulatory factor proteins CD86, CD83, CD80 and CD40 and secretion of inflammatory cytokines. The results of human umbilical cord blood monocyte-derived DC - GAD35 autoantigen incubation experiments showed that inoculation of immature DCs (iDCs), with CTB-GAD35 protein dramatically suppressed levels of CD86, CD83, CD80 and CD40 co-stimulatory factor protein biosynthesis in comparison with GAD35 alone inoculated iDCs. Surprisingly, incubation of iDCs in the presence of the CTB-autoantigen and the strong immunostimulatory molecules PMA and Ionomycin revealed that CTB-GAD35 was capable of arresting PMA + Ionomycin induced DC maturation. Consistant with this finding, CTB-GAD35 mediated suppression of DC maturation was accompanied by a dramatic decrease in the secretion of the pro-inflammatory cytokines IL-12/23p40 and IL-6 and a significant increase in secretion of the immunosuppressive cytokine IL-10. Taken together, our experimental data suggest that linkage of the weak adjuvant CTB to the dominant type 1 diabetes autoantigen GAD strongly inhibits DC

  3. Biogenic Amine Degradation by Bacillus Species Isolated from Traditional Fermented Soybean Food and Detection of Decarboxylase-Related Genes.

    PubMed

    Eom, Jeong Seon; Seo, Bo Young; Choi, Hye Sun

    2015-09-01

    Biogenic amines in some food products present considerable toxicological risks as potential human carcinogens when consumed in excess concentrations. In this study, we investigated the degradation of the biogenic amines histamine and tyramine and the presence of genes encoding histidine and tyrosine decarboxylases and amine oxidase in Bacillus species isolated from fermented soybean food. No expression of histidine and tyrosine decarboxylase genes (hdc and tydc) were detected in the Bacillus species isolated (B. subtilis HJ0-6, B. subtilis D'J53-4, and B. idriensis RD13-10), although substantial levels of amine oxidase gene (yobN) expression were observed. We also found that the three selected strains, as non-biogenic amineproducing bacteria, were significantly able to degrade the biogenic amines histamine and tyramine. These results indicated that the selected Bacillus species could be used as a starter culture for the control of biogenic amine accumulation and degradation in food. Our study findings also provided the basis for the development of potential biological control agents against these biogenic amines for use in the food preservation and food safety sectors.

  4. Characterization of a pyridoxal-5'-phosphate-dependent l-lysine decarboxylase/oxidase from Burkholderia sp. AIU 395.

    PubMed

    Sugawara, Asami; Matsui, Daisuke; Takahashi, Narumi; Yamada, Miwa; Asano, Yasuhisa; Isobe, Kimiyasu

    2014-11-01

    A novel enzyme, which catalyzed decarboxylation of l-lysine into cadaverine with release of carbon dioxide and oxidative deamination of l-lysine into l-2-aminoadipic 5-semialdehyde with release of ammonia and hydrogen peroxide, was found from a newly isolated Burkholderia sp. AIU 395. The enzyme was specific to l-lysine and did not exhibit enzyme activities for other l-amino acids, l-lysine derivatives, d-amino acids, and amines. The apparent Km values for l-lysine in the oxidation and decarboxylation reactions were estimated to be 0.44 mM and 0.84 mM, respectively. The molecular mass was estimated to be 150 kDa, which was composed of two identical subunits with molecular mass of 76.5 kDa. The enzyme contained one mol of pyridoxal 5'-phosphate per subunit as a prosthetic group. The enzyme exhibiting decarboxylase and oxidase activities for l-lysine was first reported here, while the deduced amino acid sequence was homologous to that of putative lysine decarboxylases from the genus Burkholderia.

  5. Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum

    SciTech Connect

    Krungkrai, Sudaratana R.; Tokuoka, Keiji; Kusakari, Yukiko; Inoue, Tsuyoshi; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Mori, Yusuke; Kai, Yasushi; Krungkrai, Jerapan; Horii, Toshihiro

    2006-06-01

    Orotidine 5′-monophosphate decarboxylase of human malaria parasite P. falciparum was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation. Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V{sub M} = 2.3 Å{sup 3} Da{sup −1})

  6. Hetero- and homodimerization of Arabidopsis thaliana arginine decarboxylase AtADC1 and AtADC2.

    PubMed

    Maruri-López, Israel; Jiménez-Bremont, Juan F

    2017-03-11

    The arginine decarboxylase enzyme (ADC) carries out the production of agmatine from arginine, which is the precursor of the first polyamine (PA) known as putrescine; subsequently, putrescine is turned into the higher PAs, spermidine and spermine. In Arabidopsis thaliana PA production occurs only from arginine and this step is initiated by two ADC paralogues, AtADC1 and AtADC2. PA production is essential for A. thaliana life cycle. Here, we analyzed the sub-cellular localization of AtADC1 and AtADC2 enzymes through GFP translational fusions. Our data revealed that the A. thaliana arginine decarboxylase enzymes exhibit a dual sub-cellular localization both in the cytosol and chloroplast. Moreover, we examined the protein dimer assembly using a Bimolecular Fluorescence Complementation (BiFC) approach, which showed that AtADC1 and AtADC2 proteins were able to form homodimers in the cytosol and chloroplast. Interestingly, we found the formation of AtADC1/AtADC2 heterodimers with similar sub-cellular localization than homodimers. This study reveals that both ADC proteins are located in the same cell compartments, and they are able to form protein interaction complexes with each other.

  7. Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA

    PubMed Central

    Kandiah, Eaazhisai; Carriel, Diego; Perard, Julien; Malet, Hélène; Bacia, Maria; Liu, Kaiyin; Chan, Sze W. S.; Houry, Walid A.; Ollagnier de Choudens, Sandrine; Elsen, Sylvie; Gutsche, Irina

    2016-01-01

    The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions. PMID:27080013

  8. Two UDP-glucuronic acid decarboxylases involved in the biosynthesis of a bacterial exopolysaccharide in Paenibacillus elgii.

    PubMed

    Li, Ou; Qian, Chao-Dong; Zheng, Dao-Qiong; Wang, Pin-Mei; Liu, Yu; Jiang, Xin-Hang; Wu, Xue-Chang

    2015-04-01

    Xylose is described as a component of bacterial exopolysaccharides in only a limited number of bacterial strains. A bacterial strain, Paenibacillus elgii, B69 was shown to be efficient in producing a xylose-containing exopolysaccharide. Sequence analysis was performed to identify the genes encoding the uridine diphosphate (UDP)-glucuronic acid decarboxylase required for the synthesis of UDP-xylose, the precursor of the exopolysaccharide. Two sequences, designated as Peuxs1 and Peuxs2, were found as the candidate genes for such enzymes. The activities of the UDP-glucuronic acid decarboxylases were proven by heterologous expression and real-time nuclear magnetic resonance analysis. The intracellular activity and effect of these genes on the synthesis of exopolysaccharide were further investigated by developing a thymidylate synthase based knockout system. This system was used to substitute the conventional antibiotic resistance gene system in P. elgii, a natural multi-antibiotic resistant strain. Results of intracellular nucleotide sugar analysis showed that the intracellular UDP-xylose and UDP-glucuronic acid levels were affected in Peuxs1 or Peuxs2 knockout strains. The knockout of either Peuxs1 or Peuxs2 reduced the polysaccharide production and changed the monosaccharide ratio. No polysaccharide was found in the Peuxs1/Peuxs2 double knockout strain. Our results show that P. elgii can be efficient in forming UDP-xylose, which is then used for the synthesis of xylose-containing exopolysaccharide.

  9. The role of aromatic L-amino acid decarboxylase in bacillamide C biosynthesis by Bacillus atrophaeus C89.

    PubMed

    Yuwen, Lei; Zhang, Feng-Li; Chen, Qi-Hua; Lin, Shuang-Jun; Zhao, Yi-Lei; Li, Zhi-Yong

    2013-01-01

    For biosynthesis of bacillamide C by Bacillus atrophaeus C89 associated with South China sea sponge Dysidea avara, it is hypothesized that decarboxylation from L-tryptophan to tryptamine could be performed before amidation by the downstream aromatic L-amino acid decarboxylase (AADC) to the non-ribosomal peptide synthetases (NRPS) gene cluster for biosynthesizing bacillamide C. The structural analysis of decarboxylases' known substrates in KEGG database and alignment analysis of amino acid sequence of AADC have suggested that L-tryptophan and L-phenylalanine are the potential substrates of AADC. The enzymatic kinetic experiment of the recombinant AADC proved that L-tryptophan is a more reactive substrate of AADC than L-phenylalanine. Meanwhile, the AADC-catalyzed conversion of L-tryptophan into tryptamine was confirmed by means of HPLC and LC/MS. Thus during bacillamide C biosynthesis, the decarboxylation of L-tryptophan to tryptamine is likely conducted first under AADC catalysis, followed by the amidation of tryptamine with the carboxylic product of NRPS gene cluster.

  10. Identification of a tyrosine decarboxylase gene (tdcA) in Streptococcus thermophilus 1TT45 and analysis of its expression and tyramine production in milk.

    PubMed

    La Gioia, Federica; Rizzotti, Lucia; Rossi, Franca; Gardini, Fausto; Tabanelli, Giulia; Torriani, Sandra

    2011-02-01

    In this study, a tyrosine decarboxylase gene (tdcA) was identified in 1 among 83 Streptococcus thermophilus strains tested. Its sequence, nearly identical to that of a tdcA of Lactobacillus curvatus, indicated a horizontal gene transfer event. Transcription in milk and the formation of critical levels of tyramine were observed in the presence of tyrosine.

  11. Stimulation of L-asparate beta-decarboxylase formation by L-glutamate in Pseudomonas dacunhae and Improved production of L-alanine.

    PubMed

    Shibatani, T; Kakimoto, T; Chibata, I

    1979-09-01

    The formation of L-asparate beta-decarboxylase by Pseudomonas dacunhae was compared on media containing a variety of organic acids and amino acids as a carbon source. Although the enzyme was formed constitutively when the organism was grown on basal medium or on that containing tricarboxylic acid cycle intermediates, it was induced twofold by L-glutamate and repressed one-tenth by L-serine. L-Glutamine, L-proline, L-leucine, glycine, and L-threonine also showed induction effects lower than that of L-glutamate. L-Glutamate derepressed the serine effect. This glutamate effect was observed effect was observed with other microoganisms, e.g., Achromobacter pestifer and Achromobacter liquidum. Since the intermediates from L-glutamate metabolism had no effect, this induction effect was specific to L-glutamate. The formation of some glutamate-related enzymes was measured and is discussed in relation to the formation of L-asparate beta-decarboxylase. L-Asparate beta-decarboxylase was purified to an electrophoretically homogenous state from L-glutamate-grown cells of P. dacunhae, and some properties were compared with those of the enzyme from fumarate-grown cells. The two enzymes were identical in disc electrophoresis, molecular weight, and some enzymatic properties. The industrial production of L-alanine from L-aspartic acid acid was improved by using the culture broth with highly induced L-asparate beta-decarboxylase (9.4 U/ml of broth).

  12. Pyruvate Decarboxylase Catalyzes Decarboxylation of Branched-Chain 2-Oxo Acids but Is Not Essential for Fusel Alcohol Production by Saccharomyces cerevisiae

    PubMed Central

    ter Schure, Eelko G.; Flikweert, Marcel T.; van Dijken, Johannes P.; Pronk, Jack T.; Verrips, C. Theo

    1998-01-01

    The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of pyruvate decarboxylase (EC 4.1.1.1) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three PDC genes had been disrupted. Experiments with cell extracts from S. cerevisiae mutants expressing a single PDC gene demonstrated that both PDC1- and PDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type S. cerevisiae and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production. PMID:9546164

  13. Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum

    PubMed Central

    Krungkrai, Sudaratana R.; Tokuoka, Keiji; Kusakari, Yukiko; Inoue, Tsuyoshi; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Mori, Yusuke; Kai, Yasushi; Krungkrai, Jerapan; Horii, Toshihiro

    2006-01-01

    Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V M = 2.3 Å3 Da−1). PMID:16754976

  14. Atomic Resolution Structure of the Orotidine 5′-Monophosphate Decarboxylase Product Complex Combined with Surface Plasmon Resonance Analysis

    PubMed Central

    Fujihashi, Masahiro; Mito, Kazuya; Pai, Emil F.; Miki, Kunio

    2013-01-01

    Orotidine 5′-monophosphate decarboxylase (ODCase) accelerates the decarboxylation of its substrate by 17 orders of magnitude. One argument brought forward against steric/electrostatic repulsion causing substrate distortion at the carboxylate substituent as part of the catalysis has been the weak binding affinity of the decarboxylated product (UMP). The crystal structure of the UMP complex of ODCase at atomic resolution (1.03 Å) shows steric competition between the product UMP and the side chain of a catalytic lysine residue. Surface plasmon resonance analysis indicates that UMP binds 5 orders of magnitude more tightly to a mutant in which the interfering side chain has been removed than to wild-type ODCase. These results explain the low affinity of UMP and counter a seemingly very strong argument against a contribution of substrate distortion to the catalytic reaction mechanism of ODCase. PMID:23395822

  15. Structure of lpg0406, a carboxymuconolactone decarboxylase family protein possibly involved in antioxidative response from Legionella pneumophila.

    PubMed

    Chen, Xiaofang; Hu, Yanjin; Yang, Bo; Gong, Xiaojian; Zhang, Nannan; Niu, Liwen; Wu, Yun; Ge, Honghua

    2015-12-01

    Lpg0406, a hypothetical protein from Legionella pneumophila, belongs to carboxymuconolactone decarboxylase (CMD) family. We determined the crystal structure of lpg0406 both in its apo and reduced form. The structures reveal that lpg0406 forms a hexamer and have disulfide exchange properties. The protein has an all-helical fold with a conserved thioredoxin-like active site CXXC motif and a proton relay system similar to that of alkylhydroperoxidase from Mycobacterium tuberculosis (MtAhpD), suggesting that lpg0406 might function as an enzyme with peroxidase activity and involved in antioxidant defense. A comparison of the size and the surface topology of the putative substrate-binding region between lpg0406 and MtAhpD indicates that the two enzymes accommodate the different substrate preferences. The structural findings will enhance understanding of the CMD family protein structure and its various functions.

  16. Amino acids allosterically regulate the thiamine diphosphate-dependent alpha-keto acid decarboxylase from Mycobacterium tuberculosis.

    PubMed

    Werther, Tobias; Spinka, Michael; Tittmann, Kai; Schütz, Anja; Golbik, Ralph; Mrestani-Klaus, Carmen; Hübner, Gerhard; König, Stephan

    2008-02-29

    The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.

  17. Improvement of ethanol production by recombinant expression of pyruvate decarboxylase in the white-rot fungus Phanerochaete sordida YK-624.

    PubMed

    Wang, Jianqiao; Hirabayashi, Sho; Mori, Toshio; Kawagishi, Hirokazu; Hirai, Hirofumi

    2016-07-01

    To improve ethanol production by Phanerochaete sordida YK-624, the pyruvate decarboxylase (PDC) gene was cloned from and reintroduced into this hyper lignin-degrading fungus; the gene encodes a key enzyme in alcoholic fermentation. We screened 16 transformant P. sordida YK-624 strains that each expressed a second, recombinant PDC gene (pdc) and then identified the transformant strain (designated GP7) with the highest ethanol production. Direct ethanol production from hardwood was 1.41 higher with GP7 than with wild-type P. sordida YK-624. RT-PCR analysis indicated that the increased PDC activity was caused by elevated recombinant pdc expression. Taken together, these results suggested that ethanol production by P. sordida YK-624 can be improved by the stable expression of an additional, recombinant pdc.

  18. Inhibition of placental ornithine decarboxylase by DL-alpha-difluoro-methyl ornithine causes fetal growth restriction in rat.

    PubMed

    Ishida, Makoto; Hiramatsu, Yuji; Masuyama, Hisashi; Mizutani, Yasushi; Kudo, Takafumi

    2002-02-08

    The roles of polyamines in intrauterine growth restriction (IUGR) is studied. The DL-alpha-difluoromethyl ornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) which is a rate limiting enzyme of polyamine synthesis was administrated to pregnant rats so that we obtained rat fetuses with IUGR. The changes of maternal nutrition, damage of the placenta, and the direct effect of DFMO on the fetus were examined in this IUGR model. Administration of DFMO did not induced changes of maternal nutrition except for triglyceride and the fetal metabolic state. But the placental weight, ODC activity, and DNA in the placenta were decreased significantly. The ODC activity in the total placenta decreased to less than 10% of that of the control. Depression of ODC activity in the placenta may be the major cause of IUGR induced by DFMO administration, and polyamines play important roles to carry pregnancy.

  19. Ornithine and arginine decarboxylase activities and effect of some polyamine biosynthesis inhibitors on Gigaspora rosea germinating spores.

    PubMed

    Sannazzaro, Analía I; Alvarez, Cora L; Menéndez, Ana B; Pieckenstain, Fernando L; Albertó, Edgardo O; Ruiz, Oscar A

    2004-01-15

    The pathways for putrescine biosynthesis and the effects of polyamine biosynthesis inhibitors on the germination and hyphal development of Gigaspora rosea spores were investigated. Incubation of spores with different radioactive substrates demonstrated that both arginine and ornithine decarboxylase pathways participate in putrescine biosynthesis in G. rosea. Spermidine and spermine were the most abundant polyamines in this fungus. The putrescine biosynthesis inhibitors alpha-difluoromethylarginine and alpha-difluoromethylornithine, as well as the spermidine synthase inhibitor cyclohexylamine, slightly decreased polyamine levels. However, only the latter interfered with spore germination. The consequences of the use of putrescine biosynthesis inhibitors for the control of plant pathogenic fungi on the viability of G. rosea spores in soil are discussed.

  20. Inhibition of Morganella morganii Histidine Decarboxylase Activity and Histamine Accumulation in Mackerel Muscle Derived from Filipendula ulumaria Extracts.

    PubMed

    Nitta, Yoko; Yasukata, Fumiko; Kitamoto, Noritoshi; Ito, Mikiko; Sakaue, Motoyoshi; Kikuzaki, Hiroe; Ueno, Hiroshi

    2016-03-01

    Filipendula ulmaria, also known as meadowsweet, is an herb; its extract was examined for the prevention of histamine production, primarily that caused by contaminated fish. The efficacy of meadowsweet was assessed using two parameters: inhibition of Morganella morganii histidine decarboxylase (HDC) and inhibition of histamine accumulation in mackerel. Ellagitannins from F. ulmaria (rugosin D, rugosin A methyl ester, tellimagrandin II, and rugosin A) were previously shown to be potent inhibitors of human HDC; and in the present work, these compounds inhibited M. morganii HDC, with half maximal inhibitory concentration values of 1.5, 4.4, 6.1, and 6.8 μM, respectively. Application of the extracts (at 2 wt%) to mackerel meat yielded significantly decreased histamine accumulation compared with treatment with phosphate-buffered saline as a control. Hence, F. ulmaria exhibits inhibitory activity against bacterial HDC and might be effective for preventing food poisoning caused by histamine.

  1. Aspartate 203 of the oxaloacetate decarboxylase beta-subunit catalyses both the chemical and vectorial reaction of the Na+ pump.

    PubMed Central

    Di Berardino, M; Dimroth, P

    1996-01-01

    We report here a new mode of coupling between the chemical and vectorial reaction explored for the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae. The membrane-bound beta-subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions across the membrane. The biotin prosthetic group which is attached to the alpha-subunit becomes carboxylated by carboxyltransfer from oxaloacetate. The two conserved aspartic acid residues within putative membrane-spanning domains of the beta-subunit (Asp149 and Asp203) were exchanged by site-directed mutagenesis. Mutants D149Q and D149E retained oxaloacetate decarboxylase and Na+ transport activities. Mutants D203N and D203E, however, had lost these two activities, but retained the ability to form the carboxybiotin enzyme. Direct participation of Asp203 in the catalysis of the decarboxylation reaction is therefore indicated. In addition, all previous and present data on the enzyme support a model in which the same aspartic acid residue provides a binding site for the metal ion catalysing its movement across the membrane. The model predicts that asp203 in its dissociated form binds Na+ and promotes its translocation, while the protonated residue transfers the proton to the acid-labile carboxybiotin which initiates its decarboxylation. Strong support for the model comes from the observation that Na+ transport by oxaloacetate decarboxylation is accompanied by H+ transport in the opposite direction. The inhibition of oxaloacetate decarboxylation by high Na+ concentrations in a pH-dependent manner is also in agreement with the model. Images PMID:8617230

  2. Cloning and functional analysis of the orotidine-5'-phosphate decarboxylase gene (PbrURA3) of the pathogenic fungus Paracoccidioides brasiliensis.

    PubMed

    Reinoso, Cristina; Sorais, Françoise; Niño-Vega, Gustavo A; Fermiñán, Encarnación; San-Blas, Gioconda; Domínguez, Angel

    2005-07-15

    A genomic clone encoding the Paracoccidioides brasiliensis orotidine monophosphate decarboxylase gene (PbrURA3) was isolated by screening a subgenomic plasmid DNA library of this fungus, using a PCR amplification product of the gene as a probe. Sequence analysis revealed that the gene contains an open reading frame of 855 bp with a single intron (162 bp), and encodes a putative 285 amino acids polypeptide of estimated molecular weight 31.1 kDa and isoelectric point 6.5. The deduced amino acid sequence predicted a 73.4% identity with orotidine monophosphate decarboxylase of Aspergillus nidulans. Functionality of the gene was demonstrated by transformation into a Saccharomyces cerevisiae ura3 null mutant.

  3. Endosperm-specific expression of tyramine N-hydroxycinnamoyltransferase and tyrosine decarboxylase from a single self-processing polypeptide produces high levels of tyramine derivatives in rice seeds.

    PubMed

    Park, Sangkyu; Kang, Kiyoon; Kim, Young Soon; Back, Kyoungwhan

    2009-06-01

    The plant-specific tyramine derivatives, feruloyltyramine (FT) and 4-coumaroyltyramine (CT), represent bioactive compounds found at low levels in many plant species. We generated transgenic rice seeds that produce high levels of CT (14 microg g(-1) seeds) and FT (2.7 microg g(-1) seeds) through the dual expression of tyramine N-hydroxycinnamoyltransferase and tyrosine decarboxylase, using the self-processing foot-and-mouth disease virus 2A sequence and the endosperm-specific prolamin promoter.

  4. Modulation of ornithine decarboxylase activity in the normal and regenerating rat liver by various doses of the peptide morphogen of Hydra

    SciTech Connect

    Yarygin, K.N.; Kazimirskii, A.N.; Kositskii, G.I.; Rubina, A.Yu.; Vinogradov, V.A.; Pylaev, A.S.

    1986-11-01

    In this investigation, changes in ornithine decarboxylase (ODC) activity were studied in the normal and regenerating liver of rats receiving injections of various doses of Hydra peptide morphogen (HPM). Activity of ODC was determined by a radioisotope method based on liberation of /sup 14/CO/sub 2/ from L-(1-/sup 14/C)-ornithine. The results indicate in the author's opinion that HPM may have a role in the regulation of anabolic processes and, in particular, of regenerative processes in mammals.

  5. A metabolic strategy to enhance long-term survival by Phx1 through stationary phase-specific pyruvate decarboxylases in fission yeast.

    PubMed

    Kim, Ji-Yoon; Kim, Eun-Jung; Lopez-Maury, Luis; Bähler, Jürg; Roe, Jung-Hye

    2014-07-01

    In the fission yeast Schizosaccharomyces pombe, the stationary phase-specific transcription factor Phx1 contributes to long-term survival, stress tolerance, and meiosis. We identified Phx1-dependent genes through transcriptome analysis, and further analyzed those related with carbohydrate and thiamine metabolism, whose expression decreased in ∆phx1. Consistent with mRNA changes, the level of thiamine pyrophosphate (TPP) and TPP-utilizing pyruvate decarboxylase activity that converts pyruvate to acetaldehyde were also reduced in the mutant. Therefore, Phx1 appears to shift metabolic flux by diverting pyruvate from the TCA cycle and respiration to ethanol fermentation. Among the four predicted genes for pyruvate decarboxylase, only the Phx1-dependent genes (pdc201+ and pdc202+) contributed to long-term survival as judged by mutation and overexpression studies. These findings indicate that the Phx1-mediated long-term survival is achieved primarily through increasing the synthesis and activity of pyruvate decarboxylase. Consistent with this hypothesis, we observed that Phx1 curtailed respiration when cells entered stationary phase. Introduction of Δphx1 mutation compromised the long-lived phenotypes of Δpka1 and Δsck2 mutants that are devoid of pro-aging kinases of nutrient-signalling pathways, and of the Δpyp1 mutant with constitutively activated stress-responsive kinase Sty1. Therefore, achievement of long-term viability through both nutrient limitation and anti-stress response appears to be dependent on Phx1.

  6. Cloning and sequencing of two Ceriporiopsis subvermispora bicupin oxalate oxidase allelic isoforms: implications for the reaction specificity of oxalate oxidases and decarboxylases.

    PubMed

    Escutia, Marta R; Bowater, Laura; Edwards, Anne; Bottrill, Andrew R; Burrell, Matthew R; Polanco, Rubén; Vicuña, Rafael; Bornemann, Stephen

    2005-07-01

    Oxalate oxidase is thought to be involved in the production of hydrogen peroxide for lignin degradation by the dikaryotic white rot fungus Ceriporiopsis subvermispora. This enzyme was purified, and after digestion with trypsin, peptide fragments of the enzyme were sequenced using quadrupole time-of-flight mass spectrometry. Starting with degenerate primers based on the peptide sequences, two genes encoding isoforms of the enzyme were cloned, sequenced, and shown to be allelic. Both genes contained 14 introns. The sequences of the isoforms revealed that they were both bicupins that unexpectedly shared the greatest similarity to microbial bicupin oxalate decarboxylases rather than monocupin plant oxalate oxidases (also known as germins). We have shown that both fungal isoforms, one of which was heterologously expressed in Escherichia coli, are indeed oxalate oxidases that possess < or =0.2% oxalate decarboxylase activity and that the organism is capable of rapidly degrading exogenously supplied oxalate. They are therefore the first bicupin oxalate oxidases to have been described. Heterologous expression of active enzyme was dependent on the addition of manganese salts to the growth medium. Molecular modeling provides new and independent evidence for the identity of the catalytic site and the key amino acid involved in defining the reaction specificities of oxalate oxidases and oxalate decarboxylases.

  7. Is there a difference between levodopa/ dopa-decarboxylase inhibitor and entacapone and levodopa/dopa-decarboxylase inhibitor dose fractionation strategies in Parkinson's disease patients experiencing symptom re-emergence due to wearing-off? The Honeymoon Study.

    PubMed

    Destée, Alain; Rérat, Karin; Bourdeix, Isabelle

    2009-01-01

    Two strategies to manage symptom re-emergence due to wearing-off with conventional levodopa/dopa-decarboxylase inhibitor (DDCI) therapy were compared in patients with Parkinson's disease (PD) in this randomized, open-label trial. PD patients receiving 3 daily doses of levodopa/DDCI were randomized to either levodopa/DDCI and entacapone or an increased dose frequency of levodopa/DDCI with or without an increased total daily dose (dose fractionation). After 1 month of treatment, patients were followed up for 1 year. A greater proportion of levodopa/DDCI and entacapone-treated patients had treatment success compared with dose-fractionated patients, according to investigator Clinical Global Impression of Change scores at 1 month (68 vs. 59%, respectively) and 1 year (60 vs. 51%, respectively). Mean 'off' time (time with symptoms) was improved in both groups at 1 month and 1 year, despite a reduction in the mean daily levodopa dose in the levodopa/DDCI and entacapone group at 1 month. The mean daily levodopa dose was increased in the dose fractionation group. At 1 month, there was a 4% reduction in patients experiencing dyskinesia with levodopa/DDCI and entacapone and a 3% increase with dose fractionation. These data suggest that levodopa/DDCI and entacapone reduces time with symptoms, the rate of motor complications and the daily levodopa dose compared with dose fractionation. However, as the observed differences were not statistically significant, further studies are required to confirm these results.

  8. Steady-state and transient-state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity.

    PubMed

    Flikweert, M T; Kuyper, M; van Maris, A J; Kötter, P; van Dijken, J P; Pronk, J T

    1999-01-01

    Pyruvate decarboxylase is a key enzyme in the production of low-molecular-weight byproducts (ethanol, acetate) in biomass-directed applications of Saccharomyces cerevisiae. To investigate whether decreased expression levels of pyruvate decarboxylase can reduce byproduct formation, the PDC2 gene, which encodes a positive regulator of pyruvate-decarboxylase synthesis, was inactivated in the prototrophic strain S. cerevisiae CEN. PK113-7D. This caused a 3-4-fold reduction of pyruvate-decarboxylase activity in glucose-limited, aerobic chemostat cultures grown at a dilution rate of 0.10 h(-1). Upon exposure of such cultures to a 50 mM glucose pulse, ethanol and acetate were the major byproducts formed by the wild type. In the pdc2Delta strain, formation of ethanol and acetate was reduced by 60-70%. In contrast to the wild type, the pdc2Delta strain produced substantial amounts of pyruvate after a glucose pulse. Nevertheless, its overall byproduct formation was ca. 50% lower. The specific rate of glucose consumption after a glucose pulse to pdc2Delta cultures was about 40% lower than in wild-type cultures. This suggests that, at reduced pyruvate-decarboxylase activities, glycolytic flux is controlled by NADH reoxidation. In aerobic, glucose-limited chemostat cultures, the wild type exhibited a mixed respiro-fermentative metabolism at dilution rates above 0.30 h(-1). Below this dilution rate, sugar metabolism was respiratory. At dilution rates up to 0.20 h(-1), growth of the pdc2Delta strain was respiratory and biomass yields were similar to those of wild-type cultures. Above this dilution rate, washout occurred. The low micro(max) of the pdc2Delta strain in glucose-limited chemostat cultures indicates that occurrence of respiro-fermentative metabolism in wild-type cultures is not solely caused by competition of respiration and fermentation for pyruvate. Furthermore, it implies that inactivation of PDC2 is not a viable option for reducing byproduct formation in industrial

  9. Purification, properties and cDNA cloning of glutamate decarboxylase in germinated faba bean (Vicia faba L.).

    PubMed

    Yang, Runqiang; Yin, Yongqi; Guo, Qianghui; Gu, Zhenxin

    2013-06-01

    Gamma-aminobutyric acid (GABA) is a non-protein amino acid with bioactive functions in humans. In this work, glutamate decarboxylase (EC 4.1.1.15, GAD) which is key in the GABA bioformation was purified from 5-day germinated faba beans and characterized. A single band was observed at 58 kDa using sodium dodecyl sulphate gel electrophoresis. GAD optimal activity was at pH 6.0 at 40°C with a K(m) value for glutamic acid (Glu) of 2.63 mM. The enzyme was inhibited significantly by Cu(2+), Fe(3+), Mg(2+), Ba(2+), aminoxyacetate, EGTA, Na(2)EDTA, l-cysteine and beta-mercaptoethanol; and activated at low Ca(2+) 0.2mM. Using RT-PCR, the GAD cDNA was sequenced which indicated 1787 bp long, containing a 1527 bp open reading frame (ORF) that encoded 509 amino-acid peptides with a calculated molecular weight of 57.74 kDa and a pI of 5.41 (GenBank accession number: JX444699).

  10. The HemQ coprohaem decarboxylase generates reactive oxygen species: implications for the evolution of classical haem biosynthesis

    PubMed Central

    Hobbs, Charlie; Dailey, Harry A.; Shepherd, Mark

    2016-01-01

    Bacteria require a haem biosynthetic pathway for the assembly of a variety of protein complexes, including cytochromes, peroxidases, globins, and catalase. Haem is synthesised via a series of tetrapyrrole intermediates, including non-metallated porphyrins, such as protoporphyrin IX, which is well known to generate reactive oxygen species in the presence of light and oxygen. Staphylococcus aureus has an ancient haem biosynthetic pathway that proceeds via the formation of coproporphyrin III, a less reactive porphyrin. Here, we demonstrate, for the first time, that HemY of S. aureus is able to generate both protoporphyrin IX and coproporphyrin III, and that the terminal enzyme of this pathway, HemQ, can stimulate the generation of protoporphyrin IX (but not coproporphyrin III). Assays with hydrogen peroxide, horseradish peroxidase, superoxide dismutase, and catalase confirm that this stimulatory effect is mediated by superoxide. Structural modelling reveals that HemQ enzymes do not possess the structural attributes that are common to peroxidases that form compound I [FeIV==O]+, which taken together with the superoxide data leaves Fenton chemistry as a likely route for the superoxide-mediated stimulation of protoporphyrinogen IX oxidase activity of HemY. This generation of toxic free radicals could explain why HemQ enzymes have not been identified in organisms that synthesise haem via the classical protoporphyrin IX pathway. This work has implications for the divergent evolution of haem biosynthesis in ancestral microorganisms, and provides new structural and mechanistic insights into a recently discovered oxidative decarboxylase reaction. PMID:27597779

  11. Estradiol decreases taurine level by reducing cysteine sulfinic acid decarboxylase via the estrogen receptor-α in female mice liver.

    PubMed

    Ma, Qiwang; Zhao, Jianjun; Cao, Wei; Liu, Jiali; Cui, Sheng

    2015-02-15

    Cysteine sulfinic acid decarboxylase (CSAD) and cysteine dioxygenase (CDO) are two rate-limiting enzymes in taurine de novo synthesis, and their expressions are associated with estrogen concentration. The present study was designed to determine the relationship between 17β-estradiol (E₂) and taurine in female mice liver. We initially observed the mice had lower levels of CSAD, CDO, and taurine during estrus than diestrus. We then, respectively, treated the ovariectomized mice, the cultured hepatocytes, and Hep G2 cells with different doses of E₂, and the CSAD and CDO expressions and taurine levels were analyzed. The results showed that E₂ decreased taurine level in the serum and the cultured cells by inhibiting CSAD and CDO expressions. Furthermore, we identified the molecular receptor types through which E₂ plays its role in regulating taurine synthesis, and our results showed that estrogen receptor-α (ERα) expression was much higher than estrogen receptor-β (ERβ) in the liver and hepatocytes, and the inhibiting effects of E₂ on CSAD, CDO, and taurine level were partially abrogated in the ICI-182,780-pretreated liver and hepatocytes, and in ERα knockout mice. These results indicate that estradiol decreases taurine content by reducing taurine biosynthetic enzyme expression in mice liver.

  12. Targeting ornithine decarboxylase by α-difluoromethylornithine inhibits tumor growth by impairing myeloid-derived suppressor cells

    PubMed Central

    Ye, Cong; Geng, Zhe; Dominguez, Donye; Chen, Siqi; Fan, Jie; Qin, Lei; Long, Alan; Zhang, Yi; Kuzel, Timothy M.; Zhang, Bin

    2015-01-01

    α-difluoromethylornithine (DFMO) is currently used in chemopreventive regimens primarily for its conventional direct anti-carcinogenesis activity. However, little is known about the effect of decarboxylase (ODC) inhibition by DFMO on antitumor immune responses. We showed here that pharmacologic blockade of ODC by DFMO inhibited tumor growth in intact immunocompetent mice, but abrogated in the immunodeficient Rag1−/− mice, suggesting that antitumor effect of DFMO is dependent on the induction of adaptive anti-tumor T cell immune responses. Depletion of CD8+ T cells impeded the tumor-inhibiting advantage of DFMO. Moreover, DFMO treatment enhanced antitumor CD8+ T cell infiltration and IFN-γ production, and augmented the efficacy of adoptive T cell therapy. Importantly, DFMO impaired Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs) suppressive activity through at least two mechanisms, including reducing arginase expression and activity, and inhibiting CD39/CD73-mediated pathway. MDSCs were one primary cellular target of DFMO as indicated by both adoptive transfer and MDSC depletion analyses. Our findings establish a new role of ODC inhibition by DFMO as a viable and effective immunological adjunct in effective cancer treatment, thereby adding to the growing list of chemoimmunotherapeutic applications of these agents. PMID:26663722

  13. Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli.

    PubMed

    Kang, Taek Jin; Ho, Ngoc Anh Thu; Pack, Seung Pil

    2013-08-15

    Escherichia coli glutamate decarboxylase (GAD) converts glutamate into γ-aminobutyric acid (GABA) through decarboxylation using proton as a co-substrate. Since GAD is active only at acidic conditions even though pH increases as the reaction proceeds, the conventional practice of using this enzyme involved the use of relatively high concentration of buffers, which might complicate the downstream purification steps. Here we show by simulation and experiments that the free acid substrate, glutamic acid, rather than its monosodium salt can act as a substrate and buffer at the same time. This yielded the buffer- and salt-free synthesis of GABA conveniently in a batch mode. Furthermore, we engineered GAD to hyper active ones by extending or reducing the length of the enzyme by just one residue at its C-terminus. Through the buffer-free reaction with engineered GAD, we could synthesize 1M GABA in 3h, which can be translated into a space-time yield of 34.3g/L/h.

  14. Structural Asymmetry and Disulfide Bridges among Subunits Modulate the Activity of Human Malonyl-CoA Decarboxylase*

    PubMed Central

    Aparicio, David; Pérez-Luque, Rosa; Carpena, Xavier; Díaz, Mireia; Ferrer, Joan C.; Loewen, Peter C.; Fita, Ignacio

    2013-01-01

    Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is an essential facet in the regulation of fatty acid metabolism. The structure of human peroxisomal MCD reveals a molecular tetramer that is best described as a dimer of structural heterodimers, in which the two subunits present markedly different conformations. This molecular organization is consistent with half-of-the-sites reactivity. Each subunit has an all-helix N-terminal domain and a catalytic C-terminal domain with an acetyltransferase fold (GNAT superfamily). Intersubunit disulfide bridges, Cys-206–Cys-206 and Cys-243–Cys-243, can link the four subunits of the tetramer, imparting positive cooperativity to the catalytic process. The combination of a half-of-the-sites mechanism within each structural heterodimer and positive cooperativity in the tetramer produces a complex regulatory picture that is further complicated by the multiple intracellular locations of the enzyme. Transport into the peroxisome has been investigated by docking human MCD onto the peroxisomal import protein peroxin 5, which revealed interactions that extend beyond the C-terminal targeting motif. PMID:23482565

  15. The krebs cycle enzyme α-ketoglutarate decarboxylase is an essential glycosomal protein in bloodstream African trypanosomes.

    PubMed

    Sykes, Steven; Szempruch, Anthony; Hajduk, Stephen

    2015-03-01

    α-Ketoglutarate decarboxylase (α-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei. The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, α-KDE1 was expressed in BF T. brucei and RNA interference knockdown of α-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of α-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of α-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of α-KDE1 contain overlapping mitochondrion- and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei. These results suggest that α-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei.

  16. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

    PubMed Central

    Huang, Yan; Su, Lingqia; Wu, Jing

    2016-01-01

    Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA. PMID:27438707

  17. QSAR study of malonyl-CoA decarboxylase inhibitors using GA-MLR and a new strategy of consensus modeling.

    PubMed

    Li, Jiazhong; Lei, Beilei; Liu, Huanxiang; Li, Shuyan; Yao, Xiaojun; Liu, Mancang; Gramatica, Paola

    2008-12-01

    Quantitative structure-activity relationship (QSAR) of a series of structural diverse malonyl-CoA decarboxylase (MCD) inhibitors have been investigated by using the predictive single model as well as the consensus analysis based on a new strategy proposed by us. Self-organizing map (SOM) neural network was employed to divide the whole data set into representative training set and test set. Then a multiple linear regressions (MLR) model population was built based on the theoretical molecular descriptors selected by Genetic Algorithm using the training set. In order to analyze the diversity of these models, multidimensional scaling (MDS) was employed to explore the model space based on the Hamming distance matrix calculated from each two models. In this space, Q(2) (cross-validated R(2)) guided model selection (QGMS) strategy was performed to select submodels. Then consensus modeling was built by two strategies, average consensus model (ACM) and weighted consensus model (WCM), where each submodel had a different weight according to the contribution of model expressed by MLR regression coefficients. The obtained results prove that QGMS is a reliable and practical method to guide the submodel selection in consensus modeling building and our weighted consensus model (WCM) strategy is superior to the simple ACM.

  18. Distribution of messenger RNAs encoding the enzymes glutaminase, aspartate aminotransferase and glutamic acid decarboxylase in rat brain.

    PubMed

    Najlerahim, A; Harrison, P J; Barton, A J; Heffernan, J; Pearson, R C

    1990-05-01

    In situ hybridization histochemistry (ISHH) using synthetic oligonucleotide probes has been used to identify cells containing the mRNAs coding for glutaminase (GluT), aspartate aminotransferase (AspT) and glutamic acid decarboxylase (GAD). The distribution of GAD mRNA confirms previous descriptions and matches the distribution of GAD detected using specific antibodies. AspT mRNA is widely distributed in the brain, but is present at high levels in GABAergic neuronal populations, some that may be glutamatergic, and in a subset of neurons which do not contain significant levels of either GAD or GluT mRNA. Particularly prominent are the neurons of the magnocellular division of the red nucleus, the large cells in the deep cerebellar nuclei and the vestibular nuclei and neurons of the lateral superior olivary nucleus. GluT mRNA does not appear to be present at high levels in all GAD-containing neurons, but is seen prominently in many neuronal populations that may use glutamate as a neurotransmitter, such as neocortical and hippocampal pyramidal cells, the granule cells of the cerebellum and neurons of the dentate gyrus of the hippocampus. The heaviest labelling of GluT mRNA is seen in the lateral reticular nucleus of the medulla. ISHH using probes directed against the mRNAs encoding these enzymes may be an important technique for identifying glutamate and aspartate using neuronal populations and for examining their regulation in a variety of experimental and pathological circumstances.

  19. Effects of psoralen from Psoralea corylifolia on quinone reductase, ornithine decarboxylase, and JB6 cells transformation promotion.

    PubMed

    Lee, Sung-Jin; Nam, Kung-Woo; Mar, Woongchon

    2011-01-01

    The cancer chemopreventive effect of psoralen isolated from the seeds of Psoralea corylifolia was investigated in the induction of quinone reductase (QR) activity, intracellular detoxification enzyme, inhibition of 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, a key regulatory enzyme for polyamine metabolism, and tumor promotion in mouse epidermal JB6 cells, sensitive to tumor promoters (clone 415a P+ cells), which are related to suppress multistage carcinogenesis including initiation and promotion. Psoralen was isolated and identified from the ethyl acetate-soluble fraction of the methanolic extract from the seeds. Psoralen was active in induction of QR activity, the concentration of psoralen required to induce 1.5 fold QR activity was 14.8 μg/mL. Also, this pure compound inhibited TPA-induced ODC activity by 50% (designated IC(50)) at the concentration 15.6 μg/mL and exhibited inhibition of TPA-induced tumor promotion in mouse epidermal JB6 cells with an IC(50) value of 17.1 μg/mL. Therefore, it is extrapolated that psoralen has the potential capable of inhibiting the initiation and/or promotion stage of carcinogenesis by induction of QR activity, inhibition of TPA-induced ODC activity and mouse epidermal JB6 cells tumor promotion.

  20. Crystal Structures of Malonyl-Coenzyme A Decarboxylase Provide Insights into Its Catalytic Mechanism and Disease-Causing Mutations

    PubMed Central

    Froese, D. Sean; Forouhar, Farhad; Tran, Timothy H.; Vollmar, Melanie; Kim, Yi Seul; Lew, Scott; Neely, Helen; Seetharaman, Jayaraman; Shen, Yang; Xiao, Rong; Acton, Thomas B.; Everett, John K.; Cannone, Giuseppe; Puranik, Sriharsha; Savitsky, Pavel; Krojer, Tobias; Pilka, Ewa S.; Kiyani, Wasim; Lee, Wen Hwa; Marsden, Brian D.; von Delft, Frank; Allerston, Charles K.; Spagnolo, Laura; Gileadi, Opher; Montelione, Gaetano T.; Oppermann, Udo; Yue, Wyatt W.; Tong, Liang

    2013-01-01

    Summary Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design. PMID:23791943

  1. Uroporphyrinogen decarboxylase as a potential target for specific components of traditional Chinese medicine: a virtual screening and molecular dynamics study.

    PubMed

    Tsou, Yung-An; Chen, Kuan-Chung; Lin, Hung-Che; Chang, Su-Sen; Chen, Calvin Yu-Chian

    2012-01-01

    Uroporphyrinogen decarboxylase (UROD) has been suggested as a protectant against radiation for head and neck cancer (HNC). In this study, we employed traditional Chinese medicine (TCM) compounds from TCM Database@Taiwan (http://tcm.cmu.edu.tw/) to screen for drug-like candidates with potential UROD inhibition characteristics using virtual screening techniques. Isopraeroside IV, scopolin, and nodakenin exhibited the highest Dock Scores, and were predicted to have good Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties. Two common moieties, 2H-chromen-2-one and glucoside, were observed among the top TCM candidates. Cross comparison of the docking poses indicated that candidates formed stable interactions with key binding and catalytic residues of UROD through these two moieties. The 2H-chromen-2-one moiety enabled pi-cation interactions with Arg37 and H-bonds with Tyr164. The glucoside moiety was involved in forming H-bonds with Arg37 and Asp86. From our computational results, we propose isopraeroside IV, scopolin, and nodakenin as ligands that might exhibit drug-like inhibitory effects on UROD. The glucoside and 2H-chromen-2-one moieties may potentially be used for designing inhibitors of UROD.

  2. The VP1 structural protein of enterovirus 71 interacts with human ornithine decarboxylase and gene trap ankyrin repeat.

    PubMed

    Yeo, Wee M; Chow, Vincent T K

    2007-04-01

    Enterovirus 71 (EV71) is a major etiological agent of hand, foot and mouth disease (HFMD). Several outbreaks in East Asia were associated with neurological complications and numerous deaths. EV71 possesses four structural proteins VP1-VP4 that are necessary in the formation of the pentameric icosahedral capsid. The viral capsid contributes to virulence, and VP1 is a prime target for EV71 vaccine development. Using yeast two-hybrid analysis, we demonstrated binding affinity between VP1 and three human proteins, i.e. ornithine decarboxylase (ODC1), gene trap ankyrin repeat (GTAR), and KIAA0697 expressed in brain tissue. These interactions were authenticated by co-immunoprecipitation experiments, and by indirect immunofluorescent confocal microscopy of transfected and EV71-infected Vero cells. The significant interaction between VP1 and ODC1 may compromise the latter's activity, and interfere with polyamine biosynthesis, growth and proliferation of EV71-infected cells. The interaction between VP1 and GTAR is noteworthy, since ankyrin proteins are associated with certain neural cell adhesion molecules and with the CRASH neurological syndrome. Given that VP1 is synthesized in large amounts during productive infection, these viral-host protein interactions may provide insights into the role of VP1 in the pathogenesis of EV71 disease and its neurological complications such as acute flaccid paralysis and encephalitis.

  3. Molecular identification and characterization of the pyruvate decarboxylase gene family associated with latex regeneration and stress response in rubber tree.

    PubMed

    Long, Xiangyu; He, Bin; Wang, Chuang; Fang, Yongjun; Qi, Jiyan; Tang, Chaorong

    2015-02-01

    In plants, ethanolic fermentation occurs not only under anaerobic conditions but also under aerobic conditions, and involves carbohydrate and energy metabolism. Pyruvate decarboxylase (PDC) is the first and the key enzyme of ethanolic fermentation, which branches off the main glycolytic pathway at pyruvate. Here, four PDC genes were isolated and identified in a rubber tree, and the protein sequences they encode are very similar. The expression patterns of HbPDC4 correlated well with tapping-simulated rubber productivity in virgin rubber trees, indicating it plays an important role in regulating glycometabolism during latex regeneration. HbPDC1, HbPDC2 and HbPDC3 had striking expressional responses in leaves and bark to drought, low temperature and high temperature stresses, indicating that the HbPDC genes are involve in self-protection and defense in response to various abiotic and biotic stresses during rubber tree growth and development. To understand ethanolic fermentation in rubber trees, it will be necessary to perform an in-depth study of the regulatory pathways controlling the HbPDCs in the future.

  4. Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases from Thermotoga maritima and Thermotoga hypogea.

    PubMed

    Eram, Mohammad S; Wong, Alton; Oduaran, Erica; Ma, Kesen

    2015-12-01

    Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

  5. Crystal structure of tyrosine decarboxylase and identification of key residues involved in conformational swing and substrate binding

    PubMed Central

    Zhu, Haixia; Xu, Guochao; Zhang, Kai; Kong, Xudong; Han, Ruizhi; Zhou, Jiahai; Ni, Ye

    2016-01-01

    Tyrosine decarboxylase (TDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme and is mainly responsible for the synthesis of tyramine, an important biogenic amine. In this study, the crystal structures of the apo and holo forms of Lactobacillus brevis TDC (LbTDC) were determined. The LbTDC displays only 25% sequence identity with the only reported TDC structure. Site-directed mutagenesis of the conformationally flexible sites and catalytic center was performed to investigate the potential catalytic mechanism. It was found that H241 in the active site plays an important role in PLP binding because it has different conformations in the apo and holo structures of LbTDC. After binding to PLP, H241 rotated to the position adjacent to the PLP pyridine ring. Alanine scanning mutagenesis revealed several crucial regions that determine the substrate specificity and catalytic activity. Among the mutants, the S586A variant displayed increased catalytic efficiency and substrate affinity, which is attributed to decreased steric hindrance and increased hydrophobicity, as verified by the saturation mutagenesis at S586. Our results provide structural information about the residues important for the protein engineering of TDC to improve catalytic efficiency in the green manufacturing of tyramine. PMID:27292129

  6. Reduction of Oxalate Levels in Tomato Fruit and Consequent Metabolic Remodeling Following Overexpression of a Fungal Oxalate Decarboxylase1[W

    PubMed Central

    Chakraborty, Niranjan; Ghosh, Rajgourab; Ghosh, Sudip; Narula, Kanika; Tayal, Rajul; Datta, Asis; Chakraborty, Subhra

    2013-01-01

    The plant metabolite oxalic acid is increasingly recognized as a food toxin with negative effects on human nutrition. Decarboxylative degradation of oxalic acid is catalyzed, in a substrate-specific reaction, by oxalate decarboxylase (OXDC), forming formic acid and carbon dioxide. Attempts to date to reduce oxalic acid levels and to understand the biological significance of OXDC in crop plants have met with little success. To investigate the role of OXDC and the metabolic consequences of oxalate down-regulation in a heterotrophic, oxalic acid-accumulating fruit, we generated transgenic tomato (Solanum lycopersicum) plants expressing an OXDC (FvOXDC) from the fungus Flammulina velutipes specifically in the fruit. These E8.2-OXDC fruit showed up to a 90% reduction in oxalate content, which correlated with concomitant increases in calcium, iron, and citrate. Expression of OXDC affected neither carbon dioxide assimilation rates nor resulted in any detectable morphological differences in the transgenic plants. Comparative proteomic analysis suggested that metabolic remodeling was associated with the decrease in oxalate content in transgenic fruit. Examination of the E8.2-OXDC fruit proteome revealed that OXDC-responsive proteins involved in metabolism and stress responses represented the most substantially up- and down-regulated categories, respectively, in the transgenic fruit, compared with those of wild-type plants. Collectively, our study provides insights into OXDC-regulated metabolic networks and may provide a widely applicable strategy for enhancing crop nutritional value. PMID:23482874

  7. The Krebs Cycle Enzyme α-Ketoglutarate Decarboxylase Is an Essential Glycosomal Protein in Bloodstream African Trypanosomes

    PubMed Central

    Sykes, Steven; Szempruch, Anthony

    2014-01-01

    α-Ketoglutarate decarboxylase (α-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei. The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, α-KDE1 was expressed in BF T. brucei and RNA interference knockdown of α-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of α-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of α-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of α-KDE1 contain overlapping mitochondrion- and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei. These results suggest that α-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei. PMID:25416237

  8. Genetic basis of stage-specific melanism: a putative role for a cysteine sulfinic acid decarboxylase in insect pigmentation.

    PubMed

    Saenko, S V; Jerónimo, M A; Beldade, P

    2012-06-01

    Melanism, the overall darkening of the body, is a widespread form of animal adaptation to particular environments, and includes bookcase examples of evolution by natural selection, such as industrial melanism in the peppered moth. The major components of the melanin biosynthesis pathway have been characterized in model insects, but little is known about the genetic basis of life-stage specific melanism such as cases described in some lepidopteran species. Here, we investigate two melanic mutations of Bicyclus anynana butterflies, called Chocolate and melanine, that exclusively affect pigmentation of the larval and adult stages, respectively. Our analysis of Mendelian segregation patterns reveals that the larval and adult melanic phenotypes are due to alleles at different, independently segregating loci. Our linkage mapping analysis excludes the pigmentation candidate gene black as the melanine locus, and implicates a gene encoding a putative pyridoxal phosphate-dependant cysteine sulfinic acid decarboxylase as the Chocolate locus. We show variation in coding sequence and in expression levels for this candidate larval melanism locus. This is the first study that suggests a biological function for this gene in insects. Our findings open up exciting opportunities to study the role of this locus in the evolution of adaptive variation in pigmentation, and the uncoupling of regulation of pigment biosynthesis across developmental stages with different ecologies and pressures on body coloration.

  9. Genetic basis of stage-specific melanism: a putative role for a cysteine sulfinic acid decarboxylase in insect pigmentation

    PubMed Central

    Saenko, S V; Jerónimo, M A; Beldade, P

    2012-01-01

    Melanism, the overall darkening of the body, is a widespread form of animal adaptation to particular environments, and includes bookcase examples of evolution by natural selection, such as industrial melanism in the peppered moth. The major components of the melanin biosynthesis pathway have been characterized in model insects, but little is known about the genetic basis of life-stage specific melanism such as cases described in some lepidopteran species. Here, we investigate two melanic mutations of Bicyclus anynana butterflies, called Chocolate and melanine, that exclusively affect pigmentation of the larval and adult stages, respectively. Our analysis of Mendelian segregation patterns reveals that the larval and adult melanic phenotypes are due to alleles at different, independently segregating loci. Our linkage mapping analysis excludes the pigmentation candidate gene black as the melanine locus, and implicates a gene encoding a putative pyridoxal phosphate-dependant cysteine sulfinic acid decarboxylase as the Chocolate locus. We show variation in coding sequence and in expression levels for this candidate larval melanism locus. This is the first study that suggests a biological function for this gene in insects. Our findings open up exciting opportunities to study the role of this locus in the evolution of adaptive variation in pigmentation, and the uncoupling of regulation of pigment biosynthesis across developmental stages with different ecologies and pressures on body coloration. PMID:22234245

  10. Enhanced triterpene accumulation in Panax ginseng hairy roots overexpressing mevalonate-5-pyrophosphate decarboxylase and farnesyl pyrophosphate synthase.

    PubMed

    Kim, Yong-Kyoung; Kim, Yeon Bok; Uddin, Md Romij; Lee, Sanghyun; Kim, Soo-Un; Park, Sang Un

    2014-10-17

    To elucidate the function of mevalonate-5-pyrophosphate decarboxylase (MVD) and farnesyl pyrophosphate synthase (FPS) in triterpene biosynthesis, the genes governing the expression of these enzymes were transformed into Panax ginseng hairy roots. All the transgenic lines showed higher expression levels of PgMVD and PgFPS than that by the wild-type control. Among the hairy root lines transformed with PgMVD, M18 showed the highest level of transcription compared to the control (14.5-fold higher). Transcriptions of F11 and F20 transformed with PgFPS showed 11.1-fold higher level compared with control. In triterpene analysis, M25 of PgMVD produced 4.4-fold higher stigmasterol content (138.95 μg/100 mg, dry weight [DW]) than that by the control; F17 of PgFPS showed the highest total ginsenoside (36.42 mg/g DW) content, which was 2.4-fold higher compared with control. Our results indicate that metabolic engineering in P. ginseng was successfully achieved through Agrobacterium rhizogenes-mediated transformation and that the accumulation of phytosterols and ginsenosides was enhanced by introducing the PgMVD and PgFPS genes into the hairy roots of the plant. Our results suggest that PgMVD and PgFPS play an important role in the triterpene biosynthesis of P. ginseng.

  11. Newly-diagnosed pediatric epilepsy is associated with elevated autoantibodies to glutamic acid decarboxylase but not cardiolipin.

    PubMed

    Veri, Kadi; Uibo, Oivi; Talvik, Tiina; Talvik, Inga; Metsküla, Kaja; Napa, Aita; Vaher, Ulvi; Õiglane-Šlik, Eve; Rein, Reet; Kolk, Anneli; Traat, Aili; Uibo, Raivo

    2013-07-01

    Glutamic acid decarboxylase autoantibodies (GADA) and anti-cardiolipin autoantibodies (ACA) have been detected in adult subjects with epilepsy, though the functional implications of these findings are a matter of debate. This study aimed to determine the prevalence of GADA and ACA and to investigate their clinical significance in pediatric subjects with newly-diagnosed epilepsy. For this purpose GADA and ACA were assessed by enzyme-linked immunosorbent assays in 208 pediatric patients with newly-diagnosed epilepsy and 128 controls. The clinical data (results of electroencephalography, magnetic resonance imaging, 6-month outcome etc.) was compared to antibody test results. Our study revealed GADA in 14 (6.7%) patients with epilepsy and in 1 (0.8%) control, which was a statistically significant difference (P=0.010; Chi-square test). The GADA-positive and -negative patients had similar clinical characteristics. The prevalence of ACA in patients with epilepsy (6.3%) was not significantly different than controls (2.6%). These results suggest that GADA is associated with epilepsy in a subgroup of newly-diagnosed pediatric patients. Further studies are required to determine the prognostic significance and pathogenic role of GADA among pediatric subjects with epilepsy.

  12. Ornithine decarboxylase, polyamines and CD11b expression in HL-60 cells during differentiation induced by retinoic acid.

    PubMed

    Stabellini, Giordano; Brugnoli, F; Calastrini, C; Vizzotto, L; Vertemati, M; Baroni, T; Caramelli, E; Marinucci, L; Pellati, A; Bertagnolo, V

    2004-01-01

    Polyamines (PA) and retinoic acid affect mammalian cell growth, differentiation and apoptosis. Retinoic acid induces granulocytic differentiation of mieloid cell lines and, during this process, is responsible for the expression of CD11b, a surface antigen. In this study we investigate the effects of retinoic acid on HL-60 cells, monitoring ornithine decarboxylase (ODC) activity (enzyme rate of PA), putrescine (PUT), spermidine (SPD), spermine (SPM) levels, CD11b myeloid surface marker differentiation, cell cycle, and apoptosis. ODC activity and PUT levels are correlated with mieloid cell differentiation induced by retinoic acid treatment. Only the ODC/PUT ratio is connected with retinoic acid treated HL-60 cells. Treated cultures show a decrease of proliferation and a cell block in the G0/G1 phase, with consequent diminished S phase. The G0/G1 and S phases are significantly related to ODC activity and to PUT and SPD behavior, whereas in differentiating condition only the decrease of PUT is related to the S phase. CD11b expression, stimulated by retinoic acid treatment, is associated with the SPM trend. Total PA behavior agrees with apoptotic cell increase after 96 h of stimulation. Our data show that retinoic acid treatment modifies ODC activity and the turnover of PA. PUT, SPD and SPM, therefore, have a different role, and may be involved in the differentiative/apoptotic program of retinoic acid treated HL-60 cells.

  13. Dynamic changes in gamma-aminobutyric acid and glutamate decarboxylase activity in oats (Avena nuda L.) during steeping and germination.

    PubMed

    Xu, Jian Guo; Hu, Qing Ping; Duan, Jiang Lian; Tian, Cheng Rui

    2010-09-08

    Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system and provides beneficial effects for human and other animals health. To accumulate GABA, samples from two different naked oat cultivars, Baiyan II and Bayou I, were steeped and germinated in an incubator. The content of GABA and glutamic acid as well as the activity of the glutamate decarboxylase (GAD) in oats during steeping and germination were investigated with an amino acid automatic analyzer. Compared with raw groats, an increase in GABA content of oat groats during steeping and germination was continuously observed for two oat cultivars. The activity of GAD increased greatly at the end of steeping and the second stage of germination for Baiyan II and Bayou I, respectively. Glutamic acid content of treated oat groats was significantly lower than that in raw groats until the later period of germination. GABA was correlated (p<0.01) significantly and positively with the glutamic acid rather than GAD activity in the current study. The results indicates that steeping and germination process under highly controlled conditions can effectively accumulate the GABA in oat groats for Baiyan II and Bayou I, which would greatly facilitate production of nutraceuticals or food ingredients that enable consumers to gain greater access to the health benefits of oats. However, more assays need to be further performed with more oat cultivars.

  14. Pyruvate decarboxylase and alcohol dehydrogenase overexpression in Escherichia coli resulted in high ethanol production and rewired metabolic enzyme networks.

    PubMed

    Yang, Mingfeng; Li, Xuefeng; Bu, Chunya; Wang, Hui; Shi, Guanglu; Yang, Xiushan; Hu, Yong; Wang, Xiaoqin

    2014-11-01

    Pyruvate decarboxylase and alcohol dehydrogenase are efficient enzymes for ethanol production in Zymomonas mobilis. These two enzymes were over-expressed in Escherichia coli, a promising candidate for industrial ethanol production, resulting in high ethanol production in the engineered E. coli. To investigate the intracellular changes to the enzyme overexpression for homoethanol production, 2-DE and LC-MS/MS were performed. More than 1,000 protein spots were reproducibly detected in the gel by image analysis. Compared to the wild-type, 99 protein spots showed significant changes in abundance in the recombinant E. coli, in which 46 were down-regulated and 53 were up-regulated. Most proteins related to tricarboxylic acid cycle, glycerol metabolism and other energy metabolism were up-regulated, whereas proteins involved in glycolysis and glyoxylate pathway were down-regulated, indicating the rewired metabolism in the engineered E. coli. As glycolysis is the main pathway for ethanol production, and it was inhibited significantly in engineered E. coli, further efforts should be directed at minimizing the repression of glycolysis to optimize metabolism network for higher yields of ethanol production.

  15. A polymorphic (GA/CT)n- SSR influences promoter activity of Tryptophan decarboxylase gene in Catharanthus roseus L. Don

    PubMed Central

    Kumar, Santosh; Bhatia, Sabhyata

    2016-01-01

    Simple Sequence Repeats (SSRs) of polypurine-polypyrimidine type motifs occur very frequently in the 5′ flanks of genes in plants and have recently been implicated to have a role in regulation of gene expression. In this study, 2 accessions of Catharanthus roseus having (CT)8 and (CT)21 varying motifs in the 5′UTR of Tryptophan decarboxylase (Tdc) gene, were investigated for its role in regulation of gene expression. Extensive Tdc gene expression analysis in the 2 accessions was carried out both at the level of transcription and translation. Transcript abundance was estimated using Northern analysis and qRT-PCR, whereas the rate of Tdc gene transcription was assessed using in-situ nuclear run-on transcription assay. Translation status of Tdc gene was monitored by quantification of polysome associated Tdc mRNA using qRT-PCR. These observations were validated through transient expression analysis using the fusion construct [CaM35S:(CT)8–21:GUS]. Our study demonstrated that not only does the length of (CT)n -SSRs influences the promoter activity, but the presence of SSRs per se in the 5′-UTR significantly enhances the level of gene expression. We termed this phenomenon as “microsatellite mediated enhancement” (MME) of gene expression. Results presented here will provide leads for engineering plants with enhanced amounts of medicinally important alkaloids. PMID:27623355

  16. Removal kinetics of antibodies against glutamic acid decarboxylase by various plasmapheresis modalities in the treatment of neurological disorders.

    PubMed

    Ohkubo, Atsushi; Okado, Tomokazu; Kurashima, Naoki; Maeda, Takuma; Miyamoto, Satoko; Nakamura, Ayako; Seshima, Hiroshi; Iimori, Soichiro; Sohara, Eisei; Uchida, Shinichi; Rai, Tatemitsu

    2014-06-01

    Plasmapheresis is one of the acute treatment modalities for neurological disorders associated with antibodies against glutamic acid decarboxylase (anti-GAD). However, there is little information about the removal kinetics of anti-GAD by various plasmapheresis modalities. Here, we investigated the removal rate of anti-GAD and fibrinogen (Fib) by immunoadsorption (IA), plasma exchange using a conventional plasma separator (OP-PE), and plasma exchange using a high cut-off selective membrane plasma separator (EC-PE) in two cases of anti-GAD-associated neurological diseases. In case 1, IA and OP-PE were used, and the percent reductions were as follows: anti-GAD: 38.2% and 69.1% and Fib: 67.7% and 68.2%, respectively. In case 2, OP-PE and EC-PE were used, and the percent reductions were as follows: anti-GAD: 65.8% and 48.5% and Fib: 68.5% and 19.8%, respectively. OP-PE could remove anti-GAD more efficiently than IA. Further, EC-PE could maintain coagulation factors such as Fib better than IA and OP-PE. It is important to select the appropriate plasmapheresis modality on the basis of the removal kinetics.

  17. Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution

    PubMed Central

    Sagong, Hye-Young; Kim, Kyung-Jin

    2017-01-01

    Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires continuous supplement of large amounts of PLP. In order to develop an LDC enzyme from Selenomonas ruminantium (SrLDC) with an enhanced affinity for PLP, we introduced an internal disulfide bond between Ala225 and Thr302 residues with a desire to retain the PLP binding site in a closed conformation. The SrLDCA225C/T302C mutant showed a yellow color and the characteristic UV/Vis absorption peaks for enzymes with bound PLP, and exhibited three-fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibited a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Moreover, introduction of the disulfide bond rendered SrLDC more resistant to high pH and temperature. The formation of the introduced disulfide bond and the maintenance of the PLP binding site in the closed conformation were confirmed by determination of the crystal structure of the mutant. This study shows that disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced PLP affinity. PMID:28095457

  18. Assessment of virulence factors, antibiotic resistance and amino-decarboxylase activity in Enterococcus faecium MXVK29 isolated from Mexican chorizo.

    PubMed

    Alvarez-Cisneros, Y M; Fernández, F J; Sainz-Espuñez, T; Ponce-Alquicira, E

    2017-02-01

    Enterococcus faecium MXVK29 has the ability to produce an antimicrobial compound that belongs to Class IIa of the Klaenhammer classification, and could be used as part of a biopreservation technology through direct inoculation of the strain as a starter or protective culture. However, Enterococcus is considered as an opportunistic pathogen, hence, the purpose of this work was to study the food safety determinants of E. faecium MXVK29. The strain was sensitive to all of the antibiotics tested (penicillin, tetracycline, vancomycin, erythromycin, chloramphenicol, gentamicin, neomycin, kanamycin and netilmicin) and did not demonstrate histamine, cadaverine or putrescine formation. Furthermore, tyrosine-decarboxylase activity was detected by qualitative assays and PCR. Among the virulence factors analysed for the strain, only the genes encoding the sexual pheromone cCF10 precursor lipoprotein (ccf) and cell-wall adhesion (efaAfm ) were amplified. The presence of these genes has low impact on pathogenesis, as there are no other genes encoding for virulence factors, such as aggregation proteins. Therefore, Enterococcus faecium could be employed as part of a bioconservation method, because it does not produce risk factors for consumer's health; in addition, it could be used as part of the hurdle technology in foods.

  19. Active site binding modes of inhibitors of Staphylococcus aureus mevalonate diphosphate decarboxylase from docking and molecular dynamics simulations.

    PubMed

    Addo, James K; Skaff, D Andrew; Miziorko, Henry M

    2016-01-01

    Bacterial mevalonate diphosphate decarboxylase (MDD) is an attractive therapeutic target for antibacterial drug development. In this work, we discuss a combined docking and molecular dynamics strategy toward inhibitor binding to bacterial MDD. The docking parameters utilized in this study were first validated with observations for the inhibitors 6-fluoromevalonate diphosphate (FMVAPP) and diphosphoglycolylproline (DPGP) using existing structures for the Staphylococcus epidermidis enzyme. The validated docking protocol was then used to predict structures of the inhibitors bound to Staphylococcus aureus MDD using the unliganded crystal structure of Staphylococcus aureus MDD. We also investigated a possible interactions improvement by combining this docking method with molecular dynamics simulations. Thus, the predicted docking structures were analyzed in a molecular dynamics trajectory to generate dynamic models and reinforce the predicted binding modes. FMVAPP is predicted to make more extensive contacts with S. aureus MDD, forming stable hydrogen bonds with Arg144, Arg193, Lys21, Ser107, and Tyr18, as well as making stable hydrophobic interactions with Tyr18, Trp19, and Met196. The differences in predicted binding are supported by experimentally determined Ki values of 0.23 ± 0.02 and 34 ± 8 μM, for FMVAPP and DPGP, respectively. The structural information coupled with the kinetic characterization obtained from this study should be useful in defining the requirements for inhibition as well as in guiding the selection of active compounds for inhibitor optimization.

  20. Tomato aromatic amino acid decarboxylases participate in synthesis of the flavor volatiles 2-phenylethanol and 2-phenylacetaldehyde

    PubMed Central

    Tieman, Denise; Taylor, Mark; Schauer, Nicolas; Fernie, Alisdair R.; Hanson, Andrew D.; Klee, Harry J.

    2006-01-01

    An important phenylalanine-derived volatile compound produced by plants is 2-phenylethanol. It is a major contributor to flavor in many foods, including fresh fruits, such as tomato, and an insect-attracting scent in roses and many other flowers. Despite the centrality of 2-phenylethanol to flavor and fragrance, the plant genes responsible for its synthesis have not been identified. Here, we describe a biosynthetic pathway for 2-phenylethanol and other phenylalanine-derived volatiles in tomato fruits and a small family of decarboxylases (LeAADC1A, LeAADC1B, and LeAADC2) that can mediate that pathway's first step. These enzymes each catalyze conversion of phenylalanine to phenethylamine and tyrosine to tyramine. Although tyrosine is the preferred substrate in vitro, phenylalanine levels in tomato fruits far exceed those of tyrosine, indicating that phenylalanine is a physiological substrate. Consistent with this view, overexpression of either LeAADC1A or LeAADC2 in transgenic tomato plants resulted in fruits with up to 10-fold increased emissions of the products of the pathway, including 2-phenylacetaldehyde, 2-phenylethanol, and 1-nitro-2-phenylethane. Further, antisense reduction of LeAADC2 significantly reduced emissions of these volatiles. Besides establishing a biosynthetic route, these results show that it is possible to change phenylalanine-based flavor and aroma volatiles in plants by manipulating expression of a single gene. PMID:16698923

  1. The Histidine Decarboxylase Gene Cluster of Lactobacillus parabuchneri Was Gained by Horizontal Gene Transfer and Is Mobile within the Species

    PubMed Central

    Wüthrich, Daniel; Berthoud, Hélène; Wechsler, Daniel; Eugster, Elisabeth; Irmler, Stefan; Bruggmann, Rémy

    2017-01-01

    Histamine in food can cause intolerance reactions in consumers. Lactobacillus parabuchneri (L. parabuchneri) is one of the major causes of elevated histamine levels in cheese. Despite its significant economic impact and negative influence on human health, no genomic study has been published so far. We sequenced and analyzed 18 L. parabuchneri strains of which 12 were histamine positive and 6 were histamine negative. We determined the complete genome of the histamine positive strain FAM21731 with PacBio as well as Illumina and the genomes of the remaining 17 strains using the Illumina technology. We developed the synteny aware ortholog finding algorithm SynOrf to compare the genomes and we show that the histidine decarboxylase (HDC) gene cluster is located in a genomic island. It is very likely that the HDC gene cluster was transferred from other lactobacilli, as it is highly conserved within several lactobacilli species. Furthermore, we have evidence that the HDC gene cluster was transferred within the L. parabuchneri species. PMID:28261177

  2. Chronic social subordination stress modulates glutamic acid decarboxylase (GAD) 67 mRNA expression in central stress circuits

    PubMed Central

    Makinson, Ryan; Lundgren, Kerstin H.; Seroogy, Kim B.; Herman, James P.

    2015-01-01

    Chronic social subordination is a well-known precipitant of numerous psychiatric and physiological health concerns. In this study, we examine the effects of chronic social stress in the visible burrow system (VBS) on the expression of glutamic acid decarboxylase (GAD) 67 and brain-derived neurotropic factor (BDNF) mRNA in forebrain stress circuitry. Male rats in the VBS system form a dominance hierarchy, whereby subordinate males exhibit neuroendocrine and physiological profiles characteristic of chronic exposure to stress. We found that social subordination decreases GAD67 mRNA in the peri-paraventricular nucleus region of the hypothalamus and the interfascicular nucleus of the bed nucleus of the stria terminalis (BNST), and increases in GAD67 mRNA in the hippocampus, medial prefrontal cortex, and dorsal medial hypothalamus. Expression of BDNF mRNA increased in the dorsal region of the BNST, but remained unchanged in all other regions examined. Results from this study indicate that social subordination is associated with several region-specific alterations in GAD67 mRNA expression in central stress circuits, whereas changes in the expression of BDNF mRNA are limited to the BNST. PMID:26066725

  3. Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

    PubMed

    Noda, Shuhei; Shirai, Tomokazu; Mochida, Keiichi; Matsuda, Fumio; Oyama, Sachiko; Okamoto, Mami; Kondo, Akihiko

    2015-01-01

    To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC), which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L) than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

  4. SIRT4 coordinates the balance between lipid synthesis and catabolism by repressing malonyl CoA decarboxylase

    PubMed Central

    Laurent, Gaëlle; German, Natalie J.; Saha, Asish K.; de Boer, Vincent C. J.; Davies, Michael; Koves, Timothy R.; Dephoure, Noah; Fischer, Frank; Boanca, Gina; Vaitheesvaran, Bhavapriya; Lovitch, Scott B.; Sharpe, Arlene H.; Kurland, Irwin J.; Steegborn, Clemens; Gygi, Steven P.; Muoio, Deborah M.; Ruderman, Neil B.; Haigis, Marcia C.

    2013-01-01

    Summary Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a novel regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a novel SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis. PMID:23746352

  5. p-Coumaric acid decarboxylase from Lactobacillus plantarum: structural insights into the active site and decarboxylation catalytic mechanism.

    PubMed

    Rodríguez, Héctor; Angulo, Iván; de Las Rivas, Blanca; Campillo, Nuria; Páez, Juan A; Muñoz, Rosario; Mancheño, José M

    2010-05-15

    p-Coumaric acid decarboxylases (PDCs) catalyze the nonoxidative decarboxylation of hydroxycinnamic acids to generate the corresponding vinyl derivatives. Despite the biotechnological relevance of PDCs in food industry, their catalytic mechanism remains largely unknown. Here, we report insights into the structural basis of catalysis for the homodimeric PDC from Lactobacillus plantarum (LpPDC). The global fold of LpPDC is based on a flattened beta-barrel surrounding an internal cavity. Crystallographic and functional analyses of single-point mutants of residues located within this cavity have permitted identifying a potential substrate-binding pocket and also to provide structural evidences for rearrangements of surface loops so that they can modulate the accessibility to the active site. Finally, combination of the structural and functional data with in silico results enables us to propose a two-step catalytic mechanism for decarboxylation of p-coumaric acid by PDCs where Glu71 is involved in proton transfer, and Tyr18 and Tyr20 are involved in the proper substrate orientation and in the release of the CO(2) product.

  6. Enhanced succinic acid production under acidic conditions by introduction of glutamate decarboxylase system in E. coli AFP111.

    PubMed

    Wu, Mingke; Li, Xiaozhan; Guo, Shunfeng; Lemma, Wubliker Dessie; Zhang, Wenming; Ma, Jiangfeng; Jia, Honghua; Wu, Hao; Jiang, Min; Ouyang, Pingkai

    2017-04-01

    Biological synthesis of succinic acid at low pH values was favored since it not only decreased investment cost but also simplified downstream purification process. In this study, the feasibility of using glutamate decarboxylase system to improve succinic acid production of Escherichia coli AFP111, a succinate-producing candidate with mutations in pfl, ldhA, and ptsG, under acidic conditions was investigated. By overexpressing gadBC operon in AFP111, a recombinant named as BA201 (AFP111/pMD19T-gadBC) was constructed. Fermentation at pH 5.6 showed that 30 g L(-1) glucose was consumed and 26.58 g L(-1) succinic acid was produced by BA201, which was 1.22- and 1.32-fold higher than that by the control BA200 (AFP111/pMD19T) containing the empty vector. Analysis of intracellular enzymes activities and ATP concentrations revealed that the activities of key enzymes involved in glucose uptake and products synthesis and intracellular ATP levels were all increased after overexpression of gadBC which were benefit for cell metabolism under weak acidic conditions. To further improve the succinic acid titer by recombinant BA201 at pH 5.6, the extracellular glutamate concentration was optimized and the final succinic acid titer increased 20.4% to 32.01 g L(-1). Besides, the fermentation time was prolonged by repetitive fermentation and additional 15.78 g L(-1) succinic acid was produced by recovering cells into fresh medium. The results here demonstrated a potential strategy of overexpressing gadBC for increased succinic acid production of E. coli AFP111 under weak acidic conditions.

  7. Modulation of arginine decarboxylase activity from Mycobacterium smegmatis. Evidence for pyridoxal-5'-phosphate-mediated conformational changes in the enzyme.

    PubMed

    Balasundaram, D; Tyagi, A K

    1989-08-01

    Arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19) from Mycobacterium smegmatis, TMC 1546 has been purified to homogeneity. The enzyme has a molecular mass of 232 kDa and a subunit mass of 58.9 kDa. The enzyme from mycobacteria is totally dependent on pyridoxal 5'-phosphate for its activity at its optimal pH and, unlike that from Escherichia coli, Mg2+ does not play an active role in the enzyme conformation. The enzyme is specific for arginine (Km = 1.6 mM). The holoenzyme is completely resolved in dialysis against hydroxylamine. Reconstitution of the apoenzyme with pyridoxal 5'-phosphate shows sigmoidal binding characteristics at pH 8.4 with a Hill coefficient of 2.77, whereas at pH 6.2 the binding is hyperbolic in nature. The kinetics of reconstitution at pH 8.4 are apparently sigmoidal, indicating the occurrence of two binding types of differing strengths. A low-affinity (Kd = 22.5 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations and a high-affinity (Kd = 3.0 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations. The restoration of full activity occurred in parallel with the tight binding (high affinity) of pyridoxal 5'-phosphate to the apoenzyme. Along with these characteristics, spectral analyses of holoenzyme and apoenzyme at pH 8.4 and pH 6.2 indicate a pH-dependent modulation of coenzyme function. Based on the pH-dependent changes in the polarity of the active-site environment, pyridoxal 5'-phosphate forms different Schiff-base tautomers at pH 8.4 and pH 6.2 with absorption maxima at 415 nm and 333 nm, respectively. These separate forms of Schiff-base confer different catalytic efficiencies to the enzyme.

  8. Role of ornithine decarboxylase in regulation of estrogen receptor alpha expression and growth in human breast cancer cells

    PubMed Central

    Zhu, Qingsong; Jin, Lihua; Casero, Robert A.

    2013-01-01

    Our previous studies demonstrated that specific polyamine analogues, oligoamines, down-regulated the activity of a key polyamine biosynthesis enzyme, ornithine decarboxylase (ODC), and suppressed expression of estrogen receptor alpha (ERα) in human breast cancer cells. However, the mechanism underlying the potential regulation of ERα expression by polyamine metabolism has not been explored. Here, we demonstrated that RNAi-mediated knockdown of ODC (ODC KD) down-regulated the polyamine pool, and hindered growth in ERα-positive MCF7 and T47D and ERα-negative MDA-MB-231 breast cancer cells. ODC KD significantly induced the expression and activity of the key polyamine catabolism enzymes, spermine oxidase (SMO) and spermidine/spermine N1-acetyltransferase (SSAT). However, ODC KD-induced growth inhibition could not be reversed by exogenous spermidine or overexpression of antizyme inhibitor (AZI), suggesting that regulation of ODC on cell proliferation may involve the signaling pathways independent of polyamine metabolism. In MCF7 and T47D cells, ODC KD, but not DFMO treatment, diminished the mRNA and protein expression of ERα. Overexpression of antizyme (AZ), an ODC inhibitory protein, suppressed ERα expression, suggesting that ODC plays an important role in regulation of ERα expression. Decrease of ERα expression by ODC siRNA altered the mRNA expression of a subset of ERα response genes. Our previous analysis showed that oligoamines disrupt the binding of Sp1 family members to an ERα minimal promoter element containing GC/CA-rich boxes. By using DNA affinity precipitation and mass spectrometry analysis, we identified ZBTB7A, MeCP2, PARP-1, AP2, and MAZ as co-factors of Sp1 family members that are associated with the ERα minimal promoter element. Taken together, these data provide insight into a novel antiestrogenic mechanism for polyamine biosynthesis enzymes in breast cancer. PMID:22976807

  9. Pronounced reduction in adenoma recurrence associated with aspirin use and a polymorphism in the ornithine decarboxylase gene

    PubMed Central

    Martínez, María Elena; O'Brien, Thomas G.; Fultz, Kimberly E.; Babbar, Naveen; Yerushalmi, Hagit; Qu, Ning; Guo, Yongjun; Boorman, David; Einspahr, Janine; Alberts, David S.; Gerner, Eugene W.

    2003-01-01

    Most sporadic colon adenomas acquire mutations in the adenomatous polyposis coli gene (APC) and show defects in APC-dependent signaling. APC influences the expression of several genes, including the c-myc oncogene and its antagonist Mad1. Ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is a transcriptional target of c-myc and a modifier of APC-dependent tumorigenesis. A single-nucleotide polymorphism exists in intron 1 of the human ODC gene, which lies between two myc-binding domains. This region is known to affect ODC transcription, but no data exist on the relationship of this polymorphism to risk of colorectal neoplasia in humans. We show that individuals homozygous for the minor ODC A-allele who reported using aspirin are ≈0.10 times as likely to have an adenoma recurrence as non-aspirin users homozygous for the major G-allele. Mad1 selectively suppressed the activity of the ODC promoter containing the A-allele, but not the G-allele, in a human colon cancer-derived cell line (HT29). Aspirin (≥10 μM) did not affect ODC allele-specific promoter activity but did activate polyamine catabolism and lower polyamine content in HT29 cells. We propose that the ODC polymorphism and aspirin act independently to reduce the risk of adenoma recurrence by suppressing synthesis and activating catabolism, respectively, of colonic mucosal polyamines. These findings confirm the hypothesis that the ODC polymorphism is a genetic marker for colon cancer risk, and support the use of ODC inhibitors and aspirin, or other nonsteroidal antiinflammatory drugs (NSAIDs), in combination as a strategy for colon cancer prevention. PMID:12810952

  10. The polyamine metabolism genes ornithine decarboxylase and antizyme 2 predict aggressive behavior in neuroblastomas with and without MYCN amplification

    PubMed Central

    Geerts, Dirk; Koster, Jan; Albert, David; Koomoa, Dana-Lynn T.; Feith, David J.; Pegg, Anthony E.; Volckmann, Richard; Caron, Huib; Versteeg, Rogier; Bachmann, André S.

    2010-01-01

    High polyamine (PA) levels and ornithine decarboxylase (ODC) over-expression are well-known phenomena in many aggressive cancer types. We analyzed the expression of ODC and ODC-activity regulating genes antizymes 1-3 (OAZ1-3) and AZ inhibitors 1-2 (AZ-IN1-2) in human neuroblastoma (NB) tumors and correlated these with genetic and clinical features of NB. Since ODC is a known target gene of MYCN, the correlation between ODC and MYCN was of special interest. Data were obtained from Affymetrix micro-array analysis of 88 NB tumor samples. In addition, mRNA expression levels of ODC, OAZ2, and MYCN in a MYCN-inducible NB cell line were determined by quantitative real-time RT-PCR. ODC mRNA expression in NB tumors was significantly predictive of decreased overall survival probability and correlated with several unfavorable clinical NB characteristics (all P < 0.005). Interestingly, high ODC mRNA expression also showed significant correlation with poor survival prognosis in Kaplan-Meier analyses stratified for patients without MYCN amplification, suggesting an additional role for ODC independent of MYCN. Conversely, high OAZ2 mRNA expression correlated with increased survival and with several favorable clinical NB characteristics (all P < 0.003). In addition, we provide first evidence of a role for MYCN-associated transcription factors MAD2 and MAD7 in ODC regulation. In NB cell cultures, ectopic over-expression of MYCN altered ODC, but not OAZ2 mRNA levels. In conclusion, these data suggest that elevated ODC and low OAZ2 mRNA expression levels correlate with several unfavorable genetic and clinical features in NB, offering new insights into PA pathways and PA metabolism-targeting therapy in NB. PMID:19960435

  11. Familial porphyria cutanea tarda: characterization of seven novel uroporphyrinogen decarboxylase mutations and frequency of common hemochromatosis alleles.

    PubMed Central

    Mendez, M; Sorkin, L; Rossetti, M V; Astrin, K H; del C Batlle, A M; Parera, V E; Aizencang, G; Desnick, R J

    1998-01-01

    Familial porphyria cutanea tarda (f-PCT) results from the half-normal activity of uroporphyrinogen decarboxylase (URO-D). Heterozygotes for this autosomal dominant trait are predisposed to photosensitive cutaneous lesions by various ecogenic factors, including iron overload and alcohol abuse. The 3.6-kb URO-D gene was completely sequenced, and a long-range PCR method was developed to amplify the entire gene for mutation analysis. Four missense mutations (M165R, L195F, N304K, and R332H), a microinsertion (g10insA), a deletion (g645Delta1053), and a novel exonic splicing defect (E314E) were identified. Expression of the L195F, N304K, and R332H polypeptides revealed significant residual activity, whereas reverse transcription-PCR and sequencing demonstrated that the E314E lesion caused abnormal splicing and exon 9 skipping. Haplotyping indicated that three of the four families with the g10insA mutation were unrelated, indicating that these microinsertions resulted from independent mutational events. Screening of nine f-PCT probands revealed that 44% were heterozygous or homozygous for the common hemochromatosis mutations, which suggests that iron overload may predispose to clinical expression. However, there was no clear correlation between f-PCT disease severity and the URO-D and/or hemochromatosis genotypes. These studies doubled the number of known f-PCT mutations, demonstrated that marked genetic heterogeneity underlies f-PCT, and permitted presymptomatic molecular diagnosis and counseling in these families to enable family members to avoid disease-precipitating factors. PMID:9792863

  12. Glutamic acid decarboxylase 65: a link between GABAergic synaptic plasticity in the lateral amygdala and conditioned fear generalization.

    PubMed

    Lange, Maren D; Jüngling, Kay; Paulukat, Linda; Vieler, Marc; Gaburro, Stefano; Sosulina, Ludmila; Blaesse, Peter; Sreepathi, Hari K; Ferraguti, Francesco; Pape, Hans-Christian

    2014-08-01

    An imbalance of the gamma-aminobutyric acid (GABA) system is considered a major neurobiological pathomechanism of anxiety, and the amygdala is a key brain region involved. Reduced GABA levels have been found in anxiety patients, and genetic variations of glutamic acid decarboxylase (GAD), the rate-limiting enzyme of GABA synthesis, have been associated with anxiety phenotypes in both humans and mice. These findings prompted us to hypothesize that a deficiency of GAD65, the GAD isoform controlling the availability of GABA as a transmitter, affects synaptic transmission and plasticity in the lateral amygdala (LA), and thereby interferes with fear responsiveness. Results indicate that genetically determined GAD65 deficiency in mice is associated with (1) increased synaptic length and release at GABAergic connections, (2) impaired efficacy of GABAergic synaptic transmission and plasticity, and (3) reduced spillover of GABA to presynaptic GABAB receptors, resulting in a loss of the associative nature of long-term synaptic plasticity at cortical inputs to LA principal neurons. (4) In addition, training with high shock intensities in wild-type mice mimicked the phenotype of GAD65 deficiency at both the behavioral and synaptic level, indicated by generalization of conditioned fear and a loss of the associative nature of synaptic plasticity in the LA. In conclusion, GAD65 is required for efficient GABAergic synaptic transmission and plasticity, and for maintaining extracellular GABA at a level needed for associative plasticity at cortical inputs in the LA, which, if disturbed, results in an impairment of the cue specificity of conditioned fear responses typifying anxiety disorders.

  13. MDMA decreases glutamic acid decarboxylase (GAD) 67-immunoreactive neurons in the hippocampus and increases seizure susceptibility: Role for glutamate.

    PubMed

    Huff, Courtney L; Morano, Rachel L; Herman, James P; Yamamoto, Bryan K; Gudelsky, Gary A

    2016-12-01

    3,4-Methylenedioxy-methamphetamine (MDMA) is a unique psychostimulant that continues to be a popular drug of abuse. It has been well documented that MDMA reduces markers of 5-HT axon terminals in rodents, as well as humans. A loss of parvalbumin-immunoreactive (IR) interneurons in the hippocampus following MDMA treatment has only been documented recently. In the present study, we tested the hypothesis that MDMA reduces glutamic acid decarboxylase (GAD) 67-IR, another biochemical marker of GABA neurons, in the hippocampus and that this reduction in GAD67-IR neurons and an accompanying increase in seizure susceptibility involve glutamate receptor activation. Repeated exposure to MDMA (3×10mg/kg, ip) resulted in a reduction of 37-58% of GAD67-IR cells in the dentate gyrus (DG), CA1, and CA3 regions, as well as an increased susceptibility to kainic acid-induced seizures, both of which persisted for at least 30days following MDMA treatment. Administration of the NMDA antagonist MK-801 or the glutamate transporter type 1 (GLT-1) inducer ceftriaxone prevented both the MDMA-induced loss of GAD67-IR neurons and the increased vulnerability to kainic acid-induced seizures. The MDMA-induced increase in the extracellular concentration of glutamate in the hippocampus was significantly diminished in rats treated with ceftriaxone, thereby implicating a glutamatergic mechanism in the neuroprotective effects of ceftriaxone. In summary, the present findings support a role for increased extracellular glutamate and NMDA receptor activation in the MDMA-induced loss of hippocampal GAD67-IR neurons and the subsequent increased susceptibility to evoked seizures.

  14. The YvrI Alternative σ Factor Is Essential for Acid Stress Induction of Oxalate Decarboxylase in Bacillus subtilis▿ †

    PubMed Central

    MacLellan, Shawn R.; Helmann, John D.; Antelmann, Haike

    2009-01-01

    YvrI is a recently identified alternative σ factor in Bacillus subtilis that requires the coactivator YvrHa to activate transcription. Previously, a strain engineered to overproduce YvrI was found to overproduce oxalate decarboxylase (OxdC), and further analysis identified three YvrI-activated promoters preceding the yvrI-yvrHa, yvrJ, and oxdC-yvrL operons. Independently, proteome analyses identified OxdC as a highly abundant, cell wall-associated protein that accumulated under acidic growth conditions. We show here that the accumulation of OxdC in the cell wall proteome under acidic growth conditions is absolutely dependent on YvrI and is correlated with enhanced transcription of both the yvrI-yvrHa and the oxdC-yvrL operons. Conversely, OxdC accumulates to a high level even under nonacidic growth conditions in cells lacking YvrL, a negative regulator of YvrI/YvrHa-dependent transcription. These results indicate that YvrI and its associated coregulators YvrHa and YvrL are required for the regulation of OxdC expression by acid stress. The high-level accumulation of OxdC depends, in part, on a strong oxdC promoter. A regulatory sequence with similarity to an upstream promoter element (UP) was identified upstream of the oxdC promoter and is required for high-level promoter activity. Conservation of the YvrI/YvrHa/YvrL regulatory system among related species allowed us to deduce an expanded consensus sequence for the compositionally unusual promoters recognized by this new σ factor. PMID:19047353

  15. Phloem-Specific Expression of Tyrosine/Dopa Decarboxylase Genes and the Biosynthesis of Isoquinoline Alkaloids in Opium Poppy.

    PubMed Central

    Facchini, P. J.; De Luca, V.

    1995-01-01

    Tyrosine/dopa decarboxylase (TYDC) catalyzes the formation of tyramine and dopamine and represents the first steps in the biosynthesis of the large and diverse group of tetrahydroisoquinoline alkaloids. Opium poppy accumulates morphine in aerial organs and roots, whereas sanguinarine, which is derived from a distinct branch pathway, accumulates only in roots. Expression of the TYDC gene family in opium poppy was investigated in relation to the organ-specific biosynthesis of these different types of alkaloids. Members of the TYDC gene family are classified into two groups (represented by TYDC1 and TYDC2) and are differentially expressed. In the mature plant, TYDC2-like transcripts are predominant in stems and are also present in roots, whereas TYDC1-like transcripts are abundant only in roots. In situ hybridization analysis revealed that the expression of TYDC genes is developmentally regulated. TYDC transcripts are associated with vascular tissue in mature roots and stems but are also expressed in cortical tissues at earlier stages of development. Expression of TYDC genes is restricted to metaphloem and to protoxylem in the vascular bundles of mature aerial organs. Localization of TYDC transcripts in the phloem is consistent with the expected developmental origin of laticifers, which are specialized internal secretory cells that accompany vascular tissues in all organs of select species and that contain the alkaloid-rich latex in aerial organs. The differential expression of TYDC genes and the organ-dependent accumulation of different alkaloids suggest a coordinated regulation of specific alkaloid biosynthetic genes that are ultimately controlled by specific developmental programs. PMID:12242361

  16. Assessment of the effects of glutamic acid decarboxylase antibodies and trace elements on cognitive performance in older adults

    PubMed Central

    Alghadir, Ahmad H; Gabr, Sami A; Al-Eisa, Einas S

    2015-01-01

    Background Homeostatic imbalance of trace elements such as iron (Fe), copper (Cu), and zinc (Zn) demonstrated adverse effects on brain function among older adults. Objective The present study aimed to investigate the effects of trace elements and the presence of anti-glutamic acid decarboxylase antibodies (GADAs) in human cognitive abilities among healthy older adults. Methods A total of 100 healthy subjects (65 males, 35 females; age range; 64–96 years) were recruited for this study. Based on Loewenstein Occupational Therapy Cognitive Assessment (LOTCA) score, the participants were classified according to cognitive performance into normal (n=45), moderate (n=30), and severe (n=25). Cognitive functioning, leisure-time physical activity (LTPA), serum trace elements – Fe, Cu, Zn, Zn/Cu, and GADAs were assessed using LOTCA battery, pre-validated physical activity (PA) questionnaire, atomic absorption, and immunoassay techniques, respectively. Results Approximately 45% of the study population (n=45) had normal distribution of cognitive function and 55% of the study population (n=55) had abnormal cognitive function; they were classified into moderate (score 62–92) and severe (score 31–62). There was a significant reduction in the level of Zn and Zn/Cu ratio along with an increase in the level of Fe, Cu, and anti-GADAs in subjects of severe (P=0.01) and moderate (P=0.01) cognitive performance. LOTCA-cognitive scores correlated positively with sex, HbA1c, Fe, Cu, Zn, and Zn/Cu ratio, and negatively with age, PA, body mass index, and anti-GADAs. Significant inter-correlation was reported between serum trace element concentrations and anti-GADAs which suggest producing a cognitive decline via oxidative and neural damage mechanism. Conclusion This study found significant associations among trace elements, anti-GADAs, and cognitive function in older adults. The homeostatic balance of trace elements should be recommended among older adults for better cognitive

  17. Polyamines induced by osmotic stress protect Synechocystis sp. PCC 6803 cells and arginine decarboxylase transcripts against UV-B radiation.

    PubMed

    Pothipongsa, Apiradee; Jantaro, Saowarath; Incharoensakdi, Aran

    2012-11-01

    The effect of UV-B radiation on growth and polyamines content of Synechocystis sp. PCC 6803 subjected to either NaCl or sorbitol stress was investigated. Cells could not grow in the presence of 350 mM NaCl or 500 mM sorbitol under normal white light. However, cells grown in BG11 under osmotic stress imposed by NaCl or sorbitol followed by ultraviolet-B (UV-B) irradiation for 2 h showed higher cell density than those under the same condition but no osmotic stress. The chlorophyll fluorescence parameter (F(v)/F(m)) also showed an apparent decrease upon UV-B irradiation. Intracellular polyamines increased by about 2- and 4-fold in NaCl- and sorbitol-stressed cells, respectively. When these cells were irradiated with UV-B for 1 h, a further 3-fold increase in polyamines content was detected in NaCl-stressed but not sorbitol-stressed cells. Synechocystis cells contained adc1 and adc2 genes encoding arginine decarboxylase (ADC) with only adc1 showing upregulation by NaCl or sorbitol stress. NaCl- or sorbitol-stressed cells contained about 5-fold higher level of adc1 transcript than did the unstressed cells after 1-h irradiation with UV-B, suggesting the protection of adc1 transcript by accumulated polyamines, due to NaCl or sorbitol stress, against UV-B radiation damage. ADC levels as analyzed by Western blot showed upregulation by UV-B in NaCl-stressed but not sorbitol-stressed cells.

  18. Role of protein kinase C in diacylglycerol-mediated induction of ornithine decarboxylase and reduction of epidermal growth factor binding.

    PubMed Central

    Jetten, A M; Ganong, B R; Vandenbark, G R; Shirley, J E; Bell, R M

    1985-01-01

    Tumor-promoting phorbol esters induce ornithine decarboxylase (ODCase) activity and reduce epidermal growth factor (EGF) binding in rat tracheal epithelial 2C5 cells. Phorbol esters activate protein kinase C by interacting at the same site as sn-1,2-diacylglycerols, the presumed physiological regulators. The effects of added sn-1,2-diacylglycerols and those generated by phospholipase C treatment of 2C5 cells on ODCase induction and EGF binding were investigated to establish a role for protein kinase C in these cellular responses. Treatment of 2C5 cells with phospholipase C induced ODCase activity and reduced EGF binding, whereas phospholipases A2 and D were inactive. When sn-1,2-diacylglycerols containing fatty acids 3-10 carbons in length were added to 2C5 cells, those diacylglycerols containing fatty acids 5-10 carbons in length caused ODCase induction and reduction in EGF binding. sn-1,2-Dioctanoylglycerol was one of the most active compounds tested. It induced ODCase in a dose- (50-500 microM) and time-dependent manner. The reduction of binding of 125I-labeled EGF by sn-1,2-dioctanoylglycerol was also time and dose dependent and appeared to result from a change in EGF affinity and not the number of receptor sites. This series of sn-1,2-diacylglycerols showed similar structure-function relationships in their ability to induce ODCase activity, to decrease EGF binding, to stimulate protein kinase C, and to inhibit [3H]phorbol dibutyrate binding to the phorbol ester receptor. These data demonstrate biological activities for a number of diacylglycerols and indicate that protein kinase C activation is implicated in ODCase induction and decreased EGF binding. PMID:3157191

  19. Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

    SciTech Connect

    Barta, Michael L.; Skaff, D. Andrew; McWhorter, William J.; Herdendorf, Timothy J.; Miziorko, Henry M.; Geisbrecht, Brian V.

    2011-10-28

    The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 {angstrom} resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 {angstrom} resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 {angstrom} resolution). Comparison of these structures provides a physical basis for the significant differences in K{sub i} values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser{sup 192} as making potential contributions to catalysis. Significantly, Ser {yields} Ala substitution of this side chain decreases k{sub cat} by {approx}10{sup 3}-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 {angstrom} cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.

  20. Pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon, Pyrococcus furiosus, functions as a CoA-dependent pyruvate decarboxylase.

    PubMed

    Ma, K; Hutchins, A; Sung, S J; Adams, M W

    1997-09-02

    Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from the hyperthermophilic archaeon, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C by fermenting carbohydrates and peptides. The enzyme contains thiamine pyrophosphate and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 and reduces P. furiosus ferredoxin. Here we show that this enzyme also catalyzes the formation of acetaldehyde from pyruvate in a CoA-dependent reaction. Desulfocoenzyme A substituted for CoA showing that the cofactor plays a structural rather than a catalytic role. Ferredoxin was not necessary for the pyruvate decarboxylase activity of POR, nor did it inhibit acetaldehyde production. The apparent Km values for CoA and pyruvate were 0.11 mM and 1.1 mM, respectively, and the optimal temperature for acetaldehyde formation was above 90 degrees C. These data are comparable to those previously determined for the pyruvate oxidation reaction of POR. At 80 degrees C (pH 8.0), the apparent Vm value for pyruvate decarboxylation was about 40% of the apparent Vm value for pyruvate oxidation rate (using P. furiosus ferredoxin as the electron acceptor). Tentative catalytic mechanisms for these two reactions are presented. In addition to POR, three other 2-keto acid ferredoxin oxidoreductases are involved in peptide fermentation by hyperthermophilic archaea. It is proposed that the various aldehydes produced by these oxidoreductases in vivo are used by two aldehyde-utilizing enzymes, alcohol dehydrogenase and aldehyde ferredoxin oxidoreductase, the physiological roles of which were previously unknown.

  1. Aversive odorant causing appetite decrease downregulates tyrosine decarboxylase gene expression in the olfactory receptor neuron of the blowfly, Phormia regina

    NASA Astrophysics Data System (ADS)

    Ishida, Yuko; Ozaki, Mamiko

    2012-01-01

    In the blowfly Phormia regina, exposure to d-limonene for 5 days during feeding inhibits proboscis extension reflex behavior due to decreasing tyramine (TA) titer in the brain. TA is synthesized by tyrosine decarboxylase (Tdc) and catalyzed into octopamine (OA) by TA ß-hydroxylase (Tbh). To address the mechanisms of TA titer regulation in the blowfly, we cloned Tdc and Tbh cDNAs from P. regina (PregTdc and PregTbh). The deduced amino acid sequences of both proteins showed high identity to those of the corresponding proteins from Drosophila melanogaster at the amino acid level. PregTdc was expressed in the antenna, labellum, and tarsus whereas PregTbh was expressed in the head, indicating that TA is mainly synthesized in the sensory organs whereas OA is primarily synthesized in the brain. d-Limonene exposure significantly decreased PregTdc expression in the antenna but not in the labellum and the tarsus, indicating that PregTdc expressed in the antenna is responsible for decreasing TA titer. PregTdc-like immunoreactive material was localized in the thin-walled sensillum. In contrast, the OA/TA receptor (PregOAR/TAR) was localized to the thick-walled sensillum. The results indicated that d-limonene inhibits PregTdc expression in the olfactory receptor neurons in the thin-walled sensilla, likely resulting in reduced TA levels in the receptor neurons in the antenna. TA may be transferred from the receptor neuron to the specific synaptic junction in the antennal lobe of the brain through the projection neurons and play a role in conveying the aversive odorant information to the projection and local neurons.

  2. Reducing biogenic-amine-producing bacteria, decarboxylase activity, and biogenic amines in raw milk cheese by high-pressure treatments.

    PubMed

    Calzada, Javier; del Olmo, Ana; Picón, Antonia; Gaya, Pilar; Nuñez, Manuel

    2013-02-01

    Biogenic amines may reach concentrations of public health concern in some cheeses. To minimize biogenic amine buildup in raw milk cheese, high-pressure treatments of 400 or 600 MPa for 5 min were applied on days 21 and 35 of ripening. On day 60, counts of lactic acid bacteria, enterococci, and lactobacilli were 1 to 2 log units lower in cheeses treated at 400 MPa and 4 to 6 log units lower in cheeses treated at 600 MPa than in control cheese. At that time, aminopeptidase activity was 16 to 75% lower in cheeses treated at 400 MPa and 56 to 81% lower in cheeses treated at 600 MPa than in control cheese, while the total free amino acid concentration was 35 to 53% higher in cheeses treated at 400 MPa and 3 to 15% higher in cheeses treated at 600 MPa, and decarboxylase activity was 86 to 96% lower in cheeses treated at 400 MPa and 93 to 100% lower in cheeses treated at 600 MPa. Tyramine, putrescine, and cadaverine were the most abundant amines in control cheese. The total biogenic amine concentration on day 60, which reached a maximum of 1.089 mg/g dry matter in control cheese, was 27 to 33% lower in cheeses treated at 400 MPa and 40 to 65% lower in cheeses treated at 600 MPa. On day 240, total biogenic amines attained a concentration of 3.690 mg/g dry matter in control cheese and contents 11 to 45% lower in cheeses treated at 400 MPa and 73 to 76% lower in cheeses treated at 600 MPa. Over 80% of the histidine and 95% of the tyrosine had been converted into histamine and tyramine in control cheese by day 60. Substrate depletion played an important role in the rate of biogenic amine buildup, becoming a limiting factor in the case of some amino acids.

  3. Hydrogen peroxide-independent production of α-alkenes by OleTJE P450 fatty acid decarboxylase

    PubMed Central

    2014-01-01

    Background Cytochrome P450 OleTJE from Jeotgalicoccus sp. ATCC 8456, a new member of the CYP152 peroxygenase family, was recently found to catalyze the unusual decarboxylation of long-chain fatty acids to form α-alkenes using H2O2 as the sole electron and oxygen donor. Because aliphatic α-alkenes are important chemicals that can be used as biofuels to replace fossil fuels, or for making lubricants, polymers and detergents, studies on OleTJE fatty acid decarboxylase are significant and may lead to commercial production of biogenic α-alkenes in the future, which are renewable and more environmentally friendly than petroleum-derived equivalents. Results We report the H2O2-independent activity of OleTJE for the first time. In the presence of NADPH and O2, this P450 enzyme efficiently decarboxylates long-chain fatty acids (C12 to C20) in vitro when partnering with either the fused P450 reductase domain RhFRED from Rhodococcus sp. or the separate flavodoxin/flavodoxin reductase from Escherichia coli. In vivo, expression of OleTJE or OleTJE-RhFRED in different E. coli strains overproducing free fatty acids resulted in production of variant levels of multiple α-alkenes, with a highest total hydrocarbon titer of 97.6 mg·l-1. Conclusions The discovery of the H2O2-independent activity of OleTJE not only raises a number of fundamental questions on the monooxygenase-like mechanism of this peroxygenase, but also will direct the future metabolic engineering work toward improvement of O2/redox partner(s)/NADPH for overproduction of α-alkenes by OleTJE. PMID:24565055

  4. Genetically engineered Lactobacillus plantarum WCFS1 constitutively secreting heterologous oxalate decarboxylase and degrading oxalate under in vitro.

    PubMed

    Sasikumar, Ponnusamy; Gomathi, Sivasamy; Anbazhagan, Kolandaswamy; Baby, A Ebenezer; Sangeetha, J; Selvam, Govindan Sadasivam

    2014-11-01

    Hyperoxaluria is a major risk factor for urinary stone disease, where calcium oxalate (CaOx) is the most prevalent type of kidney stones. Systemic treatments of CaOx kidney stone patients are limited and comprise drawbacks including recurrence of stone formation and kidney damages. In the present work Lactobacillus plantarum (L. plantarum) was engineered to constitutively secrete oxalate decarboxylase (OxdC) for the degradation of intestinal oxalate. The homologous promoter PldhL and signal peptide Lp_0373 of L. plantarum were used for constructing recombinant vector pLdhl0373OxdC. Results showed that homologous promoter PldhL and signal peptide Lp_0373 facilitated the production, secretion, and functional expression of OxdC protein in L. plantarum. SDS-PAGE analysis revealed that 44 kDa protein OxdC was seen exceptionally in the culture supernatant of recombinant L. plantarum (WCFS1OxdC) harboring the plasmid pLdhl0373OxdC.The culture supernatant of L. plantarum WCFS1OxdC showed OxdC activity of 0.06 U/mg of protein, whereas no enzyme activity was observed in the supernatant of the wild type WCFS1 and the recombinant NC8OxdC strains. The purified recombinant OxdC from the WCFS1OxdC strain showed an activity of 19.1 U/mg protein. The recombinant L. plantarum strain secreted 25 % of OxdC protein in the supernatant. The recombinant strain degraded more than 70 % of soluble oxalate in the culture supernatant. Plasmid segregation analysis revealed that the recombinant strain lost almost 70-89 % of plasmid in 42nd and 84th generation, respectively. In conclusion, recombinant L. plantarum strain containing plasmid pLdhl0373OxdC showed constitutive secretion of bioactive OxdC and also capable of degrading externally available oxalate under in vitro conditions.

  5. Crystal structures of Staphylococcus epidermidis mevalonate diphosphate decarboxylase bound to inhibitory analogs reveal new insight into substrate binding and catalysis.

    PubMed

    Barta, Michael L; Skaff, D Andrew; McWhorter, William J; Herdendorf, Timothy J; Miziorko, Henry M; Geisbrecht, Brian V

    2011-07-08

    The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 Å resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 Å resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 Å resolution). Comparison of these structures provides a physical basis for the significant differences in K(i) values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser(192) as making potential contributions to catalysis. Significantly, Ser → Ala substitution of this side chain decreases k(cat) by ∼10(3)-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 Å cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.

  6. Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extremely acidic conditions.

    PubMed

    Damiano, Maria Alessandra; Bastianelli, Daniela; Al Dahouk, Sascha; Köhler, Stephan; Cloeckaert, Axel; De Biase, Daniela; Occhialini, Alessandra

    2015-01-01

    Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis, and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative.

  7. Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production

    PubMed Central

    Tajabadi, Naser; Baradaran, Ali; Ebrahimpour, Afshin; Rahim, Raha A; Bakar, Fatimah A; Manap, Mohd Yazid A; Mohammed, Abdulkarim S; Saari, Nazamid

    2015-01-01

    Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products. PMID:25757029

  8. Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

    PubMed Central

    2012-01-01

    Background The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied. Results The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations. Conclusion In mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene

  9. Disease-specific monoclonal antibodies targeting glutamate decarboxylase impair GABAergic neurotransmission and affect motor learning and behavioral functions

    PubMed Central

    Manto, Mario; Honnorat, Jérôme; Hampe, Christiane S.; Guerra-Narbona, Rafael; López-Ramos, Juan Carlos; Delgado-García, José María; Saitow, Fumihito; Suzuki, Hidenori; Yanagawa, Yuchio; Mizusawa, Hidehiro; Mitoma, Hiroshi

    2015-01-01

    Autoantibodies to the smaller isoform of glutamate decarboxylase (GAD) can be found in patients with type 1 diabetes and a number of neurological disorders, including stiff-person syndrome, cerebellar ataxia and limbic encephalitis. The detection of disease-specific autoantibody epitopes led to the hypothesis that distinct GAD autoantibodies may elicit specific neurological phenotypes. We explored the in vitro/in vivo effects of well-characterized monoclonal GAD antibodies. We found that GAD autoantibodies present in patients with stiff person syndrome (n = 7) and cerebellar ataxia (n = 15) recognized an epitope distinct from that recognized by GAD autoantibodies present in patients with type 1 diabetes mellitus (n = 10) or limbic encephalitis (n = 4). We demonstrated that the administration of a monoclonal GAD antibody representing this epitope specificity; (1) disrupted in vitro the association of GAD with γ-Aminobutyric acid containing synaptic vesicles; (2) depressed the inhibitory synaptic transmission in cerebellar slices with a gradual time course and a lasting suppressive effect; (3) significantly decreased conditioned eyelid responses evoked in mice, with no modification of learning curves in the classical eyeblink-conditioning task; (4) markedly impaired the facilitatory effect exerted by the premotor cortex over the motor cortex in a paired-pulse stimulation paradigm; and (5) induced decreased exploratory behavior and impaired locomotor function in rats. These findings support the specific targeting of GAD by its autoantibodies in the pathogenesis of stiff-person syndrome and cerebellar ataxia. Therapies of these disorders based on selective removal of such GAD antibodies could be envisioned. PMID:25870548

  10. Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system

    PubMed Central

    Grassini, Gaia; Pennacchietti, Eugenia; Cappadocio, Francesca; Occhialini, Alessandra; De Biase, Daniela

    2015-01-01

    In orally acquired bacteria, the ability to counteract extreme acid stress (pH ⩽ 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known “classical” Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved. PMID:25853037

  11. Reducing Biogenic-Amine-Producing Bacteria, Decarboxylase Activity, and Biogenic Amines in Raw Milk Cheese by High-Pressure Treatments

    PubMed Central

    Calzada, Javier; del Olmo, Ana; Picón, Antonia; Gaya, Pilar

    2013-01-01

    Biogenic amines may reach concentrations of public health concern in some cheeses. To minimize biogenic amine buildup in raw milk cheese, high-pressure treatments of 400 or 600 MPa for 5 min were applied on days 21 and 35 of ripening. On day 60, counts of lactic acid bacteria, enterococci, and lactobacilli were 1 to 2 log units lower in cheeses treated at 400 MPa and 4 to 6 log units lower in cheeses treated at 600 MPa than in control cheese. At that time, aminopeptidase activity was 16 to 75% lower in cheeses treated at 400 MPa and 56 to 81% lower in cheeses treated at 600 MPa than in control cheese, while the total free amino acid concentration was 35 to 53% higher in cheeses treated at 400 MPa and 3 to 15% higher in cheeses treated at 600 MPa, and decarboxylase activity was 86 to 96% lower in cheeses treated at 400 MPa and 93 to 100% lower in cheeses treated at 600 MPa. Tyramine, putrescine, and cadaverine were the most abundant amines in control cheese. The total biogenic amine concentration on day 60, which reached a maximum of 1.089 mg/g dry matter in control cheese, was 27 to 33% lower in cheeses treated at 400 MPa and 40 to 65% lower in cheeses treated at 600 MPa. On day 240, total biogenic amines attained a concentration of 3.690 mg/g dry matter in control cheese and contents 11 to 45% lower in cheeses treated at 400 MPa and 73 to 76% lower in cheeses treated at 600 MPa. Over 80% of the histidine and 95% of the tyrosine had been converted into histamine and tyramine in control cheese by day 60. Substrate depletion played an important role in the rate of biogenic amine buildup, becoming a limiting factor in the case of some amino acids. PMID:23241980

  12. Identification of the putrescine biosynthetic genes in Pseudomonas aeruginosa and characterization of agmatine deiminase and N-carbamoylputrescine amidohydrolase of the arginine decarboxylase pathway.

    PubMed

    Nakada, Yuji; Itoh, Yoshifumi

    2003-03-01

    Putrescine can be synthesized either directly from ornithine by ornithine decarboxylase (ODC; the speC product) or indirectly from arginine via arginine decarboxylase (ADC; the speA product). The authors identified the speA and speC genes in Pseudomonas aeruginosa PAO1. The activities of the two decarboxylases were similar and each enzyme alone appeared to direct sufficient formation of the polyamine for normal growth. A mutant defective in both speA and speC was a putrescine auxotroph. In this strain, agmatine deiminase (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), which were initially identified as the catabolic enzymes of agmatine, biosynthetically convert agmatine to putrescine in the ADC pathway: a double mutant of aguAB and speC was a putrescine auxotroph. AguA was purified as a homodimer of 43 kDa subunits and AguB as a homohexamer of 33 kDa subunits. AguA specifically deiminated agmatine with K(m) and K(cat) values of 0.6 mM and 4.2 s(-1), respectively. AguB was specific to N-carbamoylputrescine and the K(m) and K(cat) values of the enzyme for the substrate were 0.5 mM and 3.3 s(-1), respectively. Whereas AguA has no structural relationship to any known C-N hydrolases, AguB is a protein of the nitrilase family that performs thiol-assisted catalysis. Inhibition by SH reagents and the conserved cysteine residue in AguA and its homologues suggested that this enzyme is also involved in thiol-mediated catalysis.

  13. Crystal Structures of Apo and Liganded 4-Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous β-Keto Acid.

    PubMed

    Guimarães, Samuel L; Coitinho, Juliana B; Costa, Débora M A; Araújo, Simara S; Whitman, Christian P; Nagem, Ronaldo A P

    2016-05-10

    The enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria Pseudomonas putida G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg(2+) as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent β-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous β-keto acid decarboxylase and the first crystal structure of a 4-OD.

  14. An organic solvent-tolerant phenolic acid decarboxylase from Bacillus licheniformis for the efficient bioconversion of hydroxycinnamic acids to vinyl phenol derivatives.

    PubMed

    Hu, Hongfei; Li, Lulu; Ding, Shaojun

    2015-06-01

    A new phenolic acid decarboxylase gene (blpad) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli. The full-length blpad encodes a 166-amino acid polypeptide with a predicted molecular mass and pI of 19,521 Da and 5.02, respectively. The recombinant BLPAD displayed maximum activity at 37 °C and pH 6.0. This enzyme possesses a broad substrate specificity and is able to decarboxylate p-coumaric, ferulic, caffeic, and sinapic acids at the relative ratios of specific activities 100:74.59:34.41:0.29. Kinetic constant K m values toward p-coumaric, ferulic, caffeic, and sinapic acids were 1.64, 1.55, 1.93, and 2.45 mM, and V max values were 268.43, 216.80, 119.07, and 0.78 U mg(-1), respectively. In comparison with other phenolic acid decarboxylases, BLPAD exhibited remarkable organic solvent tolerance and good thermal stability. BLPAD showed excellent catalytic performance in biphasic organic/aqueous systems and efficiently converted p-coumaric and ferulic acids into 4-vinylphenol and 4-vinylguaiacol. At 500 mM of p-coumaric and ferulic acids, the recombinant BLPAD produced a total 60.63 g l(-1) 4-vinylphenol and 58.30 g l(-1) 4-vinylguaiacol with the conversion yields 97.02 and 70.96 %, respectively. The low yield and product concentration are the crucial drawbacks to the practical bioproduction of vinyl phenol derivatives using phenolic acid decarboxylases. These unusual properties make BLPAD a desirable biocatalyst for commercial use in the bioconversion of hydroxycinnamic acids to vinyl phenol derivatives via enzymatic decarboxylation in a biphasic organic/aqueous reaction system.

  15. Photoaugmentation in the hairless mouse: a study using ornithine decarboxylase activity and alteration of DNA synthesis as markers of epidermal response

    SciTech Connect

    Gange, R.W.; Mendelson, I.R.

    1981-01-01

    Photoaugmentation is the potentiation of UVB-induced cutaneous erythema by UV irradiation. We have examined other cutaneous responses to UVB irradiation-the 4 hr depression of DNA synthesis, the 48 hr stimulation of DNA synthesis, and the induction of ornithine decarboxylase (ODC), to determine whether these were also susceptible to augmentation by UVA, which does not cause these responses when administered alone. No photoaugmentation of DNA synthesis, stimulation or ODC induction occurred. The early depression of DNA synthesis was slightly augmented for this did not consistently reach significance.

  16. The timing of administration, dose dependence and efficacy of dopa decarboxylase inhibitors on the reversal of motor disability produced by L-DOPA in the MPTP-treated common marmoset.

    PubMed

    Tayarani-Binazir, Kayhan A; Jackson, Michael J; Fisher, Ria; Zoubiane, Ghada; Rose, Sarah; Jenner, Peter

    2010-06-10

    Dopa decarboxylase inhibitors are routinely used to potentiate the effects of L-DOPA in the treatment of Parkinson's disease. However, neither in clinical use nor in experimental models of Parkinson's disease have the timing and dose of dopa decarboxylase inhibitors been thoroughly explored. We now report on the choice of dopa decarboxylase inhibitors, dose and the time of dosing relationships of carbidopa, benserazide and L-alpha-methyl dopa (L-AMD) in potentiating the effects of L-DOPA in the 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-treated common marmoset. Pre-treatment with benserazide for up to 3h did not alter the motor response to L-DOPA compared to simultaneous administration with L-DOPA. There was some evidence of a relationship between carbidopa and benserazide dose and increased locomotor activity and the reversal of motor disability. But in general, commonly used dose levels of dopa decarboxylase inhibitors appeared to produce a maximal motor response to L-DOPA. In contrast, dyskinesia intensity and duration continued to increase with both carbidopa and benserazide dose. The novel dopa decarboxylase inhibitor, L-AMD, increased locomotor activity and improved motor disability to the same extent as carbidopa or benserazide but importantly this was accompanied by significantly less dyskinesia. This study shows that currently, dopa decarboxylase inhibitors may be routinely employed in the MPTP-treated primate at doses which are higher than those necessary to produce a maximal potentiation of the anti-parkinsonian effect of L-DOPA. This may lead to excessive expression of dyskinesia in this model of Parkinson's disease and attention should be given to the dose regimens currently employed.

  17. Flavanols and procyanidins of cocoa and chocolate inhibit growth and polyamine biosynthesis of human colonic cancer cells.

    PubMed

    Carnésecchi, Stéphanie; Schneider, Yann; Lazarus, Sheryl A; Coehlo, David; Gossé, Francine; Raul, Francis

    2002-01-25

    The effects of cocoa powder and extracts with different amounts of flavanols and related procyanidin oligomers were investigated on the growth of Caco-2 cells. Treatment of the cells with 50 microg/ml of procyanidin-enriched (PE) extracts caused a 70% growth inhibition with a blockade of the cell cycle at the G2/M phase. PE extracts caused a significant decrease of ornithine decarboxylase and S-adenosylmethionine decarboxylase activities, two key enzymes of polyamine biosynthesis. This led to a decrease in the intracellular pool of the polyamines. These observations indicate that polyamine metabolism might be an important target in the anti-proliferative effects of cocoa polyphenols.

  18. Proteolytic degradation of glutamate decarboxylase mediates disinhibition of hippocampal CA3 pyramidal cells in cathepsin D-deficient mice.

    PubMed

    Shimizu, Tokiko; Hayashi, Yoshinori; Yamasaki, Ryo; Yamada, Jun; Zhang, Jian; Ukai, Kiyoharu; Koike, Masato; Mine, Kazunori; von Figura, Kurt; Peters, Christoph; Saftig, Paul; Fukuda, Takaichi; Uchiyama, Yasuo; Nakanishi, Hiroshi

    2005-08-01

    Although of clinical importance, little is known about the mechanism of seizure in neuronal ceroid lipofuscinosis (NCL). In the present study, we have attempted to elucidate the mechanism underlying the seizure of cathepsin D-deficient (CD-/-) mice that show a novel type of lysosomal storage disease with a phenotype resembling late infantile NCL. In hippocampal slices prepared from CD-/- mice at post-natal day (P)24, spontaneous burst discharges were recorded from CA3 pyramidal cells. At P24, the mean amplitude of IPSPs after stimulation of the mossy fibres was significantly smaller than that of wild-type mice, which was substantiated by the decreased level of gamma-aminobutyric acid (GABA) contents in the hippocampus measured by high-performance liquid chromatography (HPLC). At this stage, activated microglia were found to accumulate in the pyramidal cell layer of the hippocampal CA3 subfield of CD-/- mice. However, there was no significant change in the numerical density of GABAergic interneurons in the CA3 subfield of CD-/- mice at P24, estimated by counting the number of glutamate decarboxylase (GAD) 67-immunoreactive somata. In the hippocampus and the cortex of CD-/- mice at P24, some GABAergic interneurons displayed extremely high somatic granular immunoreactivites for GAD67, suggesting the lysosomal accumulation of GAD67. GAD67 levels in axon terminals abutting on to perisomatic regions of hippocampal CA3 pyramidal cells was not significantly changed in CD-/- mice even at P24, whereas the total protein levels of GAD67 in both the hippocampus and the cortex of CD-/- mice after P24 were significantly decreased as a result of degradation. Furthermore, the recombinant human GAD65/67 was rapidly digested by the lysosomal fraction prepared from the whole brain of wild-type and CD-/- mice. These observations strongly suggest that the reduction of GABA contents, presumably because of lysosomal degradation of GAD67 and lysosomal accumulation of its degraded forms

  19. Identification of a dominant epitope of glutamic acid decarboxylase (GAD-65) recognized by autoantibodies in stiff-man syndrome

    PubMed Central

    1993-01-01

    Glutamic acid decarboxylase (GAD) is the enzyme that synthesizes the neurotransmitter gamma-aminobutyric acid (GABA) in neurons and in pancreatic beta cells. It is a major target of autoimmunity in Stiff- Man syndrome (SMS), a rare neurological disease, and in insulin- dependent diabetes mellitus. The two GAD isoforms, GAD-65 and GAD-67, are the products of two different genes. GAD-67 and GAD-65 are very similar to each other in amino acid sequence and differ substantially only at their NH2-terminal region. We have investigated the reactivity of autoantibodies of 30 Stiff-Man syndrome patients to GAD. All patient sera contained antibodies that recognize strongly GAD-65, but also GAD- 67, when tested by immunoprecipitation on brain extracts and by immunoprecipitation or immunocytochemistry on cells transfected with either the GAD-65 or the GAD-67 gene. When tested by Western blotting, all patient sera selectively recognized GAD-65. Western blot analysis of deletion mutants of GAD-65 demonstrated that autoantibodies are directed predominantly against two regions of the GAD-65 molecule. All SMS sera strongly recognized a fragment contained between amino acid 475 and the COOH terminus (amino acid 585). Within this region, amino acids 475-484 and 571-585 were required for reactivity. The requirement of these two discontinuous segments implies that the epitope is influenced by conformation. This reactivity is similar to that displayed by the monoclonal antibody GAD 6, suggesting the presence of a single immunodominant epitope (SMS-E1) in this region of GAD-65. In addition, most SMS sera recognized at least one epitope (SMS-E2) in the NH2-terminal domain of GAD-65 (amino acids 1-95). The demonstration in SMS patients of a strikingly homogeneous humoral autoimmune response against GAD and the identification of dominant autoreactive target regions may help to elucidate the molecular mechanisms of GAD processing and presentation involved in GAD autoimmunity. Moreover, the

  20. Biochemical, Mutational and In Silico Structural Evidence for a Functional Dimeric Form of the Ornithine Decarboxylase from Entamoeba histolytica

    PubMed Central

    Preeti; Tapas, Satya; Kumar, Pravindra; Madhubala, Rentala; Tomar, Shailly

    2012-01-01

    Background Entamoeba histolytica is responsible for causing amoebiasis. Polyamine biosynthesis pathway enzymes are potential drug targets in parasitic protozoan diseases. The first and rate-limiting step of this pathway is catalyzed by ornithine decarboxylase (ODC). ODC enzyme functions as an obligate dimer. However, partially purified ODC from E. histolytica (EhODC) is reported to exist in a pentameric state. Methodology and Results In present study, the oligomeric state of EhODC was re-investigated. The enzyme was over-expressed in Escherichia coli and purified. Pure protein was used for determination of secondary structure content using circular dichroism spectroscopy. The percentages of α-helix, β-sheets and random coils in EhODC were estimated to be 39%, 25% and 36% respectively. Size-exclusion chromatography and mass spectrophotometry analysis revealed that EhODC enzyme exists in dimeric form. Further, computational model of EhODC dimer was generated. The homodimer contains two separate active sites at the dimer interface with Lys57 and Cys334 residues of opposite monomers contributing to each active site. Molecular dynamic simulations were performed and the dimeric structure was found to be very stable with RMSD value ∼0.327 nm. To gain insight into the functional role, the interface residues critical for dimerization and active site formation were identified and mutated. Mutation of Lys57Ala or Cys334Ala completely abolished enzyme activity. Interestingly, partial restoration of the enzyme activity was observed when inactive Lys57Ala and Cys334Ala mutants were mixed confirming that the dimer is the active form. Furthermore, Gly361Tyr and Lys157Ala mutations at the dimer interface were found to abolish the enzyme activity and destabilize the dimer. Conclusion To our knowledge, this is the first report which demonstrates that EhODC is functional in the dimeric form. These findings and availability of 3D structure model of EhODC dimer opens up

  1. Distribution and development of glutamic acid decarboxylase immunoreactivity in the spinal cord of the dogfish Scyliorhinus canicula (elasmobranchs).

    PubMed

    Sueiro, Catalina; Carrera, Iván; Molist, Pilar; Rodríguez-Moldes, Isabel; Anadón, Ramón

    2004-10-11

    The adult distribution and development of gamma-aminobutyric acid (GABA)-synthesizing cells and fibers in the spinal cord of the lesser spotted dogfish (Scyliorhinus canicula L.) was studied by means of immunohistochemistry using antibodies against glutamic acid decarboxylase (GAD). Complementary immunostaining with antibodies against GABA, tyrosine hydroxylase (TH), and HuC/HuD (members of the Hu/Elav family of RNA-associated proteins) and staining with a reduced silver procedure ("en bloc" Bielschowski method), Nissl, and hematoxylin were also used. In adults, GAD-immunoreactive (GAD-ir) cells were observed in the ventral horns, in the spinal nucleus of the dorsal horn, at the base of the dorsal horns, and around the central canal, where some GAD-ir cells were cerebrospinal fluid-contacting (CSF-c). In addition, a few GAD-ir cells were observed in the lateral funiculus between the ventral horn and the marginal nucleus. The adult spinal cord was richly innervated by GAD-ir fibers. Large numbers of GAD-ir fibers and boutons were observed in the dorsal and ventral horns and also interstitially in the dorsal, lateral, and ventral funiculi. In addition, a rich GAD-ir innervation was observed in the marginal nucleus of the spinal cord. In the embryonic spinal cord, GAD-ir cells develop very early: The earliest cells were observed in the very thin mantle/marginal layer of stage 22 embryos in a short length of the spinal cord. At stages 25 and 26, several types of GAD-ir cells (commissural and noncommissural) were distinguished, and two of these cells were of CSF-c type. At stages 28, 30, and 31, the GAD-ir populations exhibited a marked longitudinal columnar organization. Double-immunolabeling experiments in embryos showed the presence of two different GAD-ir CSF-c cell populations, one ventral that is simultaneously TH-ir and other more dorsal that is TH-negative. By stage 33 (prehatching), GAD-expressing cells are present in virtually all loci, as in adults

  2. Use of isotope effects to determine the chemical mechanism of oxidative decarboxylases: NADP malic enzyme and NADP isocitrate dehydrogenase

    SciTech Connect

    Grisson, C.B.

    1985-01-01

    The chemical mechanism of the NADP-linked oxidative decarboxylases chicken liver malic enzyme and pig heart isocitrate dehydrogenase has been examined using carbon and deuterium isotope effects and their variation with pH, metal ions, and solution viscosity. The following /sup 13/C isotope effects on V/K for malate are observed with the stated metal ion at pH 8.0 and 25/sup 0/C: Mg/sup 2 +/, 1.0336; Mn/sup 2 +/, 1.0365; Cd/sup 2 +/, 1.0366; Zn/sup 2 +/, 1.03373; Co/sup 2 +/, 1.0283; and Ni/sup 2 +/, 1.025. The /sup 13/C isotope effect on nonenzymatic decarboxylation of dibasic oxalacetate at 25/sup 0/C and the stated metal ion is : Mg/sup 2 +/, 1.0489; Mn/sup 2 +/, 1.0505; Ni/sup 2 +/, 1.044; Cd/sup 2 +/, 1.0492; Zn/sup 2 +/, 1.0504; and Co/sup 2 +/, 1.0480. By quantitating the partitioning of the 2-ketocarboxylic acid reaction intermediate between reverse hydride transfer and decarboxylation, it is possible to solve for the intrinsic isotope effects in both reactions. With malic enzyme activated by Mg/sup 2 +/, the partitioning ratio of oxalacetate between pyruvate and malate formation is 0.47. This gives an intrinsic deuterium and /sup 13/C isotope effect of 5.6 and 1.0493, respectively. The /sup 13/C isotope effect for the Mg/sup 2 +/ catalyzed nonenzymatic decarboxylation of oxalacetate is 1.0489, thereby suggesting a similarity of transition states between the enzymatic and nonenzymatic processes. The observed /sup 13/C primary isotope effect for isocitrate in the isocitrate dehydrogenase reaction is obscured by the stickiness of isocitrate at neutral pH. At low pH, the external commitment is eliminated and the observed /sup 13/C isotope effect increases to a limiting value of 1.0353. The pK of the pH dependence of /sup 13/(V/K) is 4.7.

  3. An assembly of proteins and lipid domains regulates transport of phosphatidylserine to phosphatidylserine decarboxylase 2 in Saccharomyces cerevisiae.

    PubMed

    Riekhof, Wayne R; Wu, Wen-I; Jones, Jennifer L; Nikrad, Mrinalini; Chan, Mallory M; Loewen, Christopher J R; Voelker, Dennis R

    2014-02-28

    Saccharomyces cerevisiae uses multiple biosynthetic pathways for the synthesis of phosphatidylethanolamine. One route involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), the transport of this lipid to endosomes, and decarboxylation by PtdSer decarboxylase 2 (Psd2p) to produce phosphatidylethanolamine. Several proteins and protein motifs are known to be required for PtdSer transport to occur, namely the Sec14p homolog PstB2p/Pdr17p; a PtdIns 4-kinase, Stt4p; and a C2 domain of Psd2p. The focus of this work is on defining the protein-protein and protein-lipid interactions of these components. PstB2p interacts with a protein encoded by the uncharacterized gene YPL272C, which we name Pbi1p (PstB2p-interacting 1). PstB2p, Psd2, and Pbi1p were shown to be lipid-binding proteins specific for phosphatidic acid. Pbi1p also interacts with the ER-localized Scs2p, a binding determinant for several peripheral ER proteins. A complex between Psd2p and PstB2p was also detected, and this interaction was facilitated by a cryptic C2 domain at the extreme N terminus of Psd2p (C2-1) as well the previously characterized C2 domain of Psd2p (C2-2). The predicted N-terminal helical region of PstB2p was necessary and sufficient for promoting the interaction with both Psd2p and Pbi1p. Taken together, these results support a model for PtdSer transport involving the docking of a PtdSer donor membrane with an acceptor via specific protein-protein and protein-lipid interactions. Specifically, our model predicts that this process involves an acceptor membrane complex containing the C2 domains of Psd2p, PstB2p, and Pbi1p that ligate to Scs2p and phosphatidic acid present in the donor membrane, forming a zone of apposition that facilitates PtdSer transfer.

  4. Mechanism of the Orotidine 5’-Monophosphate Decarboxylase-Catalyzed Reaction: Importance of Residues in the Orotate Binding Site†

    PubMed Central

    Iiams,