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Sample records for acid decarboxylase aaad

  1. Investigation of a substrate-specifying residue within Papaver somniferum and Catharanthus roseus aromatic amino acid decarboxylases.

    PubMed

    Torrens-Spence, Michael P; Lazear, Michael; von Guggenberg, Renee; Ding, Haizhen; Li, Jianyong

    2014-10-01

    Plant aromatic amino acid decarboxylases (AAADs) catalyze the decarboxylation of aromatic amino acids with either benzene or indole rings. Because the substrate selectivity of AAADs is intimately related to their physiological functions, primary sequence data and their differentiation could provide significant physiological insights. However, due to general high sequence identity, plant AAAD substrate specificities have been difficult to identify through primary sequence comparison. In this study, bioinformatic approaches were utilized to identify several active site residues within plant AAAD enzymes that may impact substrate specificity. Next a Papaver somniferum tyrosine decarboxylase (TyDC) was selected as a model to verify our putative substrate-dictating residues through mutation. Results indicated that mutagenesis of serine 372 to glycine enables the P. somniferum TyDC to use 5-hydroxytryptophan as a substrate, and reduces the enzyme activity toward 3,4-dihydroxy-L-phenylalanine (dopa). Additionally, the reverse mutation in a Catharanthus roseus tryptophan decarboxylase (TDC) enables the mutant enzyme to utilize tyrosine and dopa as substrates with a reduced affinity toward tryptophan. Molecular modeling and molecular docking of the P. somniferum TyDC and the C. roseus TDC enzymes provided a structural basis to explain alterations in substrate specificity. Identification of an active site residue that impacts substrate selectivity produces a primary sequence identifier that may help differentiate the indolic and phenolic substrate specificities of individual plant AAADs.

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

    MedlinePlus

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

  3. 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.

  4. 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.

  5. 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. PMID:2742363

  6. 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.

  7. Cerebellar Ataxia and Glutamic Acid Decarboxylase Antibodies

    PubMed Central

    Ariño, Helena; Gresa-Arribas, Nuria; Blanco, Yolanda; Martínez-Hernández, Eugenia; Sabater, Lidia; Petit-Pedrol, Mar; Rouco, Idoia; Bataller, Luis; Dalmau, Josep O.; Saiz, Albert; Graus, Francesc

    2016-01-01

    IMPORTANCE Current clinical and immunologic knowledge on cerebellar ataxia (CA) with glutamic acid decarboxylase 65 antibodies (GAD65-Abs) is based on case reports and small series with short-term follow-up data. OBJECTIVE To report the symptoms, additional antibodies, prognostic factors, and long-term outcomes in a cohort of patients with CA and GAD65-Abs. DESIGN, SETTING, AND PARTICIPANTS Retrospective cohort study and laboratory investigations at a center for autoimmune neurologic disorders among 34 patients with CA and GAD65-Abs, including 25 with long-term follow-up data (median, 5.4 years; interquartile range, 3.1-10.3 years). MAIN OUTCOMES AND MEASURES Analysis of clinicoimmunologic features and predictors of response to immunotherapy. Immunochemistry on rat brain, cultured neurons, and human embryonic kidney cells expressing GAD65, GAD67, α1-subunit of the glycine receptor, and a repertoire of known cell surface autoantigens were used to identify additional antibodies. Twenty-eight patients with stiff person syndrome and GAD65-Abs served as controls. RESULTS The median age of patients was 58 years (range, 33-80 years); 28 of 34 patients (82%) were women. Nine patients (26%) reported episodes of brainstem and cerebellar dysfunction or persistent vertigo several months before developing CA. The clinical presentation was subacute during a period of weeks in 13 patients (38%). Nine patients (26%) had coexisting stiff person syndrome symptoms. Systemic organ-specific autoimmunities (type 1 diabetes mellitus and others) were present in 29 patients (85%). Twenty of 25 patients with long-term follow-up data received immunotherapy (intravenous immunoglobulin in 10 and corticosteroids and intravenous immunoglobulin or other immunosuppressors in 10), and 7 of them (35%) improved. Predictors of clinical response included subacute onset of CA (odds ratio [OR], 0.50; 95% CI, 0.25-0.99; P = .047) and prompt immunotherapy (OR, 0.98; 95% CI, 0.96-0.99; P = .01). Similar

  8. Retinoic acid modulation of ultraviolet light-induced epidermal ornithine decarboxylase activity

    SciTech Connect

    Lowe, N.J.; Breeding, J.

    1982-02-01

    Irradiation of skin with ultraviolet light of sunburn range (UVB) leads to a large and rapid induction of the polyamine biosynthetic enzyme ornithine decarboxylase in the epidermis. Induction of epidermal ornithine decarboxylase also occurs following application of the tumor promoting agent 12-0-tetradecanoylphorbol-13 acetate and topical retinoic acid is able to block both this ornithine decarboxylase induction and skin tumor promotion. In the studies described below, topical application of retinoic acid to hairless mouse skin leads to a significant inhibition of UVB-induced epidermal ornithine decarboxylase activity. The degree of this inhibition was dependent on the dose, timing, and frequency of the application of retinoic acid. To show significant inhibition of UVB-induced ornithine decarboxylase the retinoic acid had to be applied within 5 hr of UVB irradiation. If retinoic acid treatment was delayed beyond 7 hr following UVB, then no inhibition of UVB-induced ornithine decarboxylase was observed. The quantities of retinoic acid used (1.7 nmol and 3.4 nmol) have been shown effective at inhibiting 12-0-tetradecanoyl phorbol-13 acetate induced ornithine decarboxylase. The results show that these concentrations of topical retinoic acid applied either before or immediately following UVB irradiation reduces the UVB induction of epidermal ornithine decarboxylase. The effect of retinoic acid in these regimens on UVB-induced skin carcinogenesis is currently under study.

  9. 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)

  10. Anti-glutamic acid decarboxylase antibody positive neurological syndromes.

    PubMed

    Tohid, Hassaan

    2016-07-01

    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 calledhyperexcitability 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

  11. 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. PMID:26643381

  12. 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.

  13. Isofunctional enzymes PAD1 and UbiX catalyze formation of a novel cofactor required by ferulic acid decarboxylase and 4-hydroxy-3-polyprenylbenzoic acid decarboxylase.

    PubMed

    Lin, Fengming; Ferguson, Kyle L; Boyer, David R; Lin, Xiaoxia Nina; Marsh, E Neil G

    2015-04-17

    The decarboxylation of antimicrobial aromatic acids such as phenylacrylic acid (cinnamic acid) and ferulic acid by yeast requires two enzymes described as phenylacrylic acid decarboxylase (PAD1) and ferulic acid decarboxylase (FDC). These enzymes are of interest for various biotechnological applications, such as the production of chemical feedstocks from lignin under mild conditions. However, the specific role of each protein in catalyzing the decarboxylation reaction remains unknown. To examine this, we have overexpressed and purified both PAD1 and FDC from E. coli. We demonstrate that PAD1 is a flavin mononucleotide (FMN)-containing protein. However, it does not function as a decarboxylase. Rather, PAD1 catalyzes the formation of a novel, diffusible cofactor required by FDC for decarboxylase activity. Coexpression of FDC and PAD1 results in the production of FDC with high levels cofactor bound. Holo-FDC catalyzes the decarboxylation of phenylacrylic acid, coumaric acid and ferulic acid with apparent kcat ranging from 1.4-4.6 s(-1). The UV-visible and mass spectra of the cofactor indicate that it appears to be a novel, modified form of reduced FMN; however, its instability precluded determination of its structure. The E. coli enzymes UbiX and UbiD are related by sequence to PAD1 and FDC respectively and are involved in the decarboxylation of 4-hydroxy-3-octaprenylbenzoic acid, an intermediate in ubiquinone biosynthesis. We found that endogenous UbiX can also activate FDC. This implies that the same cofactor is required for decarboxylation of 4-hydroxy-3-polyprenylbenzoic acid by UbiD and suggests a wider role for this cofactor in metabolism.

  14. Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella.

    PubMed

    Viala, Julie P M; Méresse, Stéphane; Pocachard, Bérengère; Guilhon, Aude-Agnès; Aussel, Laurent; Barras, Frédéric

    2011-01-01

    During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.

  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. Pyruvate decarboxylase from Pisum sativum. Properties, nucleotide and amino acid sequences.

    PubMed

    Mücke, U; Wohlfarth, T; Fiedler, U; Bäumlein, H; Rücknagel, K P; König, S

    1996-04-15

    To study the molecular structure and function of pyruvate decarboxylase (PDC) from plants the protein was isolated from pea seeds and partially characterised. The active enzyme which occurs in the form of higher oligomers consists of two different subunits appearing in SDS/PAGE and mass spectroscopy experiments. For further experiments, like X-ray crystallography, it was necessary to elucidate the protein sequence. Partial cDNA clones encoding pyruvate decarboxylase from seeds of Pisum sativum cv. Miko have been obtained by means of polymerase chain reaction techniques. The first sequences were found using degenerate oligonucleotide primers designated according to conserved amino acid sequences of known pyruvate decarboxylases. The missing parts of one cDNA were amplified applying the 3'- and 5'-rapid amplification of cDNA ends systems. The amino acid sequence deduced from the entire cDNA sequence displays strong similarity to pyruvate decarboxylases from other organisms, especially from plants. A molecular mass of 64 kDa was calculated for this protein correlating with estimations for the smaller subunit of the oligomeric enzyme. The PCR experiments led to at least three different clones representing the middle part of the PDC cDNA indicating the existence of three isozymes. Two of these isoforms could be confirmed on the protein level by sequencing tryptic peptides. Only anaerobically treated roots showed a positive signal for PDC mRNA in Northern analysis although the cDNA from imbibed seeds was successfully used for PCR.

  17. Volatile Organic Compounds Derived from 2-Keto-Acid Decarboxylase in Microcystis aeruginosa

    PubMed Central

    Hasegawa, Masateru; Nishizawa, Akito; Tsuji, Kiyomi; Kimura, Shigenobu; Harada, Ken-ichi

    2012-01-01

    Volatile organic compounds (VOCs), 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol, were detected together with β-cyclocitral from the cyanobacterium Microcystis aeruginosa NIES-843. These alcohols were optimally produced after 35 d of culture, during which nitrate nitrogen in the cultured broth became exhausted. Additionally, these alcohols were definitely produced using the 2-keto-acid decarboxylase (MaKDC) in Microcystis strains. These results suggested that these VOCs from Microcystis are significant for their lifecycle, because these compounds are not produced by any other genus of cyanobacteria. This is the first report of 2-keto-acid decarboxylase producing 3-methyl-1-butanol and 2-phenylethanol by an oxygenic photosynthetic microorganism. PMID:23047148

  18. Overexpression of PAD1 and FDC1 results in significant cinnamic acid decarboxylase activity in Saccharomyces cerevisiae.

    PubMed

    Richard, Peter; Viljanen, Kaarina; Penttilä, Merja

    2015-01-01

    The S. cerevisiae PAD1 gene had been suggested to code for a cinnamic acid decarboxylase, converting trans-cinnamic acid to styrene. This was suggested for the reason that the over-expression of PAD1 resulted in increased tolerance toward cinnamic acid, up to 0.6 mM. We show that by over-expression of the PAD1 together with the FDC1 the cinnamic acid decarboxylase activity can be increased significantly. The strain over-expressing PAD1 and FDC1 tolerated cinnamic acid concentrations up to 10 mM. The cooperation of Pad1p and Fdc1p is surprising since the PAD1 has a mitochondrial targeting sequence and the FDC1 codes for a cytosolic protein. The cinnamic acid decarboxylase activity was also seen in the cell free extract. The activity was 0.019 μmol per minute and mg of extracted protein. The overexpression of PAD1 and FDC1 resulted also in increased activity with the hydroxycinnamic acids ferulic acid, p-coumaric acid and caffeinic acid. This activity was not seen when FDC1 was overexpressed alone. An efficient cinnamic acid decarboxylase is valuable for the genetic engineering of yeast strains producing styrene. Styrene can be produced from endogenously produced L-phenylalanine which is converted by a phenylalanine ammonia lyase to cinnamic acid and then by a decarboxylase to styrene.

  19. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases.

    PubMed

    Liu, Pingyang; Torrens-Spence, Michael P; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2013-02-01

    Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development. PMID:22718265

  20. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases.

    PubMed

    Liu, Pingyang; Torrens-Spence, Michael P; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2013-02-01

    Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development.

  1. Tissue and regional distribution of cysteic acid decarboxylase. A new assay method.

    PubMed

    Wu, J Y; Moss, L G; Chen, M S

    1979-04-01

    A sensitive and rapid assay method method for cysteic acid decarboxylase was develped which combined the selectivity of ion exchange resin (a complete retention of the substrate, cysteic acid, and exclusion of the product, taurine) with the speed of a vacuum filtration. The synthesis and purification of 35S-labeled cysteic acid were described. The validity of the assay was established by the identification of the reaction product as taurine. With this new method, the decarboxylase activity was measured in discrete regions of bovine brain. Putamen had the highest activity, 172 pmol taurine formed/min/mg protein (100%), followed by caudate nucleus, 90%; cerebral cortex, 82%; hypothalamus, 81%; cerebellar cortex, 79%; cerebellar peduncle, 59%; thalamus, 42%; brain stem, 25%; pons, 10%; and corpus callosum, 3%. The decarboxylase activity in various mouse tissues was also determined as follows: liver, 403; brain, 145; kidney, 143; spinal cord, 59; lung, 21; and spleen, 10 pmol taurine formed/min/mg. No activity could be detected in skeleton muscle and heart, suggesting a different biosynthetic pathway for taurine synthesis in these tissues. The advantages and disadvantages of the new assay method are also discussed.

  2. 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. PMID:21957966

  3. 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.

  4. 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.

  5. Alternating skew deviation in association with anti-glutamic acid decarboxylase antibodies

    PubMed Central

    Farooq, Asim V.; Soin, Ketki; Moss, Heather E.

    2015-01-01

    The presence of an elevated anti-glutamic acid decarboxylase (GAD) antibody level has been associated with a number of eye movement abnormalities, as well as other findings including cerebellar ataxia and insulin dependent diabetes mellitus. Skew deviation in association with anti-GAD antibodies has not been previously reported. Here we report a case of alternating skew deviation along with cerebellar-brainstem signs in a patient with an elevated anti-GAD antibody titer. Follow-up neurologic evaluation after treatment with intravenous immunoglobulin revealed improvement in cerebellar-brainstem signs, while ophthalmic evaluation was stable. PMID:26594078

  6. 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; 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 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

  7. A glutamic acid decarboxylase (CgGAD) highly expressed in hemocytes of Pacific oyster Crassostrea gigas.

    PubMed

    Li, Meijia; Wang, Lingling; Qiu, Limei; Wang, Weilin; Xin, Lusheng; Xu, Jiachao; Wang, Hao; Song, Linsheng

    2016-10-01

    Glutamic acid decarboxylase (GAD), a rate-limiting enzyme to catalyze the reaction converting the excitatory neurotransmitter glutamate to inhibitory neurotransmitter γ-aminobutyric acid (GABA), not only functions in nervous system, but also plays important roles in immunomodulation in vertebrates. However, GAD has rarely been reported in invertebrates, and never in molluscs. In the present study, one GAD homologue (designed as CgGAD) was identified from Pacific oyster Crassostrea gigas. The full length cDNA of CgGAD was 1689 bp encoding a polypeptide of 562 amino acids containing a conserved pyridoxal-dependent decarboxylase domain. CgGAD mRNA and protein could be detected in ganglion and hemocytes of oysters, and their abundance in hemocytes was unexpectedly much higher than those in ganglion. More importantly, CgGAD was mostly located in those granulocytes without phagocytic capacity in oysters, and could dynamically respond to LPS stimulation. Further, after being transfected into HEK293 cells, CgGAD could promote the production of GABA. Collectively, these findings suggested that CgGAD, as a GABA synthase and molecular marker of GABAergic system, was mainly distributed in hemocytes and ganglion and involved in neuroendocrine-immune regulation network in oysters, which also provided a novel insight to the co-evolution between nervous system and immune system. PMID:27208883

  8. Effects of glutamate decarboxylase and gamma-aminobutyric acid (GABA) transporter on the bioconversion of GABA in engineered Escherichia coli.

    PubMed

    Le Vo, Tam Dinh; Kim, Tae Wan; Hong, Soon Ho

    2012-05-01

    Gamma-aminobutyric acid (GABA) is a non-essential amino acid and a precursor of pyrrolidone, a monomer of nylon 4. GABA can be biosynthesized through the decarboxylation of L: -glutamate by glutamate decarboxylase. In this study, the effects of glutamate decarboxylase (gadA, gadB), glutamate/GABA antiporter (gadC) and GABA aminotransferase (gabT) on GABA production were investigated in Escherichia coli. Glutamate decarboxylase was overexpressed alone or with the glutamate/GABA antiporter to enhance GABA synthesis. GABA aminotransferase, which redirects GABA into the TCA cycle, was knock-out mutated. When gadB and gadC were co-overexpressed in the gabT mutant strain, a final GABA concentration of 5.46 g/l was obtained from 10 g/l of monosodium glutamate (MSG), which corresponded to a GABA yield of 89.5%.

  9. Structure and Mechanism of Ferulic Acid Decarboxylase (FDC1) from Saccharomyces cerevisiae

    PubMed Central

    Bhuiya, Mohammad Wadud; Lee, Soon Goo

    2015-01-01

    The nonoxidative decarboxylation of aromatic acids occurs in a range of microbes and is of interest for bioprocessing and metabolic engineering. Although phenolic acid decarboxylases provide useful tools for bioindustrial applications, the molecular bases for how these enzymes function are only beginning to be examined. Here we present the 2.35-Å-resolution X-ray crystal structure of the ferulic acid decarboxylase (FDC1; UbiD) from Saccharomyces cerevisiae. FDC1 shares structural similarity with the UbiD family of enzymes that are involved in ubiquinone biosynthesis. The position of 4-vinylphenol, the product of p-coumaric acid decarboxylation, in the structure identifies a large hydrophobic cavity as the active site. Differences in the β2e-α5 loop of chains in the crystal structure suggest that the conformational flexibility of this loop allows access to the active site. The structure also implicates Glu285 as the general base in the nonoxidative decarboxylation reaction catalyzed by FDC1. Biochemical analysis showed a loss of enzymatic activity in the E285A mutant. Modeling of 3-methoxy-4-hydroxy-5-decaprenylbenzoate, a partial structure of the physiological UbiD substrate, in the binding site suggests that an ∼30-Å-long pocket adjacent to the catalytic site may accommodate the isoprenoid tail of the substrate needed for ubiquinone biosynthesis in yeast. The three-dimensional structure of yeast FDC1 provides a template for guiding protein engineering studies aimed at optimizing the efficiency of aromatic acid decarboxylation reactions in bioindustrial applications. PMID:25862228

  10. Aromatic L-amino acid decarboxylase (AADC) is crucial for brain development and motor functions.

    PubMed

    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

  11. GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop.

    PubMed

    Fenalti, Gustavo; Law, Ruby H P; Buckle, Ashley M; Langendorf, Christopher; Tuck, Kellie; Rosado, Carlos J; Faux, Noel G; Mahmood, Khalid; Hampe, Christiane S; Banga, J Paul; Wilce, Matthew; Schmidberger, Jason; Rossjohn, Jamie; El-Kabbani, Ossama; Pike, Robert N; Smith, A Ian; Mackay, Ian R; Rowley, Merrill J; Whisstock, James C

    2007-04-01

    Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5'-phosphate-dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form. We have determined the crystal structures of N-terminal truncations of both GAD isoforms. The structure of GAD67 shows a tethered loop covering the active site, providing a catalytic environment that sustains GABA production. In contrast, the same catalytic loop is inherently mobile in GAD65. Kinetic studies suggest that mobility in the catalytic loop promotes a side reaction that results in cofactor release and GAD65 autoinactivation. These data reveal the molecular basis for regulation of GABA homeostasis.

  12. Conversion of levulinic acid to 2-butanone by acetoacetate decarboxylase from Clostridium acetobutylicum.

    PubMed

    Min, Kyoungseon; Kim, Seil; Yum, Taewoo; Kim, Yunje; Sang, Byoung-In; Um, Youngsoon

    2013-06-01

    In this study, a novel system for synthesis of 2-butanone from levulinic acid (γ-keto-acid) via an enzymatic reaction was developed. Acetoacetate decarboxylase (AADC; E.C. 4.1.1.4) from Clostridium acetobutylicum was selected as a biocatalyst for decarboxylation of levulinic acid. The purified recombinant AADC from Escherichia coli successfully converted levulinic acid to 2-butanone with a conversion yield of 8.4-90.3 % depending on the amount of AADC under optimum conditions (30 °C and pH 5.0) despite that acetoacetate, a β-keto-acid, is a natural substrate of AADC. In order to improve the catalytic efficiency, an AADC-mediator system was tested using methyl viologen, methylene blue, azure B, zinc ion, and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as mediators. Among them, methyl viologen showed the best performance, increasing the conversion yield up to 6.7-fold in comparison to that without methyl viologen. The results in this study are significant in the development of a renewable method for the synthesis of 2-butanone from biomass-derived chemical, levulinic acid, through enzymatic decarboxylation. PMID:23624707

  13. Immunotherapy-responsive limbic encephalitis with antibodies to glutamic acid decarboxylase.

    PubMed

    Markakis, Ioannis; Alexopoulos, Harry; Poulopoulou, Cornelia; Akrivou, Sofia; Papathanasiou, Athanasios; Katsiva, Vassiliki; Lyrakos, Georgios; Gekas, Georgios; Dalakas, Marinos C

    2014-08-15

    Glutamic acid decarboxylase (GAD) has been recently identified as a target of humoral autoimmunity in a small subgroup of patients with non-paraneoplastic limbic encephalitis (NPLE). We present a patient with NPLE and positive anti-GAD antibodies who showed significant improvement after long-term immunotherapy. A 48-year old female was admitted with a two-year history of anterograde amnesia and seizures. Brain MRI revealed bilateral lesions of medial temporal lobes. Screening for anti-neuronal antibodies showed high anti-GAD titers in both serum and cerebrospinal fluid (CSF) with strong evidence of intrathecal production. The patient received treatment with prednisolone and long-term plasma exchange. During a 12-month follow-up, she exhibited complete seizure remission and an improvement in memory and visuo-spatial skills. Anti-GAD antibodies may serve as a useful marker to identify a subset of NPLE patients that respond to immunoregulatory treatment.

  14. Intrathecal-specific glutamic acid decarboxylase antibodies at low titers in autoimmune neurological disorders.

    PubMed

    Sunwoo, Jun-Sang; Chu, Kon; Byun, Jung-Ick; Moon, Jangsup; Lim, Jung-Ah; Kim, Tae-Joon; Lee, Soon-Tae; Jung, Keun-Hwa; Park, Kyung-Il; Jeon, Daejong; Jung, Ki-Young; Kim, Manho; Lee, Sang Kun

    2016-01-15

    Autoantibodies to glutamic acid decarboxylase (Gad-Abs) are implicated in various neurological syndromes. The present study aims to identify intrathecal-specific GAD-Abs and to determine clinical manifestations and treatment outcomes. Nineteen patients had GAD-Abs in cerebrospinal fluid but not in paired serum samples. Neurological syndromes included limbic encephalitis, temporal lobe epilepsy, cerebellar ataxia, autonomic dysfunction, and stiff-person syndrome. Immunotherapy had beneficial effects in 57.1% of patients, and the patients with limbic encephalitis responded especially well to immunotherapy. Intrathecal-specific antibodies to GAD at low titers may appear as nonspecific markers of immune activation within the central nervous system rather than pathogenic antibodies causing neuronal dysfunction. PMID:26711563

  15. 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.

  16. Cysteine Sulfinic Acid Decarboxylase Regulation: A Role for FXR and SHP in Murine Hepatic Taurine Metabolism

    PubMed Central

    Kerr, Thomas A.; Matsumoto, Yuri; Matsumoto, Hitoshi; Xie, Yan; Hirschberger, Lawrence L.; Stipanuk, Martha H.; Anakk, Sayeepriyadarshini; Moore, David D.; Watanabe, Mitsuhiro; Kennedy, Susan

    2014-01-01

    Background Bile acid synthesis is regulated by nuclear receptors including farnesoid X receptor (FXR) and small heterodimer partner (SHP), and by fibroblast growth factor15/19 (FGF15/19). Because bile acid synthesis involves amino acid conjugation, we hypothesized that hepatic cysteine sulfinic acid decarboxylase (CSAD) (a key enzyme in taurine synthesis) is regulated by bile acids. Aims To investigate CSAD regulation by bile acids and CSAD regulatory mechanisms. Methods Mice were fed a control diet or a diet supplemented with either 0.5% cholate or 2% cholestyramine. To gain mechanistic insight into CSAD regulation, we utilized GW4064 (FXR agonist), FGF19, or T-0901317 (LXR agonist) and Shp−/− mice. Tissue mRNA expression was determined by qRT-PCR. Amino acids were measured by HPLC. Results Mice supplemented with dietary cholate exhibited reduced hepatic CSAD mRNA expression while those receiving cholestyramine exhibited increased hepatic CSAD mRNA expression. Activation of FXR suppressed CSAD mRNA expression whereas hepatic CSAD mRNA expression was increased in Shp−/− mice. Hepatic hypotaurine concentration (the product of CSAD) was higher in Shp−/− mice with a corresponding increase in serum (but not hepatic) taurine-conjugated bile acids. FGF19 administration suppressed hepatic CYP7A1 mRNA but did not change CSAD mRNA expression. LXR activation induced CYP7A1 mRNA yet failed to induce CSAD mRNA expression. Conclusion CSAD mRNA expression is physiologically regulated by bile acids in a feedback fashion via mechanisms involving SHP and FXR but not FGF15/19 or LXR. These novel findings implicate bile acids as regulators of CSAD mRNA via mechanisms shared in part with CYP7A1. PMID:24033844

  17. Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase.

    PubMed

    Kanjee, Usheer; Gutsche, Irina; Alexopoulos, Eftichia; Zhao, Boyu; El Bakkouri, Majida; Thibault, Guillaume; Liu, Kaiyin; Ramachandran, Shaliny; Snider, Jamie; Pai, Emil F; Houry, Walid A

    2011-03-01

    The Escherichia coli inducible lysine decarboxylase, LdcI/CadA, together with the inner-membrane lysine-cadaverine antiporter, CadB, provide cells with protection against mild acidic conditions (pH∼5). To gain a better understanding of the molecular processes underlying the acid stress response, the X-ray crystal structure of LdcI was determined. The structure revealed that the protein is an oligomer of five dimers that associate to form a decamer. Surprisingly, LdcI was found to co-crystallize with the stringent response effector molecule ppGpp, also known as the alarmone, with 10 ppGpp molecules in the decamer. ppGpp is known to mediate the stringent response, which occurs in response to nutrient deprivation. The alarmone strongly inhibited LdcI enzymatic activity. This inhibition is important for modulating the consumption of lysine in cells during acid stress under nutrient limiting conditions. Hence, our data provide direct evidence for a link between the bacterial acid stress and stringent responses. PMID:21278708

  18. 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...

  19. Amino acids regulate expression of antizyme-1 to modulate ornithine decarboxylase activity.

    PubMed

    Ray, Ramesh M; Viar, Mary Jane; Johnson, Leonard R

    2012-02-01

    In a glucose-salt solution (Earle's balanced salt solution), asparagine (Asn) stimulates ornithine decarboxylase (ODC) activity in a dose-dependent manner, and the addition of epidermal growth factor (EGF) potentiates the effect of Asn. However, EGF alone fails to activate ODC. Thus, the mechanism by which Asn activates ODC is important for understanding the regulation of ODC activity. Asn reduced antizyme-1 (AZ1) mRNA and protein. Among the amino acids tested, Asn and glutamine (Gln) effectively inhibited AZ1 expression, suggesting a differential role for amino acids in the regulation of ODC activity. Asn decreased the putrescine-induced AZ1 translation. The absence of amino acids increased the binding of eukaryotic initiation factor 4E-binding protein (4EBP1) to 5'-mRNA cap and thereby inhibited global protein synthesis. Asn failed to prevent the binding of 4EBP1 to mRNA, and the bound 4EBP1 was unphosphorylated, suggesting the involvement of the mammalian target of rapamycin (mTOR) in the regulation of AZ1 synthesis. Rapamycin treatment (4 h) failed to alter the expression of AZ1. However, extending the treatment (24 h) allowed expression in the presence of amino acids, indicating that AZ1 is expressed when TORC1 signaling is decreased. This suggests the involvement of cap-independent translation. However, transient inhibition of mTORC2 by PP242 completely abolished the phosphorylation of 4EBP1 and decreased basal as well as putrescine-induced AZ1 expression. Asn decreased the phosphorylation of mTOR-Ser(2448) and AKT-Ser(473), suggesting the inhibition of mTORC2. In the absence of amino acids, mTORC1 is inhibited, whereas mTORC2 is activated, leading to the inhibition of global protein synthesis and increased AZ1 synthesis via a cap-independent mechanism. PMID:22157018

  20. Selective loss of Purkinje cells in a patient with anti‐glutamic acid decarboxylase antibody‐associated cerebellar ataxia

    PubMed Central

    Ishida, Kazuyuki; Mitoma, Hiroshi; Wada, Yoshiaki; Oka, Teruaki; Shibahara, Junji; Saito, Yuko; Murayama, Shigeo; Mizusawa, Hidehiro

    2007-01-01

    Anti‐glutamic acid decarboxylase antibody is associated with the development of progressive cerebellar ataxia and slowly progressive insulin‐dependent diabetes mellitus. Previously, the neurophysiological characteristics of IgG in the cerebrospinal fluid of a patient with anti‐glutamic acid decarboxylase antibody‐associated progressive cerebellar ataxia and slowly progressive insulin‐dependent diabetes mellitus were reported. Using a voltage‐gated whole‐cell recording technique, it was observed that the IgG in the cerebrospinal fluid of the patient selectively suppressed the inhibitory postsynaptic currents in the Purkinje cells. The patient died from aspiration pneumonia. Postmortem examination showed almost complete depletion of the Purkinje cells with Bergmann gliosis. Therefore, the main cause of cerebellar ataxia observed in this case may be attributed to the near‐complete depletion of the Purkinje cells. In this paper, the pathomechanisms underlying Purkinje cell damage are discussed. PMID:17119008

  1. Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines

    PubMed Central

    Izzo, Emanuela; Auta, James; Impagnatiello, Francesco; Pesold, Christine; Guidotti, Alessandro; Costa, Erminio

    2001-01-01

    Protracted administration of diazepam elicits tolerance, whereas discontinuation of treatment results in signs of dependence. Tolerance to the anticonvulsant action of diazepam is present in an early phase (6, 24, and 36 h) but disappears in a late phase (72–96 h) of withdrawal. In contrast, signs of dependence such as decrease in open-arm entries on an elevated plus-maze and increased susceptibility to pentylenetetrazol-induced seizures were apparent 96 h (but not 12, 24, or 48 h) after diazepam withdrawal. During the first 72 h of withdrawal, tolerance is associated with changes in the expression of GABAA (γ-aminobutyric acid type A) receptor subunits (decrease in γ2 and α1; increase in α5) and with an increase of mRNA expression of the most abundant form of glutamic acid decarboxylase (GAD), GAD67. In contrast, dl-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit mRNA and cognate protein, which are normal during the early phase of diazepam withdrawal, increase by approximately 30% in cortex and hippocampus in association with the appearance of signs of dependence 96 h after diazepam withdrawal. Immunohistochemical studies of GluR1 subunit expression with gold-immunolabeling technique reveal that the increase of GluR1 subunit protein is localized to layer V pyramidal neurons and their apical dendrites in the cortex, and to pyramidal neurons and in their dendritic fields in hippocampus. The results suggest an involvement of GABA-mediated processes in the development and maintenance of tolerance to diazepam, whereas excitatory amino acid-related processes (presumably via AMPA receptors) may be involved in the expression of signs of dependence after withdrawal. PMID:11248104

  2. Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines.

    PubMed

    Izzo, E; Auta, J; Impagnatiello, F; Pesold, C; Guidotti, A; Costa, E

    2001-03-13

    Protracted administration of diazepam elicits tolerance, whereas discontinuation of treatment results in signs of dependence. Tolerance to the anticonvulsant action of diazepam is present in an early phase (6, 24, and 36 h) but disappears in a late phase (72-96 h) of withdrawal. In contrast, signs of dependence such as decrease in open-arm entries on an elevated plus-maze and increased susceptibility to pentylenetetrazol-induced seizures were apparent 96 h (but not 12, 24, or 48 h) after diazepam withdrawal. During the first 72 h of withdrawal, tolerance is associated with changes in the expression of GABA(A) (gamma-aminobutyric acid type A) receptor subunits (decrease in gamma(2) and alpha(1); increase in alpha(5)) and with an increase of mRNA expression of the most abundant form of glutamic acid decarboxylase (GAD), GAD(67). In contrast, dl-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit mRNA and cognate protein, which are normal during the early phase of diazepam withdrawal, increase by approximately 30% in cortex and hippocampus in association with the appearance of signs of dependence 96 h after diazepam withdrawal. Immunohistochemical studies of GluR1 subunit expression with gold-immunolabeling technique reveal that the increase of GluR1 subunit protein is localized to layer V pyramidal neurons and their apical dendrites in the cortex, and to pyramidal neurons and in their dendritic fields in hippocampus. The results suggest an involvement of GABA-mediated processes in the development and maintenance of tolerance to diazepam, whereas excitatory amino acid-related processes (presumably via AMPA receptors) may be involved in the expression of signs of dependence after withdrawal.

  3. Aromatic L-amino acid decarboxylase deficiency diagnosed by clinical metabolomic profiling of plasma.

    PubMed

    Atwal, Paldeep S; Donti, Taraka R; Cardon, Aaron L; Bacino, C A; Sun, Qin; Emrick, L; Reid Sutton, V; Elsea, Sarah H

    2015-01-01

    Aromatic L-amino acid decarboxylase (AADC) deficiency is an inborn error of metabolism affecting the biosynthesis of serotonin, dopamine, and catecholamines. We report a case of AADC deficiency that was detected using the Global MAPS platform. This is a novel platform that allows for parallel clinical testing of hundreds of metabolites in a single plasma specimen. It uses a state-of-the-art mass spectrometry platform, and the resulting spectra are compared against a library of ~2500 metabolites. Our patient is now a 4 year old boy initially seen at 11 months of age for developmental delay and hypotonia. Multiple tests had not yielded a diagnosis until exome sequencing revealed compound heterozygous variants of uncertain significance (VUS), c.286G>A (p.G96R) and c.260C>T (p.P87L) in the DDC gene, causal for AADC deficiency. CSF neurotransmitter analysis confirmed the diagnosis with elevated 3-methoxytyrosine (3-O-methyldopa). Metabolomic profiling was performed on plasma and revealed marked elevation in 3-methoxytyrosine (Z-score +6.1) consistent with the diagnosis of AADC deficiency. These results demonstrate that the Global MAPS platform is able to diagnose AADC deficiency from plasma. In summary, we report a novel and less invasive approach to diagnose AADC deficiency using plasma metabolomic profiling.

  4. Taurine homeostasis requires de novo synthesis via cysteine sulfinic acid decarboxylase during zebrafish early embryogenesis.

    PubMed

    Chang, Yen-Chia; Ding, Shih-Torng; Lee, Yen-Hua; Wang, Ya-Ching; Huang, Ming-Feng; Liu, I-Hsuan

    2013-02-01

    Cysteine sulfinic acid decarboxylase (Csad) is the rate-limiting enzyme in the de novo biosynthesis of taurine. There are a number of physiological roles of taurine, such as bile salt synthesis, osmoregulation, lipid metabolism, and oxidative stress inhibition. To investigate the role of de novo synthesis of taurine during embryonic development, zebrafish csad was cloned and functionally analyzed. Semi-quantitative RT-PCR showed that csad transcripts are maternally deposited, while whole-mount in situ hybridization demonstrated that csad is expressed in yolk syncytial layer and various embryonic tissues such as notochord, brain, retina, pronephric duct, liver, and pancreas. Knockdown of csad significantly reduced the embryonic taurine level, and the affected embryos had increased early mortality and cardiac anomalies. mRNA coinjection and taurine supplementation rescued the cardiac phenotypes suggesting that taurine originating from the de novo synthesis pathway plays a role in cardiac development. Our findings indicated that the de novo synthesis pathway via Csad plays a critical role in taurine homeostasis and cardiac development in zebrafish early embryos. PMID:22907836

  5. Production of Dopamine by Aromatic l-Amino Acid Decarboxylase Cells after Spinal Cord Injury.

    PubMed

    Ren, Li-Qun; Wienecke, Jacob; Hultborn, Hans; Zhang, Mengliang

    2016-06-15

    Aromatic l-amino acid decarboxylase (AADC) cells are widely distributed in the spinal cord, and their functions are largely unknown. We have previously found that AADC cells in the spinal cord could increase their ability to produce serotonin (5-hydroxytryptamine) from 5-hydroxytryptophan after spinal cord injury (SCI). Because AADC is a common enzyme catalyzing 5-hydroxytryptophan to serotonin and l-3,4-dihydroxyphenylalanine (l-dopa) to dopamine (DA), it seems likely that the ability of AADC cells using l-dopa to synthesize DA is also increased. To prove whether or not this is the case, a similar rat sacral SCI model and a similar experimental paradigm were adopted as that which we had used previously. In the chronic SCI rats (> 45 days), no AADC cells expressed DA if there was no exogenous l-dopa application. However, following administration of a peripheral AADC inhibitor (carbidopa) with or without a monoamine oxidase inhibitor (pargyline) co-application, systemic administration of l-dopa resulted in ∼94% of AADC cells becoming DA-immunopositive in the spinal cord below the lesion, whereas in normal or sham-operated rats none or very few of AADC cells became DA-immunopositive with the same treatment. Using tail electromyography, spontaneous tail muscle activity was increased nearly fivefold over the baseline level. When pretreated with a central AADC inhibitor (NSD-1015), further application of l-dopa failed to increase the motoneuron activity although the expression of DA in the AADC cells was not completely inhibited. These findings demonstrate that AADC cells in the spinal cord below the lesion gain the ability to produce DA from its precursor in response to SCI. This ability also enables the AADC cells to produce 5-HT and trace amines, and likely contributes to the development of hyperexcitability. These results might also be implicated for revealing the pathological mechanisms underlying l-dopa-induced dyskinesia in Parkinson's disease. PMID:26830512

  6. Production of Dopamine by Aromatic l-Amino Acid Decarboxylase Cells after Spinal Cord Injury.

    PubMed

    Ren, Li-Qun; Wienecke, Jacob; Hultborn, Hans; Zhang, Mengliang

    2016-06-15

    Aromatic l-amino acid decarboxylase (AADC) cells are widely distributed in the spinal cord, and their functions are largely unknown. We have previously found that AADC cells in the spinal cord could increase their ability to produce serotonin (5-hydroxytryptamine) from 5-hydroxytryptophan after spinal cord injury (SCI). Because AADC is a common enzyme catalyzing 5-hydroxytryptophan to serotonin and l-3,4-dihydroxyphenylalanine (l-dopa) to dopamine (DA), it seems likely that the ability of AADC cells using l-dopa to synthesize DA is also increased. To prove whether or not this is the case, a similar rat sacral SCI model and a similar experimental paradigm were adopted as that which we had used previously. In the chronic SCI rats (> 45 days), no AADC cells expressed DA if there was no exogenous l-dopa application. However, following administration of a peripheral AADC inhibitor (carbidopa) with or without a monoamine oxidase inhibitor (pargyline) co-application, systemic administration of l-dopa resulted in ∼94% of AADC cells becoming DA-immunopositive in the spinal cord below the lesion, whereas in normal or sham-operated rats none or very few of AADC cells became DA-immunopositive with the same treatment. Using tail electromyography, spontaneous tail muscle activity was increased nearly fivefold over the baseline level. When pretreated with a central AADC inhibitor (NSD-1015), further application of l-dopa failed to increase the motoneuron activity although the expression of DA in the AADC cells was not completely inhibited. These findings demonstrate that AADC cells in the spinal cord below the lesion gain the ability to produce DA from its precursor in response to SCI. This ability also enables the AADC cells to produce 5-HT and trace amines, and likely contributes to the development of hyperexcitability. These results might also be implicated for revealing the pathological mechanisms underlying l-dopa-induced dyskinesia in Parkinson's disease.

  7. Spinal cord injury enables aromatic L-amino acid decarboxylase cells to synthesize monoamines.

    PubMed

    Wienecke, Jacob; Ren, Li-Qun; Hultborn, Hans; Chen, Meng; Møller, Morten; Zhang, Yifan; Zhang, Mengliang

    2014-09-01

    Serotonin (5-HT), an important modulator of both sensory and motor functions in the mammalian spinal cord, originates mainly in the raphe nuclei of the brainstem. However, following complete transection of the spinal cord, small amounts of 5-HT remain detectable below the lesion. It has been suggested, but not proven, that this residual 5-HT is produced by intraspinal 5-HT neurons. Here, we show by immunohistochemical techniques that cells containing the enzyme aromatic l-amino acid decarboxylase (AADC) occur not only near the central canal, as reported by others, but also in the intermediate zone and dorsal horn of the spinal gray matter. We show that, following complete transection of the rat spinal cord at S2 level, AADC cells distal to the lesion acquire the ability to produce 5-HT from its immediate precursor, 5-hydroxytryptophan. Our results indicate that this phenotypic change in spinal AADC cells is initiated by the loss of descending 5-HT projections due to spinal cord injury (SCI). By in vivo and in vitro electrophysiology, we show that 5-HT produced by AADC cells increases the excitability of spinal motoneurons. The phenotypic change in AADC cells appears to result from a loss of inhibition by descending 5-HT neurons and to be mediated by 5-HT1B receptors expressed by AADC cells. These findings indicate that AADC cells are a potential source of 5-HT at spinal levels below an SCI. The production of 5-HT by AADC cells, together with an upregulation of 5-HT2 receptors, offers a partial explanation of hyperreflexia below a chronic SCI. PMID:25186745

  8. Single amino-acid replacement is responsible for the stabilization of ornithine decarboxylase in HMOA cells.

    PubMed

    Miyazaki, Y; Matsufuji, S; Murakami, Y; Hayashi, S

    1993-06-15

    The half-life of ornithine decarboxylase (ODC) in HMOA cells, a variant cell line derived from hepatoma tissue culture (HTC) cells, is markedly increased compared with that in the parental cell line. In the present study, we examined which of the three relevant factors is responsible for the ODC stabilization in HMOA cells, namely ODC itself, a regulatory protein antizyme and an ODC-degrading activity. SDS/PAGE analysis of radiolabeled ODC revealed that ODC from HMOA cells migrated somewhat faster than that from HTC cells, suggesting that HMOA ODC was structurally altered. Direct sequencing of reverse-transcription/polymerase-chain-reaction (RT-PCR) products of ODC mRNA from HMOA cells revealed a T to G replacement, causing a Cys441-->Trp replacement near the C-terminus. No alteration was found in the whole coding region of antizyme mRNA. An authentic mutant ODC cDNA with the same replacement was transfected and expressed in C55.7 ODC-deficient Chinese hamster ovary cells. Upon cycloheximide treatment, the mutant ODC activity did not decrease appreciably for at least 3 h, whereas wild-type ODC activity decreased with a half-life of 1 h. In-vitro-synthesized mutant ODC with the Cys441-->Trp (or Ala) replacement was also stable in a reticulocyte-lysate ODC-degradation system. Metabolically labeled and purified mouse ODC was degraded in HMOA cell extracts in the presence of ATP and antizyme as rapidly as in HTC cell extracts, indicating that HMOA cells have a normal ODC degrading activity. These results indicated that the single amino acid replacement, Cys441-->Trp, is responsible for the stabilization of ODC in HMOA cells and that Cys441 is important for rapid ODC turnover.

  9. Structural Basis of Enzymatic Activity for the Ferulic Acid Decarboxylase (FADase) from Enterobacter sp. Px6-4

    PubMed Central

    Liang, Lianming; Sun, Yuna; Huang, Jingwen; Li, Xuemei; Cao, Yi; Meng, Zhaohui; Zhang, Ke-Qin

    2011-01-01

    Microbial ferulic acid decarboxylase (FADase) catalyzes the transformation of ferulic acid to 4-hydroxy-3-methoxystyrene (4-vinylguaiacol) via non-oxidative decarboxylation. Here we report the crystal structures of the Enterobacter sp. Px6-4 FADase and the enzyme in complex with substrate analogues. Our analyses revealed that FADase possessed a half-opened bottom β-barrel with the catalytic pocket located between the middle of the core β-barrel and the helical bottom. Its structure shared a high degree of similarity with members of the phenolic acid decarboxylase (PAD) superfamily. Structural analysis revealed that FADase catalyzed reactions by an “open-closed” mechanism involving a pocket of 8×8×15 Å dimension on the surface of the enzyme. The active pocket could directly contact the solvent and allow the substrate to enter when induced by substrate analogues. Site-directed mutagenesis showed that the E134A mutation decreased the enzyme activity by more than 60%, and Y21A and Y27A mutations abolished the enzyme activity completely. The combined structural and mutagenesis results suggest that during decarboxylation of ferulic acid by FADase, Trp25 and Tyr27 are required for the entering and proper orientation of the substrate while Glu134 and Asn23 participate in proton transfer. PMID:21283705

  10. Aromatic L-amino acid decarboxylase deficiency with hyperdopaminuria. Clinical and laboratory findings in response to different therapies.

    PubMed

    Fiumara, A; Bräutigam, C; Hyland, K; Sharma, R; Lagae, L; Stoltenborg, B; Hoffmann, G F; Jaeken, J; Wevers, R A

    2002-08-01

    Aromatic L-amino acid decarboxylase (AADC - E.C. 4.1.1.28) converts L-dopa to dopamine and 5-hydroxytryptophan to serotonin. Inherited deficiency of this enzyme leads to decreased brain levels of these neurotransmitters. Clinically this results in the development of a progressive neurometabolic disorder characterized by severe hypotonia, dystonic and choreoathetoid movements, oculogyric crises, and hypothermia from infancy. Here we describe the clinical, biochemical and molecular details of two affected brothers, one of whom, despite the lack of AADC, presented with hyperdopaminuria. In addition, we detail his reactions to treatment with dopaminergic agonists, monoamine oxidase inhibitors and pyridoxine.

  11. Wound-Inducible Biosynthesis of Phytoalexin Hydroxycinnamic Acid Amides of Tyramine in Tryptophan and Tyrosine Decarboxylase Transgenic Tobacco Lines1

    PubMed Central

    Guillet, Gabriel; De Luca, Vincenzo

    2005-01-01

    The wound-activated biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine was compared in untransformed and transgenic tobacco (Nicotiana tabacum) lines that express tryptophan decarboxylase (TDC), tyrosine decarboxylase (TYDC), or both activities. Transgenic in vitro-grown tobacco lines expressing TDC activity accumulated high levels of tryptamine but not hydroxycinnamic amides of tryptamine. In contrast, transgenic tobacco lines expressing TYDC accumulated tyramine as well as p-coumaroyltyramine and feruloyltyramine. The MeOH-soluble and cell wall fractions showed higher concentrations of wound-inducible p-coumaroyltyramine and feruloyltyramine, especially at and around wound sites, in TYDC and TDC ×TYDC tobacco lines compared to wild-type or TDC lines. All the enzymes involved in the biosynthesis of hydroxycinnamic acid amides of tyramine were found to be similarly wound inducible in all tobacco genotypes investigated. These results provide experimental evidence that, under some circumstances, TYDC activity can exert a rate-limiting control over the carbon flux allocated to the biosynthesis of hydroxycinnamic acid amides of tyramine. PMID:15665252

  12. Amino acid decarboxylase activity and other chemical characteristics as related to freshness loss in iced cod (Gadus morhua).

    PubMed

    Hernández-Herrero, M Manuela; Duflos, Guillaume; Malle, Pierre; Bouquelet, Stéphane

    2002-07-01

    Biogenic amine levels and other biochemical indicators were measured to study the safety of and the loss of freshness in iced Atlantic cod. Biogenic amine content exhibited high variability during iced storage of Atlantic cod. Ornithine and lysine decarboxylase activity apparently increased at the end of the storage period. Amino acid activity was probably generated by endogenous amino acid decarboxylases of raw fish. No statistical differences were observed in the total volatile base fraction or in the ammonia or monomethylamine contents during iced storage. However, trimethylamine contents showed a significant exponential relationship with time and sensory score. Cod formed inosine as the major metabolite of IMP. The H and G indices showed a linear relationship with time and sensory score and served as good indicators of cod freshness quality. However, the K, Ki, and P indices showed a logarithmic relationship with time and sensory score. IMP, K, Ki, and P served as indicators of freshness lost during the early stages of chilled storage of cod. PMID:12117250

  13. 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.

  14. Inhibition of ornithine decarboxylase induction by retinobenzoic acids in relation to their binding affinities to cellular retinoid-binding proteins.

    PubMed

    Takagi, K; Suganuma, M; Kagechika, H; Shudo, K; Ninomiya, M; Muto, Y; Fujiki, H

    1988-01-01

    Retinobenzoic acids induce differentiation of human promyelocytic leukemia cells (HL-60). Like retinoic acid, 14 retinobenzoic acids inhibited the induction of ornithine decarboxylase (ODC) by teleocidin in mouse skin. The mechanism(s) of inhibition of ODC induction by 7 retinobenzoic acids, Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, and Ch 55 was compared with those by all-trans-retinoic acid and the arotinoid compound 19. Application of 114 nmol of Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, or Ch 55, 10 min before 11.4 nmol of teleocidin, resulted in 76.7%, 82.0%, 76.2%, 28.3%, 48.4%, 58.6%, and 85.1% inhibition of ODC induction, respectively. Since all-trans-retinoic acid and compound 19 were also inhibitory, we determined whether retinobenzoic acids bind to cellular retinoic acid-binding protein (CRABP) isolated from bovine adrenal glands. Am 80 and Am 580 inhibited the specific binding of 3H-retinoic acid to CRABP, but also showed less affinity than authentic unlabeled retinoic acid and compound 19. Am 81, Am 590, Am 68, Sa 80, and Ch 55 at up to 10 microM were not effective competitors of the binding of either 3H-retinoic acid or 3H-retinol. These results suggest that the inhibition of ODC induction can be mediated by pathways that do not involve CRABP or the cellular retinol-binding protein.

  15. 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. PMID:25547838

  16. Co-localization of glutamic acid decarboxylase and vesicular GABA transporter in cytochrome oxidase patches of macaque striate cortex.

    PubMed

    Adams, Daniel L; Economides, John R; Horton, Jonathan C

    2015-01-01

    The patches in primary visual cortex constitute hot spots of metabolic activity, manifested by enhanced levels of cytochrome oxidase (CO) activity. They are also labeled preferentially by immunostaining for glutamic acid decarboxylase (GAD), γ-aminobutyric acid (GABA), and parvalbumin. However, calbindin shows stronger immunoreactivity outside patches. In light of this discrepancy, the distribution of the vesicular GABA transporter (VGAT) was examined in striate cortex of two normal macaques. VGAT immunoreactivity was strongest in layers 4B, 4Cα, and 5. In tangential sections, the distribution of CO, GAD, and VGAT was compared in layer 2/3. There was a close match between all three labels. This finding indicates that GABA synthesis is enriched in patches, and that inhibitory synapses are more active in patches than interpatches. PMID:26579566

  17. Terminal Olefin (1-Alkene) Biosynthesis by a Novel P450 Fatty Acid Decarboxylase from Jeotgalicoccus Species ▿ †

    PubMed Central

    Rude, Mathew A.; Baron, Tarah S.; Brubaker, Shane; Alibhai, Murtaza; Del Cardayre, Stephen B.; Schirmer, Andreas

    2011-01-01

    Terminal olefins (1-alkenes) are natural products that have important industrial applications as both fuels and chemicals. However, their biosynthesis has been largely unexplored. We describe a group of bacteria, Jeotgalicoccus spp., which synthesize terminal olefins, in particular 18-methyl-1-nonadecene and 17-methyl-1-nonadecene. These olefins are derived from intermediates of fatty acid biosynthesis, and the key enzyme in Jeotgalicoccus sp. ATCC 8456 is a terminal olefin-forming fatty acid decarboxylase. This enzyme, Jeotgalicoccus sp. OleT (OleTJE), was identified by purification from cell lysates, and its encoding gene was identified from a draft genome sequence of Jeotgalicoccus sp. ATCC 8456 using reverse genetics. Heterologous expression of the identified gene conferred olefin biosynthesis to Escherichia coli. OleTJE is a P450 from the cyp152 family, which includes bacterial fatty acid hydroxylases. Some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in α- and/or β-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. The discovery of these terminal olefin-forming P450 enzymes represents a third biosynthetic pathway (in addition to alkane and long-chain olefin biosynthesis) to convert fatty acid intermediates into hydrocarbons. Olefin-forming fatty acid decarboxylation is a novel reaction that can now be added to the catalytic repertoire of the versatile cytochrome P450 enzyme family. PMID:21216900

  18. On the biosynthesis of free and covalently bound PQQ. Glutamic acid decarboxylase from Escherichia coli is a pyridoxo-quinoprotein.

    PubMed

    van der Meer, R A; Groen, B W; Duine, J A

    1989-03-27

    Analysis of glutamic acid decarboxylase (GDC) (EC 4.1.1.15) from Escherichia coli ATCC 11246 revealed the presence of six pyridoxal phosphates (PLPs) as well as six covalently bound pyrroloquinoline quinones (PQQs) per hexameric enzyme molecule. This is the second example of a pyridoxo-quinoprotein, suggesting that other atypical pyridoxoproteins (PLP-containing enzymes) have similar cofactor composition. Since the organism did not produce free PQQ and its quinoprotein glucose dehydrogenase was present in the apo form, free PQQ is not used in the assemblage of GDC. Most probably, biosynthesis of covalently bound cofactor occurs in situ via a route which is different from that of free PQQ. Thus, organisms previously believed to be unable to synthesize (free) PQQ could in fact be able to produce quinoproteins with covalently bound cofactor. Implications for the role of PQQ in eukaryotic cells are discussed.

  19. Aromatic amino Acid decarboxylase deficiency not responding to pyridoxine and bromocriptine therapy: case report and review of response to treatment.

    PubMed

    Alfadhel, Majid; Kattan, Rana

    2014-01-01

    Aromatic L-amino acid decarboxylase (AADC) deficiency (MIM #608643) is an autosomal recessive inborn error of monoamines. It is caused by a mutation in the DDC gene that leads to a deficiency in the AADC enzyme. The clinical features of this condition include a combination of dopamine, noradrenaline, and serotonin deficiencies, and a patient may present with hypotonia, oculogyric crises, sweating, hypersalivation, autonomic dysfunction, and progressive encephalopathy with severe developmental delay. We report the case of an 8-month-old boy who presented with the abovementioned symptoms and who was diagnosed with AADC deficiency based on clinical, biochemical, and molecular investigations. Treatment with bromocriptine and pyridoxine showed no improvement. These data support the findings observed among previously reported cohorts that showed poor response of this disease to current regimens. Alternative therapies are needed to ameliorate the clinical complications associated with this disorder.

  20. 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

    2010-09-01

    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.

  1. Molecular and Functional Analyses of Amino Acid Decarboxylases Involved in Cuticle Tanning in Tribolium castaneum*

    PubMed Central

    Arakane, Yasuyuki; Lomakin, Joseph; Beeman, Richard W.; Muthukrishnan, Subbaratnam; Gehrke, Stevin H.; Kanost, Michael R.; Kramer, Karl J.

    2009-01-01

    Aspartate 1-decarboxylase (ADC) and 3,4-dihydroxyphenylalanine decarboxylase (DDC) provide β-alanine and dopamine used in insect cuticle tanning. β-Alanine is conjugated with dopamine to yield N-β-alanyldopamine (NBAD), a substrate for the phenol oxidase laccase that catalyzes the synthesis of cuticle protein cross-linking agents and pigment precursors. We identified ADC and DDC genes in the red flour beetle, Tribolium castaneum (Tc), and investigated their functions. TcADC mRNA was most abundant prior to the pupal-adult molt. Injection of TcADC double-stranded (ds) RNA (dsTcADC) into mature larvae resulted in depletion of NBAD in pharate adults, accumulation of dopamine, and abnormally dark pigmentation of the adult cuticle. Injection of β-alanine, the expected product of ADC, into dsTcADC-treated pupae rescued the pigmentation phenotype, resulting in normal rust-red color. A similar pattern of catechol content consisting of elevated dopamine and depressed NBAD was observed in the genetic black mutants of Tribolium, in which levels of TcADC mRNA were drastically reduced. Furthermore, from the Tribolium black mutant and dsTcADC-injected insects both exhibited similar changes in material properties. Dynamic mechanical analysis of elytral cuticle from beetles with depleted TcADC transcripts revealed diminished cross-linking of cuticular components, further confirming the important role of oxidation products of NBAD as cross-linking agents during cuticle tanning. Injection of dsTcDDC into larvae produced a lethal pupal phenotype, and the resulting grayish pupal cuticle exhibited many small patches of black pigmentation. When dsTcDDC was injected into young pupae, the resulting adults had abnormally dark brown body color, but there was little mortality. Injection of dsTcDDC resulted in more than a 5-fold increase in levels of DOPA, indicating that lack of TcDDC led to accumulation of its substrate, DOPA. PMID:19366687

  2. 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.

  3. Gamma-aminobutyric acid production using immobilized glutamate decarboxylase followed by downstream processing with cation exchange chromatography.

    PubMed

    Lee, Seungwoon; Ahn, Jungoh; Kim, Yeon-Gu; Jung, Joon-Ki; Lee, Hongweon; Lee, Eun Gyo

    2013-01-15

    We have developed a gamma-aminobutyric acid (GABA) production technique using his-tag mediated immobilization of Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamate to GABA. The GAD was obtained at 1.43 g/L from GAD-overexpressed E. coli fermentation and consisted of 59.7% monomer, 29.2% dimer and 2.3% tetramer with a 97.6% soluble form of the total GAD. The harvested GAD was immobilized to metal affinity gel with an immobilization yield of 92%. Based on an investigation of specific enzyme activity and reaction characteristics, glutamic acid (GA) was chosen over monosodium glutamate (MSG) as a substrate for immobilized GAD, resulting in conversion of 2.17 M GABA in a 1 L reactor within 100 min. The immobilized enzymes retained 58.1% of their initial activities after ten consecutive uses. By using cation exchange chromatography followed by enzymatic conversion, GABA was separated from the residual substrate and leached GAD. As a consequence, the glutamic acid was mostly removed with no detectable GAD, while 91.2% of GABA was yielded in the purification step.

  4. Identification of the Enterobacteriaceae in Montasio cheese and assessment of their amino acid decarboxylase activity.

    PubMed

    Maifreni, Michela; Frigo, Francesca; Bartolomeoli, Ingrid; Innocente, Nadia; Biasutti, Marialuisa; Marino, Marilena

    2013-02-01

    The aim of the study was to identify the species of Enterobacteriaceae present in Montasio cheese and to assess their potential to produce biogenic amines. Plate count methods and an Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) approach, combined with 16S rDNA sequencing, were used to investigate the Enterobacteriaceae community present during the cheesemaking and ripening of 6 batches of Montasio cheese. Additionally, the potential decarboxylation abilities of selected bacterial isolates were qualitatively and quantitatively assessed against tyrosine, histidine, ornithine and lysine. The most predominant species detected during cheese manufacturing and ripening were Enterobacter cloacae, Escherichia coli and Hafnia alvei. The non-limiting physico-chemical conditions (pH, NaCl% and a(w)) during ripening were probably the cause of the presence of detectable levels of Enterobacteriaceae up to 120 d of ripening. The HPLC test showed that cadaverine and putrescine were the amines produced in higher amounts by almost all isolates, indicating that the presence of these amines in cheese can be linked to the presence of high counts of Enterobacteriaceae. 44 isolates produced low amounts of histamine (<300 ppm), and four isolates produced more than 1000 ppm of this amine. Only 9 isolates, belonging to the species Citrobacter freundii, Esch. coli and Raoultella ornithinolytica, appeared to produce tyramine. These data provided new information regarding the decarboxylase activity of some Enterobacteriaceae species, including Pantoea agglomerans, Esch. fergusonii and R. ornithinolytica. PMID:23298547

  5. Knockout of the p-Coumarate Decarboxylase Gene from Lactobacillus plantarum Reveals the Existence of Two Other Inducible Enzymatic Activities Involved in Phenolic Acid Metabolism

    PubMed Central

    Barthelmebs, Lise; Divies, Charles; Cavin, Jean-François

    2000-01-01

    Lactobacillus plantarum NC8 contains a pdc gene coding for p-coumaric acid decarboxylase activity (PDC). A food grade mutant, designated LPD1, in which the chromosomal pdc gene was replaced with the deleted pdc gene copy, was obtained by a two-step homologous recombination process using an unstable replicative vector. The LPD1 mutant strain remained able to weakly metabolize p-coumaric and ferulic acids into vinyl derivatives or into substituted phenyl propionic acids. We have shown that L. plantarum has a second acid phenol decarboxylase enzyme, better induced with ferulic acid than with p-coumaric acid, which also displays inducible acid phenol reductase activity that is mostly active when glucose is added. Those two enzymatic activities are in competition for p-coumaric and ferulic acid degradation, and the ratio of the corresponding derivatives depends on induction conditions. Moreover, PDC appeared to decarboxylate ferulic acid in vitro with a specific activity of about 10 nmol · min−1 · mg−1 in the presence of ammonium sulfate. Finally, PDC activity was shown to confer a selective advantage on LPNC8 grown in acidic media supplemented with p-coumaric acid, compared to the LPD1 mutant devoid of PDC activity. PMID:10919793

  6. 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

  7. Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production.

    PubMed

    Tajabadi, Naser; Baradaran, Ali; Ebrahimpour, Afshin; Rahim, Raha A; Bakar, Fatimah A; Manap, Mohd Yazid A; Mohammed, Abdulkarim S; Saari, Nazamid

    2015-07-01

    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.

  8. Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production.

    PubMed

    Tajabadi, Naser; Baradaran, Ali; Ebrahimpour, Afshin; Rahim, Raha A; Bakar, Fatimah A; Manap, Mohd Yazid A; Mohammed, Abdulkarim S; Saari, Nazamid

    2015-07-01

    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

  9. The selective conversion of glutamic acid in amino acid mixtures using glutamate decarboxylase--a means of separating amino acids for synthesizing biobased chemicals.

    PubMed

    Teng, Yinglai; Scott, Elinor L; Sanders, Johan P M

    2014-01-01

    Amino acids (AAs) derived from hydrolysis of protein rest streams are interesting feedstocks for the chemical industry due to their functionality. However, separation of AAs is required before they can be used for further applications. Electrodialysis may be applied to separate AAs, but its efficiency is limited when separating AAs with similar isoelectric points. To aid the separation, specific conversion of an AA to a useful product with different charge behavior to the remaining compounds is desired. Here the separation of L-aspartic acid (Asp) and L-glutamic acid (Glu) was studied. L-Glutamate α-decarboxylase (GAD, Type I, EC 4.1.1.15) was applied to specifically convert Glu into γ-aminobutyric acid (GABA). GABA has a different charge behavior from Asp therefore allowing a potential separation by electrodialysis. Competitive inhibition and reduced operational stability caused by Asp could be eliminated by maintaining a sufficiently high concentration of Glu. Immobilization of GAD does not reduce the enzyme's initial activity. However, the operational stability was slightly reduced. An initial study on the reaction operating in a continuous mode was performed using a column reactor packed with immobilized GAD. As the reaction mixture was only passed once through the reactor, the conversion of Glu was lower than expected. To complete the conversion of Glu, the stream containing Asp and unreacted Glu might be recirculated back to the reactor after GABA has been removed. Overall, the reaction by GAD is specific to Glu and can be applied to aid the electrodialysis separation of Asp and Glu. PMID:24616376

  10. The selective conversion of glutamic acid in amino acid mixtures using glutamate decarboxylase--a means of separating amino acids for synthesizing biobased chemicals.

    PubMed

    Teng, Yinglai; Scott, Elinor L; Sanders, Johan P M

    2014-01-01

    Amino acids (AAs) derived from hydrolysis of protein rest streams are interesting feedstocks for the chemical industry due to their functionality. However, separation of AAs is required before they can be used for further applications. Electrodialysis may be applied to separate AAs, but its efficiency is limited when separating AAs with similar isoelectric points. To aid the separation, specific conversion of an AA to a useful product with different charge behavior to the remaining compounds is desired. Here the separation of L-aspartic acid (Asp) and L-glutamic acid (Glu) was studied. L-Glutamate α-decarboxylase (GAD, Type I, EC 4.1.1.15) was applied to specifically convert Glu into γ-aminobutyric acid (GABA). GABA has a different charge behavior from Asp therefore allowing a potential separation by electrodialysis. Competitive inhibition and reduced operational stability caused by Asp could be eliminated by maintaining a sufficiently high concentration of Glu. Immobilization of GAD does not reduce the enzyme's initial activity. However, the operational stability was slightly reduced. An initial study on the reaction operating in a continuous mode was performed using a column reactor packed with immobilized GAD. As the reaction mixture was only passed once through the reactor, the conversion of Glu was lower than expected. To complete the conversion of Glu, the stream containing Asp and unreacted Glu might be recirculated back to the reactor after GABA has been removed. Overall, the reaction by GAD is specific to Glu and can be applied to aid the electrodialysis separation of Asp and Glu.

  11. Japanese cases of acute onset diabetic ketosis without acidosis in the absence of glutamic acid decarboxylase autoantibody.

    PubMed

    Iwasaki, Yorihiro; Hamamoto, Yoshiyuki; Kawasaki, Yukiko; Ikeda, Hiroki; Honjo, Sachiko; Wada, Yoshiharu; Koshiyama, Hiroyuki

    2010-04-01

    We report consecutive Japanese patients presented with acute onset diabetic ketosis who had negative glutamic acid decarboxylase autoantibody (GADAb) to clarify the clinical characteristics of them. A total of consecutive 1,296 in-patients with newly diagnosed diabetes mellitus, who were admitted to our center from April 2003 to October 2008, were analyzed. Among them, 17 patients who presented with acute onset diabetic ketosis without acidosis, and found to be negative for GADAb, were included. They showed male preponderance (n = 15). Ten patients had history of excessive ingestion of sugar-containing soft drink. Patients who successfully withdrew insulin therapy by 6 months (n = 7) showed significantly higher insulin secretion capacity and higher body mass index at the time of diagnosis than those who continued insulin therapy at least for 6 months (n = 10). These findings suggest that some of Japanese patients who presented with acute onset diabetic ketosis and negative for GADAb share several clinical characteristics with atypical type 2 diabetes such as ketosis-prone diabetes and "soft-drink ketosis," but others do not. PMID:20960264

  12. Characterization of bovine aromatic L-amino acid decarboxylase expressed in a mouse cell line: comparison with native enzyme.

    PubMed

    Park, D H; Kim, K T; Choi, M U; Samanta, H; Joh, T H

    1992-12-01

    Bovine aromatic L-amino acid decarboxylase (AADC) was expressed in a mouse cell line, using a bovine papilloma virus-derived expression vector containing the full coding region of bovine AADC. The recombinant bovine AADC was characterized biochemically and immunochemically and compared with the native bovine AADC. The specific activity of crude recombinant bovine AADC was 30-fold higher than that of crude native AADC. With regard to optimal pH, effects of pyridoxal phosphate concentration and Km for 3,4-dihydroxyphenylalanine as a substrate, both native and recombinant enzymes were essentially identical. Rabbit polyclonal antiserum directed against bovine adrenal AADC recognized on Western blot a single protein band (molecular mass = 55,000 Dalton) in both native and recombinant bovine AADC crude extracts. Furthermore, double immunodiffusion analysis showed a single precipitin line of confluence with both enzyme preparations, indicating immunological identity of native and recombinant bovine AADC. Northern blot analysis identified a single mRNA species (2.2 kb) from native and recombinant bovine AADC preparations. The recombinant bovine AADC has two charge isozymes corresponding to those of the native bovine enzyme, although their relative abundances are different between native and recombinant enzymes. Taken together, our results show that recombinant bovine AADC, expressed from bovine AADC cDNA in a mouse cell line is not only enzymatically active, but also shares many biochemical and immunochemical common features with native bovine AADC.

  13. 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

  14. Inhibition of ultraviolet-B skin carcinogenesis by all-trans-retinoic acid regimens that inhibit ornithine decarboxylase induction

    SciTech Connect

    Connor, M.J.; Lowe, N.J.; Breeding, J.H.; Chalet, M.

    1983-01-01

    There is a correlation between the ability to induce the polyamine-biosynthetic enzyme ornithine decarboxylase (ODC) and the tumor-promoting ability of various carcinogens in mouse epidermis. Some agents which inhibit skin carcinogenesis also inhibit ODC induction. In this study, all-trans-retinoic acid (RA) regimens that inhibited the induction of epidermal ODC by ultraviolet-B (UVB) were tested for their ability to inhibit UVB skin carcinogenesis. Hairless mice were irradiated once daily with UVB for 20 days, receiving a total dose of UVB (17.1 kJ/sq m). Topical RA was applied immediately (RA, one dose) or applied 0, 1, 2, 3, and 4 hr (RA, five doses) after each irradiance. The mice were maintained for 52 weeks and then sacrificed. Groups treated with RA tended to have fewer mice with tumors, fewer tumors per mouse, smaller tumor diameters, and slower growing tumors than did appropriate irradiated control groups. RA given five times was more effective than was RA given one time at inhibiting UVB skin carcinogenesis. These results show that RA treatments that inhibit epidermal ODC induction may be effective in reducing the carcinogenicity of UVB.

  15. 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

  16. Japanese cases of acute onset diabetic ketosis without acidosis in the absence of glutamic acid decarboxylase autoantibody.

    PubMed

    Iwasaki, Yorihiro; Hamamoto, Yoshiyuki; Kawasaki, Yukiko; Ikeda, Hiroki; Honjo, Sachiko; Wada, Yoshiharu; Koshiyama, Hiroyuki

    2010-04-01

    We report consecutive Japanese patients presented with acute onset diabetic ketosis who had negative glutamic acid decarboxylase autoantibody (GADAb) to clarify the clinical characteristics of them. A total of consecutive 1,296 in-patients with newly diagnosed diabetes mellitus, who were admitted to our center from April 2003 to October 2008, were analyzed. Among them, 17 patients who presented with acute onset diabetic ketosis without acidosis, and found to be negative for GADAb, were included. They showed male preponderance (n = 15). Ten patients had history of excessive ingestion of sugar-containing soft drink. Patients who successfully withdrew insulin therapy by 6 months (n = 7) showed significantly higher insulin secretion capacity and higher body mass index at the time of diagnosis than those who continued insulin therapy at least for 6 months (n = 10). These findings suggest that some of Japanese patients who presented with acute onset diabetic ketosis and negative for GADAb share several clinical characteristics with atypical type 2 diabetes such as ketosis-prone diabetes and "soft-drink ketosis," but others do not.

  17. 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.

  18. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid.

    PubMed

    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

  19. 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.

  20. Multiplicity of glutamic acid decarboxylases (GAD) in vertebrates: molecular phylogeny and evidence for a new GAD paralog.

    PubMed

    Bosma, P T; Blázquez, M; Collins, M A; Bishop, J D; Drouin, G; Priede, I G; Docherty, K; Trudeau, V L

    1999-03-01

    The evolution of chordate glutamic acid decarboxylase (GAD; EC 4.1.1.15), a key enzyme in the central nervous system synthesizing the neurotransmitter gamma-amino-butyric acid (GABA) from glutamate, was studied. Prior to this study, molecular data of GAD had been restricted to mammals, which express two distinct forms, GAD65 and GAD67. These are the products of separate genes and probably are derived from a common ancestral GAD following gene duplication at some point during vertebrate evolution. To enable a comprehensive phylogenetic analysis, molecular information of GAD forms in other vertebrate classes was essential. By reverse transcriptase-polymerase chain reaction (RT-PCR), partial nucleotide sequences of GAD were cloned from brains of zebra finch (Taeniopygia guttata), turtle (Trachemys scripta), goldfish (Carassius auratus), zebrafish (Danio rerio), and armoured grenadier (Coryphaenoides (Nematonurus) armatus, a deep-sea fish), and from the cerebral ganglion plus neural gland of Ciona intestinalis, a protochordate. Whereas GAD65 and GAD67 homologs were expressed in birds, reptiles, and fish, only a single GAD cDNA with equal similarities to both vertebrate GAD forms was found in the protochordate. This indicates that the duplication of the vertebrate GAD gene occurred between 400 and 560 million years ago. For both GAD65 and GAD67, the generated phylogenetic tree followed the general tree topology for the major vertebrate classes. In turtle, an alternative spliced form of GAD65, putatively encoding a truncated, nonactive GAD, was found. Furthermore, a third GAD form, which is equally divergent from both GAD65 and GAD67, is expressed in C. (N.) armatus. This third form might have originated from an ancient genome duplication specific to modern ray-finned fishes.

  1. Enhancement of the catalytic activity of ferulic acid decarboxylase from Enterobacter sp. Px6-4 through random and site-directed mutagenesis.

    PubMed

    Lee, Hyunji; Park, Jiyoung; Jung, Chaewon; Han, Dongfei; Seo, Jiyoung; Ahn, Joong-Hoon; Chong, Youhoon; Hur, Hor-Gil

    2015-11-01

    The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass.

  2. Enhancement of the catalytic activity of ferulic acid decarboxylase from Enterobacter sp. Px6-4 through random and site-directed mutagenesis.

    PubMed

    Lee, Hyunji; Park, Jiyoung; Jung, Chaewon; Han, Dongfei; Seo, Jiyoung; Ahn, Joong-Hoon; Chong, Youhoon; Hur, Hor-Gil

    2015-11-01

    The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass. PMID:26059194

  3. High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase.

    PubMed

    Schwartz, H L; Chandonia, J M; Kash, S F; Kanaani, J; Tunnell, E; Domingo, A; Cohen, F E; Banga, J P; Madec, A M; Richter, W; Baekkeskov, S

    1999-04-16

    The smaller isoform of the GABA-synthesizing enzyme, glutamic acid decarboxylase 65 (GAD65), is unusually susceptible to becoming a target of autoimmunity affecting its major sites of expression, GABA-ergic neurons and pancreatic beta-cells. In contrast, a highly homologous isoform, GAD67, is not an autoantigen. We used homolog-scanning mutagenesis to identify GAD65-specific amino acid residues which form autoreactive B-cell epitopes in this molecule. Detailed mapping of 13 conformational epitopes, recognized by human monoclonal antibodies derived from patients, together with two and three-dimensional structure prediction led to a model of the GAD65 dimer. GAD65 has structural similarities to ornithine decarboxylase in the pyridoxal-5'-phosphate-binding middle domain (residues 201-460) and to dialkylglycine decarboxylase in the C-terminal domain (residues 461-585). Six distinct conformational and one linear epitopes cluster on the hydrophilic face of three amphipathic alpha-helices in exons 14-16 in the C-terminal domain. Two of those epitopes also require amino acids in exon 4 in the N-terminal domain. Two distinct epitopes reside entirely in the N-terminal domain. In the middle domain, four distinct conformational epitopes cluster on a charged patch formed by amino acids from three alpha-helices away from the active site, and a fifth epitope resides at the back of the pyridoxal 5'-phosphate binding site and involves amino acid residues in exons 6 and 11-12. The epitopes localize to multiple hydrophilic patches, several of which also harbor DR*0401-restricted T-cell epitopes, and cover most of the surface of the protein. The results reveal a remarkable spectrum of human autoreactivity to GAD65, targeting almost the entire surface, and suggest that native folded GAD65 is the immunogen for autoreactive B-cells. PMID:10222205

  4. 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

  5. Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse.

    PubMed

    Liu, W S; Pesold, C; Rodriguez, M A; Carboni, G; Auta, J; Lacor, P; Larson, J; Condie, B G; Guidotti, A; Costa, E

    2001-03-13

    Heterozygous reeler mice (HRM) haploinsufficient for reelin express approximately 50% of the brain reelin content of wild-type mice, but are phenotypically different from both wild-type mice and homozygous reeler mice. They exhibit, (i) a down-regulation of glutamic acid decarboxylase 67 (GAD(67))-positive neurons in some but not every cortical layer of frontoparietal cortex (FPC), (ii) an increase of neuronal packing density and a decrease of cortical thickness because of neuropil hypoplasia, (iii) a decrease of dendritic spine expression density on basal and apical dendritic branches of motor FPC layer III pyramidal neurons, and (iv) a similar decrease in dendritic spines expressed on the basal dendrite branches of CA1 pyramidal neurons of the hippocampus. To establish whether the defect of GAD(67) down-regulation observed in HRM is responsible for neuropil hypoplasia and decreased dendritic spine density, we studied heterozygous GAD(67) knockout mice (HG(67)M). These mice exhibited a down-regulation of GAD(67) mRNA expression in FPC (about 50%), but they expressed normal amounts of reelin and had no neuropil hypoplasia or down-regulation of dendritic spine expression. These findings, coupled with electron-microscopic observations that reelin colocalizes with integrin receptors on dendritic spines, suggest that reelin may be a factor in the dynamic expression of cortical dendritic spines perhaps by promoting integrin receptor clustering. These findings are interesting because the brain neurochemical and neuroanatomical phenotypic traits exhibited by the HRM are in several ways similar to those found in postmortem brains of psychotic patients.

  6. Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse

    PubMed Central

    Liu, Wen Sheng; Pesold, Christine; Rodriguez, Miguel A.; Carboni, Giovanni; Auta, James; Lacor, Pascal; Larson, John; Condie, Brian G.; Guidotti, Alessandro; Costa, Erminio

    2001-01-01

    Heterozygous reeler mice (HRM) haploinsufficient for reelin express ≈50% of the brain reelin content of wild-type mice, but are phenotypically different from both wild-type mice and homozygous reeler mice. They exhibit, (i) a down-regulation of glutamic acid decarboxylase 67 (GAD67)-positive neurons in some but not every cortical layer of frontoparietal cortex (FPC), (ii) an increase of neuronal packing density and a decrease of cortical thickness because of neuropil hypoplasia, (iii) a decrease of dendritic spine expression density on basal and apical dendritic branches of motor FPC layer III pyramidal neurons, and (iv) a similar decrease in dendritic spines expressed on the basal dendrite branches of CA1 pyramidal neurons of the hippocampus. To establish whether the defect of GAD67 down-regulation observed in HRM is responsible for neuropil hypoplasia and decreased dendritic spine density, we studied heterozygous GAD67 knockout mice (HG67M). These mice exhibited a down-regulation of GAD67 mRNA expression in FPC (about 50%), but they expressed normal amounts of reelin and had no neuropil hypoplasia or down-regulation of dendritic spine expression. These findings, coupled with electron-microscopic observations that reelin colocalizes with integrin receptors on dendritic spines, suggest that reelin may be a factor in the dynamic expression of cortical dendritic spines perhaps by promoting integrin receptor clustering. These findings are interesting because the brain neurochemical and neuroanatomical phenotypic traits exhibited by the HRM are in several ways similar to those found in postmortem brains of psychotic patients. PMID:11248103

  7. 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. PMID:27082660

  8. HosA, a MarR Family Transcriptional Regulator, Represses Nonoxidative Hydroxyarylic Acid Decarboxylase Operon and Is Modulated by 4-Hydroxybenzoic Acid.

    PubMed

    Roy, Ajit; Ranjan, Akash

    2016-02-23

    Members of the Multiple antibiotic resistance Regulator (MarR) family of DNA binding proteins regulate transcription of a wide array of genes required for virulence and pathogenicity of bacteria. The present study reports the molecular characterization of HosA (Homologue of SlyA), a MarR protein, with respect to its target gene, DNA recognition motif, and nature of its ligand. Through a comparative genomics approach, we demonstrate that hosA is in synteny with nonoxidative hydroxyarylic acid decarboxylase (HAD) operon and is present exclusively within the mutS-rpoS polymorphic region in nine different genera of Enterobacteriaceae family. Using molecular biology and biochemical approach, we demonstrate that HosA binds to a palindromic sequence downstream to the transcription start site of divergently transcribed nonoxidative HAD operon and represses its expression. Furthermore, in silico analysis showed that the recognition motif for HosA is highly conserved in the upstream region of divergently transcribed operon in different genera of Enterobacteriaceae family. A systematic chemical search for the physiological ligand revealed that 4-hydroxybenzoic acid (4-HBA) interacts with HosA and derepresses HosA mediated repression of the nonoxidative HAD operon. Based on our study, we propose a model for molecular mechanism underlying the regulation of nonoxidative HAD operon by HosA in Enterobacteriaceae family. PMID:26818787

  9. Role of glutamic acid decarboxylase 67 in regulating cortical parvalbumin and GABA membrane transporter 1 expression: Implications for schizophrenia

    PubMed Central

    Curley, Allison A.; Eggan, Stephen M.; Lazarus, Matt S.; Huang, Z. Josh; Volk, David W.; Lewis, David A.

    2012-01-01

    Markers of GABA neurotransmission are altered in multiple regions of the neocortex in individuals with schizophrenia. Lower levels of glutamic acid decarboxylase 67 (GAD67) mRNA and protein, which is responsible for most cortical GABA synthesis, are accompanied by lower levels of GABA membrane transporter 1 (GAT1) mRNA. These alterations are thought to be most prominent in the parvalbumin (PV)-containing subclass of interneurons, which also contain lower levels of PV mRNA. Since GAT1 and PV each reduce the availability of GABA at postsynaptic receptors, lower levels of GAT1 and PV mRNAs have been hypothesized to represent compensatory responses to an upstream reduction in cortical GABA synthesis in schizophrenia. However, such cause-and-effect hypotheses cannot be directly tested in a human illness. Consequently, we used two mouse models with reduced GAD67 expression specifically in PV neurons (PVGAD67+/−) or in all interneurons (GABAGAD67+/−) and quantified GAD67, GAT1 and PV mRNA levels using methods identical to those employed in studies of schizophrenia. Cortical levels of PV or GAT1 mRNAs were not altered in PVGAD67+/− mice during postnatal development or in adulthood. Furthermore, cellular analyses confirmed the predicted reduction in GAD67 mRNA, but failed to show a deficit in PV mRNA in these animals. Levels of PV and GAT1 mRNAs were also unaltered in GABAGAD67+/− mice. Thus, mouse lines with cortical reductions in GAD67 mRNA that match or exceed those present in schizophrenia, and that differ in the developmental timing and cell typespecificity of the GAD67 deficit, failed to provide proof-of-concept evidence that lower PV and GAT1 expression in schizophrenia are a consequence of lower GAD67 expression. Together, these findings suggest that the correlated decrements in cortical GAD67, PV and GAT1 mRNAs in schizophrenia may be a common consequence of some other upstream factor. PMID:23103418

  10. Glutamic acid decarboxylase (anti-GAD) & tissue transglutaminase (anti-TTG) antibodies in patients with thyroid autoimmunity

    PubMed Central

    Marwaha, R.K.; Garg, M.K.; Tandon, N.; Kanwar, Ratnesh; Narang, A.; Sastry, A.; Saberwal, A.; Bhadra, Kuntal

    2013-01-01

    Background & objectives: Several autoimmune disorders have been reported to be associated with autoimmune thyroiditis and may coexist with other organ-specific autoantibodies. The aim of the present study was to evaluate the presence of tissue transglutaminase (anti-TTG) and glutamic acid decarboxylase (anti-GAD) antibodies in patients suffering from autoimmune thyroiditis as diagnosed by anti-thyroid peroxidase (anti-TPO) antibodies, which may indicate high risk for developing celiac disease or type 1 diabetes mellitus. Methods: Five thousand children and 2800 adults were screening as part of a general health examination done on a voluntary basis in four different parts of Delhi. A total of 577 subjects positive for anti-TPO antibody constituted the cases. Equal number of age and sex matched anti-TPO antibody negative controls were randomly selected from the same cohort to form paired case control study. The cases and controls were further divided into two groups as follows: group-1 (children and adolescent <18 yr), group-2 (adults >18 yr). Serum samples of cases and controls were analysed for thyroid function test (FT3, FT4, and TSH), anti-TTG and anti-GAD antibodies. Results: A total of 1154 subjects (577 cases and 577 controls) were included in this study. Hypothyroidism was present in 40.2 per cent (232) cases compared to only 4.7 per cent (27) in controls (P<0.001). Anti-TTG and anti-GAD antibodies were present in 6.9 and 12.5 per cent subjects among cases compared to 3.5 per cent (P=0.015) and 4.3 per cent (P=0.001) in controls, respectively. Only anti-GAD antibody were significantly positive in cases among children and adolescents (P =0.0044) and adult (P=0.001) compared to controls. Levels of anti-TTG and anti-GAD antibodies increased with increasing titre of anti-TPO antibody. Interpretation & conclusions: Our findings showed high positivity of anti-GAD and anti-TTG antibodies among subjects with thyroid autoimmunity. It is, therefore, important to have

  11. Progesterone receptor isoforms differentially regulate the expression of tryptophan and tyrosine hydroxylase and glutamic acid decarboxylase in the rat hypothalamus.

    PubMed

    González-Flores, Oscar; Gómora-Arrati, Porfirio; García-Juárez, Marcos; Miranda-Martínez, Alfredo; Armengual-Villegas, Alejandra; Camacho-Arroyo, Ignacio; Guerra-Araiza, Christian

    2011-10-01

    Progesterone exerts a variety of actions in the brain through the interaction with its receptors (PR) which have two isoforms with different function and regulation: PR-A and PR-B. Progesterone may modulate neurotransmission by regulating the expression of neurotransmitters synthesizing enzymes or their receptors in several brain regions. The role of PR isoforms in this modulation is unknown. We explored the role of PR isoforms in the regulation of tryptophan (TPH) and tyrosine (TH) hydroxylase, and glutamic acid decarboxylase (GAD) expression in the hypothalamus of ovariectomized rats. Two weeks after ovariectomy, animals were subcutaneously injected with 5 μg of estradiol benzoate (EB), and 40 h later, progesterone (P) was intracerebroventricularly (ICV) injected. Each animal received two ICV injections of 1 μg/μl (4 nmol) of PR-B and total PR (PR-A+PR-B) sense or antisense (As) oligonucleotides (ODNs). First injection was made immediately before sc EB injection, and 24h later animals received the second one. Twenty-four hours after P administration, rats were euthanized and brains removed to measure the expression of PR-A and PR-B, TPH, TH and GAD by Western blot. We observed that sense ODNs modified neither PR isoforms nor enzymes expression in the hypothalamus, whereas PR A+B antisense (PR A+B As) clearly decreased the expression of both PR isoforms in this region. ICV administration of PR-B As only decreased PR-B isoform expression with no significant effects on PR-A expression. A differential protein expression of TPH, TH and GAD was observed after PR isoforms antisense administration. PR-B As administration decreased the expression of TPH (65% with respect to control). In contrast, PR A+B As and PR-B As administration increased (51.6% and 34.4%, respectively) TH expression. The administration of PR A+B As and PR-B As diminished GAD expression (33.4% and 41.6%, respectively). Our findings indicate that PR isoforms play a differential role in the

  12. Elimination of islet cell antibodies and glutamic acid decarboxylase antibodies II in a patient with newly diagnosed insulin-dependent diabetes mellitus.

    PubMed

    Richter, W O; Donner, M G; Schwandt, P

    1997-01-01

    Islet cell antibodies and glutamic acid decarboxylase II (GAD II) antibodies have been discussed in the autoimmune pathogenesis of insulin-dependent diabetes mellitus (IDDM). Hence, immunosuppressants, intravenous immunoglobulins, and plasmapheresis have been used in an effort to modulate autoimmune activity and thereby prevent the destruction of pancreatic beta-cells. We describe the autoantibody (islet cell antibody and GAD II) kinetics and clinical course in a patient with newly diagnosed IDDM treated with a specific immunoglobulin apheresis technique. Five days after the initial diagnosis a 37-year-old patient with IDDM underwent a series of seven immunoglobulin aphereses. Immunoglobulin (IgG, IgA, IgM), islet cell antibody, GAD II, and C-peptide concentrations were monitored for a time course of 74 days. Daily insulin requirements were recorded. One single immunoglobulin apheresis decreased IgG by 66.2 +/- 9.1%, IgA by 66.8 +/- 8.7%, and IgM by 57.7 +/- 12.9%. GAD II antibodies were reduced by 61.9 +/- 12.4%. The islet cell antibody titer declined from 1:32 to 1:4 after the treatment series. There were no relevant changes in the safety parameters determined nor were there any clinical side effects. The efficient decrease in islet cell antibodies and glutamic acid decarboxylase II antibodies in a patient with IDDM encourages further investigations into the impact of this treatment on the clinical course of this autoimmune disorder.

  13. Dual mechanisms regulating glutamate decarboxylases and accumulation of gamma-aminobutyric acid in tea (Camellia sinensis) leaves exposed to multiple stresses.

    PubMed

    Mei, Xin; Chen, Yiyong; Zhang, Lingyun; Fu, Xiumin; Wei, Qing; Grierson, Don; Zhou, Ying; Huang, Yahui; Dong, Fang; Yang, Ziyin

    2016-01-01

    γ-Aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in the central nervous system. It has multiple positive effects on mammalian physiology and is an important bioactive component of tea (Camellia sinensis). GABA generally occurs at a very low level in plants but GABA content increases substantially after exposure to a range of stresses, especially oxygen-deficiency. During processing of tea leaves, a combination of anoxic stress and mechanical damage are essential for the high accumulation of GABA. This is believed to be initiated by a change in glutamate decarboxylase activity, but the underlying mechanisms are unclear. In the present study we characterized factors regulating the expression and activity of three tea glutamate decarboxylase genes (CsGAD1, 2, and 3), and their encoded enzymes. The results suggests that, unlike the model plant Arabidopsis thaliana, there are dual mechanisms regulating the accumulation of GABA in tea leaves exposed to multiple stresses, including activation of CsGAD1 enzymatic activity by calmodulin upon the onset of the stress and accumulation of high levels of CsGAD2 mRNA induced by a combination of anoxic stress and mechanical damage. PMID:27021285

  14. Dual mechanisms regulating glutamate decarboxylases and accumulation of gamma-aminobutyric acid in tea (Camellia sinensis) leaves exposed to multiple stresses

    PubMed Central

    Mei, Xin; Chen, Yiyong; Zhang, Lingyun; Fu, Xiumin; Wei, Qing; Grierson, Don; Zhou, Ying; Huang, Yahui; Dong, Fang; Yang, Ziyin

    2016-01-01

    γ-Aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in the central nervous system. It has multiple positive effects on mammalian physiology and is an important bioactive component of tea (Camellia sinensis). GABA generally occurs at a very low level in plants but GABA content increases substantially after exposure to a range of stresses, especially oxygen-deficiency. During processing of tea leaves, a combination of anoxic stress and mechanical damage are essential for the high accumulation of GABA. This is believed to be initiated by a change in glutamate decarboxylase activity, but the underlying mechanisms are unclear. In the present study we characterized factors regulating the expression and activity of three tea glutamate decarboxylase genes (CsGAD1, 2, and 3), and their encoded enzymes. The results suggests that, unlike the model plant Arabidopsis thaliana, there are dual mechanisms regulating the accumulation of GABA in tea leaves exposed to multiple stresses, including activation of CsGAD1 enzymatic activity by calmodulin upon the onset of the stress and accumulation of high levels of CsGAD2 mRNA induced by a combination of anoxic stress and mechanical damage. PMID:27021285

  15. 4-Vinylphenol biosynthesis from cellulose as the sole carbon source using phenolic acid decarboxylase- and tyrosine ammonia lyase-expressing Streptomyces lividans.

    PubMed

    Noda, Shuhei; Kawai, Yoshifumi; Tanaka, Tsutomu; Kondo, Akihiko

    2015-03-01

    Streptomyces lividans was adopted as a host strain for 4-vinylphenol (4VPh) production directly from cellulose. In order to obtain novel phenolic acid decarboxylase (PAD) expressed in S. lividans, PADs distributed among Streptomyces species were screened. Three novel PADs, derived from Streptomycessviceus, Streptomyceshygroscopicus, and Streptomycescattleya, were successfully obtained and expressed in S. lividans. S. sviceus PAD (SsPAD) could convert p-hydroxycinnamic acid (pHCA) to 4VPh more efficiently than the others both in vitro and in vivo. For 4VPh production directly from cellulose, l-tyrosine ammonia lyase derived from Rhodobacter sphaeroides and SsPAD were introduced into endoglucanase-secreting S. lividans, and the 4VPh biosynthetic pathway was constructed therein. The created transformants successfully produced 4VPh directly from cellulose.

  16. Immunocytochemical localization of glutamic acid decarboxylase (GAD) and glutamine synthetase (GS) in the area postrema of the cat. Light and electron microscopy

    NASA Technical Reports Server (NTRS)

    D'Amelio, Fernando E.; Mehler, William R.; Gibbs, Michael A.; Eng, Lawrence F.; Wu, Jang-Yen

    1987-01-01

    Morphological evidence is presented of the existence of the putative neurotransmitter gamma-aminobutyric acid (GABA) in axon terminals and of glutamine synthetase (GS) in ependymoglial cells and astroglial components of the area postrema (AP) of the cat. Purified antiserum directed against the GABA biosynthetic enzyme glutamic acid decarboxylase (GAD) and GS antiserum were used. The results showed that punctate structures of variable size corresponding to axon terminals exhibited GAD-immunoreactivity and were distributed in varying densities. The greatest accumulation occurred in the caudal and middle segment of the AP and particularly in the area subpostrema, where the aggregation of terminals was extremely dense. The presence of both GAD-immunoreactive profiles and GS-immunostained ependymoglial cells and astrocytes in the AP provide further evidence of the functional correlation between the two enzymes.

  17. Molecular cloning and analysis of cDNA encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases.

    PubMed Central

    De Luca, V; Marineau, C; Brisson, N

    1989-01-01

    The sequence of a cDNA clone that includes the complete coding region of tryptophan decarboxylase (EC 4.1.1.28, formerly EC 4.1.1.27) from periwinkle (Catharanthus roseus) is reported. The cDNA clone (1747 base pairs) was isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA found in developing seedlings of C. roseus. The clone hybridized to a 1.8-kilobase mRNA from developing seedlings and from young leaves of mature plants. The identity of the clone was confirmed when extracts of transformed Escherichia coli expressed a protein containing tryptophan decarboxylase enzyme activity. The tryptophan decarboxylase cDNA clone encodes a protein of 500 amino acids with a calculated molecular mass of 56,142 Da. The amino acid sequence shows a high degree of similarity with the aromatic L-amino acid decarboxylase (dopa decarboxylase) and the alpha-methyldopa-hypersensitive protein of Drosophila melanogaster. The tryptophan decarboxylase sequence also showed significant similarity to feline glutamate decarboxylase and mouse ornithine decarboxylase, suggesting a possible evolutionary link between these amino acid decarboxylases. Images PMID:2704736

  18. Efficient production of gamma-aminobutyric acid using Escherichia coli by co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter.

    PubMed

    Dung Pham, Van; Somasundaram, Sivachandiran; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

    2016-01-01

    Gamma-aminobutyric acid (GABA) is an important bio-product, which is used in pharmaceutical formulations, nutritional supplements, and biopolymer monomer. The traditional GABA process involves the decarboxylation of glutamate. However, the direct production of GABA from glucose is a more efficient process. To construct the recombinant strains of Escherichia coli, a novel synthetic scaffold was introduced. By carrying out the co-localization of glutamate synthase, glutamate decarboxylase, and GABA transporter, we redirected the TCA cycle flux to GABA pathway. The genetically engineered E. coli strain produced 1.08 g/L of GABA from 10 g/L of initial glucose. Thus, with the introduction of a synthetic scaffold, we increased GABA production by 2.2-fold. The final GABA concentration was increased by 21.8% by inactivating competing pathways.

  19. Production of gamma-aminobutyric acid from glucose by introduction of synthetic scaffolds between isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase in recombinant Escherichia coli.

    PubMed

    Pham, Van Dung; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

    2015-08-10

    Escherichia coli were engineered for the direct production of gamma-aminobutyric acid from glucose by introduction of synthetic protein scaffold. In this study, three enzymes consisting GABA pathway (isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase) were connected via synthetic protein scaffold. By introduction of scaffold, 0.92g/L of GABA was produced from 10g/L of glucose while no GABA was produced in wild type E. coli. The optimum pH and temperature for GABA production were 4.5 and 30°C, respectively. When competing metabolic network was inactivated by knockout mutation, maximum GABA concentration of 1.3g/L was obtained from 10g/L glucose. The recombinant E. coli strain which produces GABA directly from glucose was successfully constructed by introduction of protein scaffold.

  20. Exogenous γ-aminobutyric acid (GABA) affects pollen tube growth via modulating putative Ca2+-permeable membrane channels and is coupled to negative regulation on glutamate decarboxylase.

    PubMed

    Yu, Guang-Hui; Zou, Jie; Feng, Jing; Peng, Xiong-Bo; Wu, Ju-You; Wu, Ying-Liang; Palanivelu, Ravishankar; Sun, Meng-Xiang

    2014-07-01

    γ-Aminobutyric acid (GABA) is implicated in pollen tube growth, but the molecular and cellular mechanisms that it mediates are largely unknown. Here, it is shown that exogenous GABA modulates putative Ca(2+)-permeable channels on the plasma membranes of tobacco pollen grains and pollen tubes. Whole-cell voltage-clamp experiments and non-invasive micromeasurement technology (NMT) revealed that the influx of Ca(2+) increases in pollen tubes in response to exogenous GABA. It is also demonstrated that glutamate decarboxylase (GAD), the rate-limiting enzyme of GABA biosynthesis, is involved in feedback controls of Ca(2+)-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca(2+)-permeable channels relays an extracellular GABA signal and integrates multiple signal pathways to modulate tobacco pollen tube growth. Thus, the data explain how GABA mediates the communication between the style and the growing pollen tubes.

  1. Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase.

    PubMed

    Burbaeva, G Sh; Boksha, I S; Tereshkina, E B; Savushkina, O K; Prokhorova, T A; Vorobyeva, E A

    2014-10-01

    Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

  2. Increased levels of tyrosine hydroxylase and glutamic acid decarboxylase in locus coeruleus neurons after rapid eye movement sleep deprivation in rats.

    PubMed

    Majumdar, S; Mallick, B N

    2003-03-01

    Norepinephrine, acetylcholine and GABA levels alter during rapid eye movement (REM) sleep and its deprivation. Increased synthesis of those neurotransmitters is necessary for their sustained release. Hence, in this study, the concentrations of tyrosine hydroxylase (TH), choline acetyl transferase (ChAT) and glutamic acid decarboxylase (GAD), the enzymes responsible for their synthesis, were immunohistochemically estimated within the neurons in locus coeruleus, laterodorsal tegmentum and pedunculopontine tegmentum and medial preoptic area in REM sleep deprived and control rats. It was observed that as compared to controls, deprivation increased TH and GAD significantly in the locus coeruleus only, while in other areas, they remained unchanged. The findings help explaining the mechanism of increase in neurotransmitter levels in the brain after REM sleep deprivation and their significance has been discussed.

  3. NF-Y binding is required for transactivation of neuronal aromatic L-amino acid decarboxylase gene promoter by the POU-domain protein Brn-2.

    PubMed

    Dugast, C; Weber, M J

    2001-04-18

    We have previously characterized binding sites for the NF-Y transcription factor (-71/-52) and Brn-2 POU-domain protein (-92/-71) in the neuronal promoter of the human aromatic L-amino acid decarboxylase gene [Mol. Brain Res. 56 (1998) 227]. We have now explored the functional role of these binding sites in transfected SK-N-BE neuroblastoma cells. Mutations of the NF-Y site that abolish binding depressed expression of a luciferase reporter gene up to 25-fold. The overexpression of a dominant negative mutant of NF-YA subunit depressed expression by 60%. Promoter activity was increased by the overexpression of Brn-2. Mutations or deletion of the binding site of Brn-2 did not suppress transcriptional activation by overexpressed Brn-2, while promoters defective in NF-Y binding were not transactivated by Brn-2. A GST-pulldown experiment showed that recombinant human Brn-2 protein weakly interacts with recombinant NF-Y outside of DNA. Cooperative binding of recombinant NF-Y and GST--Brn-2 proteins on the neuronal promoter was evidenced by an electrophoretic mobility shift assay. The POU-domain of Brn-2 was sufficient for such interaction. The results thus suggest that the activation of the neuronal promoter of the aromatic L-amino acid decarboxylase gene requires a direct interaction between the ubiquitous NF-Y factor and a cell-specific POU-domain protein. The NF-Y, but not the Brn-2 binding site, is essential for the recruitment of the NF-Y/Brn-2 complex on the promoter. PMID:11311976

  4. Loss of Autonoetic Awareness of Recent Autobiographical Episodes and Accelerated Long-Term Forgetting in a Patient with Previously Unrecognized Glutamic Acid Decarboxylase Antibody Related Limbic Encephalitis

    PubMed Central

    Witt, Juri-Alexander; Vogt, Viola Lara; Widman, Guido; Langen, Karl-Josef; Elger, Christian Erich; Helmstaedter, Christoph

    2015-01-01

    We describe a 35-year-old male patient presenting with depressed mood and emotional instability, who complained about severe anterograde and retrograde memory deficits characterized by accelerated long-term forgetting and loss of autonoetic awareness regarding autobiographical memories of the last 3 years. Months before he had experienced two breakdowns of unknown etiology giving rise to the differential diagnosis of epileptic seizures after various practitioners and clinics had suggested different etiologies such as a psychosomatic condition, burnout, depression, or dissociative amnesia. Neuropsychological assessment indicated selectively impaired figural memory performance. Extended diagnostics confirmed accelerated forgetting of previously learned and retrievable verbal material. Structural imaging showed bilateral swelling and signal alterations of temporomesial structures (left >right). Video-EEG monitoring revealed a left temporal epileptic focus and subclincal seizure, but no overt seizures. Antibody tests in serum and liquor were positive for glutamic acid decarboxylase antibodies. These findings led to the diagnosis of glutamic acid decarboxylase antibody related limbic encephalitis. Monthly steroid pulses over 6 months led to recovery of subjective memory and to intermediate improvement but subsequent worsening of objective memory performance. During the course of treatment, the patient reported de novo paroxysmal non-responsive states. Thus, antiepileptic treatment was started and the patient finally became seizure free. At the last visit, vocational reintegration was successfully in progress. In conclusion, amygdala swelling, retrograde biographic memory impairment, accelerated long-term forgetting, and emotional instability may serve as indicators of limbic encephalitis, even in the absence of overt epileptic seizures. The monitoring of such patients calls for a standardized and concerted multilevel diagnostic approach with repeated assessments

  5. 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

  6. Efficient gamma-aminobutyric acid bioconversion by employing synthetic complex between glutamate decarboxylase and glutamate/GABA antiporter in engineered Escherichia coli.

    PubMed

    Le Vo, Tam Dinh; Ko, Ji-seun; Park, Si Jae; Lee, Seung Hwan; Hong, Soon Ho

    2013-08-01

    Gamma-aminobutyric acid (GABA) is a precursor of one of the most promising heat-resistant biopolymers, Nylon-4, and can be produced by the decarboxylation of monosodium glutamate (MSG). In this study, a synthetic protein complex was applied to improve the GABA conversion in engineered Escherichia coli. Complexes were constructed by assembling a single protein-protein interaction domain SH3 to the glutamate decarboxylase (GadA and GadB) and attaching a cognate peptide ligand to the glutamate/GABA antiporter (GadC) at the N-terminus, C-terminus, and the 233rd amino acid residue. When GadA and GadC were co-overexpressed via the C-terminus complex, a GABA concentration of 5.65 g/l was obtained from 10 g/l MSG, which corresponds to a GABA yield of 93 %. A significant increase of the GABA productivity was also observed where the GABA productivity increased 2.5-fold in the early culture period due to the introduction of the synthetic protein complex. The GABA pathway efficiency and GABA productivity were enhanced by the introduction of the complex between Gad and glutamate/GABA antiporter.

  7. Glutamic acid decarboxylase activity is stimulated in quail retina neuronal cells transformed by Rous sarcoma virus and is regulated by pp60v-src.

    PubMed Central

    Crisanti, P; Lorinet, A M; Calothy, G; Pessac, B

    1985-01-01

    Rous sarcoma virus (RSV) stimulates in quail embryo neuro-retina (NR) cultures the specific activity of glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of gamma-aminobutyric acid, a major inhibitory neurotransmitter in NR and in central nervous system. In quail embryo NR cultures transformed by ts NY-68, a thermodependent transformation-defective mutant of RSV, stimulation of GAD activity is regulated by pp60v-src, the product of the src gene of RSV. Fibroblasts and myoblasts have a very low GAD activity that is not stimulated after transformation by RSV. Neuronal clones, previously derived from ts NY-68-transformed established NR cell lines, have a high GAD activity which is regulated by pp60v-src, while other clones have a low GAD activity apparently not regulated by pp60v-src. These data indicate that pp60v-src selectively activates the expression of GAD in distinct neuronal cells of quail embryo NR cultures transformed by RSV. GAD activity is also stimulated in NR cells infected with viruses containing v-mil. PMID:2992933

  8. [Critical amino acids of ornitin decarboxylase degron: the presence and C-terminal arrangement is insufficient for alfa-fetoprotein degradation].

    PubMed

    Morozov, A V; Timofeev, A V; Morozov, V A; Karpov, V L

    2011-01-01

    Mouse ornithine decarboxylase (ODC) degrades in proteasome in an ubiquitin-independent manner with an averagehalf-life of 2 h. The 37 amino acid long C-terminal fragment known as a degradation signal (degron) is responsible for the effective degradation of ODC. Recently, amino acids being critical for degradation in the ODC-degron have been mapped. Mutations of Cys441 and Ala442 led to protein stabilization, while a substitution of other amino acids composing ODC-degron had almost no effect on the protein turnover; whereas insertions or deletions in region between Ala442 and ODC C-terminus diminished greatly rate of protein degradation, e.g. positioning of the key amino acids from the C-terminus was shown to be crucial. Using these data we introduced both key amino acids into the alfa-fetoprotein with truncated exportation signal (deltaAFP), at the same distance from the C-terminus as they being in the ODC (deltaAFPCAG and deltaAFPLCAG). Removal of N-terminal exportation signal prevented secretion of modified proteins. Using in silico approach we demonstrated no significant difference in hydrophobicity or secondary structure between C-terminus of deltaAFP and mutated proteins. The degradation kinetics of deltaAFP, deltaAFPCAG, deltaAFPLCAG in cyloheximide-chase and proteasome inhibition assay (using MG132) was identical. Obtained results suggest that introduced substitutions are insufficient for effective recognition of mutated deltaAFP by26S proteasome. We assume thatadditional amino aci ds composing ODC-degron or their combine action could also affect degradation. Besides that, one cannot exclude that conformation of the mutated deltaAFP limits its C-terminus accessibility to proteasome. PMID:21790016

  9. Molecular cloning of genomic DNA and chromosomal assignment of the gene for human aromatic L-amino acid decarboxylase, the enzyme for catecholamine and serotonin biosynthesis

    SciTech Connect

    Sumi-Ichinose, Chiho ); Ichinose, Hiroshi; Nagatsu, Toshiharu ); Takahashi, Eiichi; Hori, Tadaaki )

    1992-03-03

    Aromatic L-amino acid decarboxylase (AADC) catalyzes the decarboxylation of both L-3,4-dihydroxyphenylalanine and L-5-hydroxytryptophan to dopamine and serotonin, respectively, which are major mammalian neurotransmitters and hormones belonging to catecholamines and indoleamines. This report describes the organization of the human AADC gene. The authors proved that the gene of human AADC consists of 15 exons spanning more than 85 kilobases and exists as a single copy in the haploid genome. The boundaries between exon and intron followed the AG/GT rule. The sizes of exons and introns ranged from 20 to 400 bp and from 1.0 to 17.7 kb, respectively, while the sizes of four introns were not determined. Untranslated regions located in the 5{prime} region of mRNA were encoded by two exons, exons 1 and 2. The transcriptional starting point was determined around G at position {minus}111 by primer extension and S1 mapping. There were no typical TATA box' and CAAT box' within 540 bp from the transcriptional starting point. The human AADC gene was mapped to chromosome band 7p12.1-p12.3 by fluorescence in situ hybridization. This is the first report on the genomic structure and chromosomal localization of the AADC gene in mammals.

  10. Exponential increase of glutamic acid decarboxylase (GAD) antibody titer after initiating and stopping insulin in a patient with slowly progressive type 1 diabetes.

    PubMed

    Nishimura, Akihiro; Nagasawa, Kaoru; Okubo, Minoru; Kobayashi, Tetsuro; Mori, Yasumichi

    2015-01-01

    Few articles have described fluctuations in glutamic acid decarboxylase antibody (GADAb) levels after a diagnosis of slowly progressive type 1 diabetes (SPIDDM). Here, we present a case in which GADAb levels exponentially increased after initiating and stopping insulin. A 64-year-old female patient newly diagnosed with SPIDDM was admitted and started multiple daily insulin injections. The patient's GADAb titer was 6.9 U/mL (normal: <1.4 U/mL) and the patient had a type 1 diabetes susceptible HLA class II haplotype known in the Japanese population as: DRB1*04:05-DQB1*04:01. When the patient's "honeymoon period" set in, hypoglycemia was observed and the dose of insulin was reduced. Two months after the diagnosis, 1 unit of insulin glargine/day was being injected and the patient demonstrated good glycemic control. Subsequently, the patient's home doctor recommended that insulin injections be stopped. Three months after the diagnosis, the patient's GADAb titer suddenly increased to 1600 U/mL. The patient's GADAb titer decreased but was still positive (40 U/mL) 36 months after diagnosis. HbA1c levels were maintained below 7%, and oral glucose tolerance tests at 10, 26, and 36 months after diagnosis suggested that the patient had preserved insulin secretion. To the best of our knowledge, this is the first report that describes exponential increases in GADAb after initiating and stopping insulin in a patient with SPIDDM.

  11. Characterization of CD4+ T cells specific for glutamic acid decarboxylase (GAD65) and proinsulin in a patient with stiff-person syndrome but without type 1 diabetes

    PubMed Central

    Hänninen, Arno; Soilu-Hänninen, Merja; Hampe, Christiane S.; Deptula, Angie; Geubtner, Kelly; Ilonen, Jorma; Knip, Mikael; Reijonen, Helena

    2010-01-01

    Glutamic acid decarboxylase GAD is a rate-limiting enzyme in the synthesis of GABA and an important autoantigen in both type 1 diabetes (T1D) and in stiff-person syndrome (SPS). Autoantibodies (GADA) to the 65kD isoform of GAD are a characteristic feature in both diseases. Approximately 30% of SPS patients develop diabetes, yet, it is unclear to which extent co-existing autoimmunity to GAD65 and other islet autoantigens determines the risk of developing T1D. In this study we monitored CD4+ T-cell responses to GAD65 and proinsulin in a patient with SPS who remained normoglycemic during the 46-month follow-up. Fluctuating but persistent T-cell reactivity to GAD65 was identified, as well as T-cell reactivity to proinsulin at one time point. The majority of the T-cell clones isolated from the SPS patient produced high levels of Th2 cytokines (IL-13, IL-5 and IL-4). We also examined levels of GADA, insulin and IA-2 autoantibodies, and epitope specificity of GADA. In both serum and cerebrospinal fluid GADA levels were high, and GADA persisted throughout the follow-up. Despite T-cell reactivity to both GAD65 and proinsulin, autoantibodies to other islet autoantigens did not develop. Further follow-up will determine whether or not the beta-cell autoimmunity observed in this patient will eventually lead to T1D. PMID:20503259

  12. Immunocytochemical localization of glutamic acid decarboxylase (GAD) and substance P in neural areas mediating motion-induced emesis: Effects of vagal stimulation on GAD immunoreactivity

    NASA Technical Reports Server (NTRS)

    Damelio, F.; Gibbs, M. A.; Mehler, W. R.; Daunton, Nancy G.; Fox, Robert A.

    1991-01-01

    Immunocytochemical methods were employed to localize the neurotransmitter amino acid gamma-aminobutyric acid (GABA) by means of its biosynthetic enzyme glutamic acid decarboxylase (GAD) and the neuropeptide substance P in the area postrema (AP), area subpostrema (ASP), nucleus of the tractus solitarius (NTS), and gelatinous nucleus (GEL). In addition, electrical stimulation was applied to the night vagus nerve at the cervical level to assess the effects on GAD-immunoreactivity (GAR-IR). GAD-IR terminals and fibers were observed in the AP, ASP, NTS, and GEL. They showed pronounced density at the level of the ASP and gradual decrease towards the solitary complex. Nerve cells were not labelled in our preparations. Ultrastructural studies showed symmetric or asymmetric synaptic contracts between labelled terminals and non-immunoreactive dendrites, axons, or neurons. Some of the labelled terminals contained both clear- and dense-core vesicles. Our preliminary findings, after electrical stimulation of the vagus nerve, revealed a bilateral decrease of GAD-IR that was particularly evident at the level of the ASP. SP-immunoreactive (SP-IR) terminals and fibers showed varying densities in the AP, ASP, NTS, and GEL. In our preparations, the lateral sub-division of the NTS showed the greatest accumulation. The ASP showed medium density of immunoreactive varicosities and terminals and the AP and GEL displayed scattered varicose axon terminals. The electron microscopy revealed that all immunoreactive terminals contained clear-core vesicles which make symmetric or asymmetric synaptic contact with unlabelled dendrites. It is suggested that the GABAergic terminals might correspond to vagal afferent projections and that GAD/GABA and substance P might be co-localized in the same terminal allowing the possibility of a regulated release of the transmitters in relation to demands.

  13. High-yield production of vanillin from ferulic acid by a coenzyme-independent decarboxylase/oxygenase two-stage process.

    PubMed

    Furuya, Toshiki; Miura, Misa; Kuroiwa, Mari; Kino, Kuniki

    2015-05-25

    Vanillin is one of the world's most important flavor and fragrance compounds in foods and cosmetics. Recently, we demonstrated that vanillin could be produced from ferulic acid via 4-vinylguaiacol in a coenzyme-independent manner using the decarboxylase Fdc and the oxygenase Cso2. In this study, we investigated a new two-pot bioprocess for vanillin production using the whole-cell catalyst of Escherichia coli expressing Fdc in the first stage and that of E. coli expressing Cso2 in the second stage. We first optimized the second-step Cso2 reaction from 4-vinylguaiacol to vanillin, a rate-determining step for the production of vanillin. Addition of FeCl2 to the cultivation medium enhanced the activity of the resulting E. coli cells expressing Cso2, an iron protein belonging to the carotenoid cleavage oxygenase family. Furthermore, a butyl acetate-water biphasic system was effective in improving the production of vanillin. Under the optimized conditions, we attempted to produce vanillin from ferulic acid by a two-pot bioprocess on a flask scale. In the first stage, E. coli cells expressing Fdc rapidly decarboxylated ferulic acid and completely converted 75 mM of this substrate to 4-vinylguaiacol within 2 h at pH 9.0. After the first-stage reaction, cells were removed from the reaction mixture by centrifugation, and the pH of the resulting supernatant was adjusted to 10.5, the optimal pH for Cso2. This solution was subjected to the second-stage reaction. In the second stage, E. coli cells expressing Cso2 efficiently oxidized 4-vinylguaiacol to vanillin. The concentration of vanillin reached 52 mM (7.8 g L(-1)) in 24 h, which is the highest level attained to date for the biotechnological production of vanillin using recombinant cells.

  14. Enhancement of γ-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis.

    PubMed

    Shi, Feng; Jiang, Junjun; Li, Yongfu; Li, Youxin; Xie, Yilong

    2013-11-01

    γ-Aminobutyric acid (GABA), a non-protein amino acid, is a bioactive component in the food, feed and pharmaceutical fields. To establish an effective single-step production system for GABA, a recombinant Corynebacterium glutamicum strain co-expressing two glutamate decarboxylase (GAD) genes (gadB1 and gadB2) derived from Lactobacillus brevis Lb85 was constructed. Compared with the GABA production of the gadB1 or gadB2 single-expressing strains, GABA production by the gadB1-gadB2 co-expressing strain increased more than twofold. By optimising urea supplementation, the total production of L-glutamate and GABA increased from 22.57 ± 1.24 to 30.18 ± 1.33 g L⁻¹, and GABA production increased from 4.02 ± 0.95 to 18.66 ± 2.11 g L⁻¹ after 84-h cultivation. Under optimal urea supplementation, L-glutamate continued to be consumed, GABA continued to accumulate after 36 h of fermentation, and the pH level fluctuated. GABA production increased to a maximum level of 27.13 ± 0.54 g L⁻¹ after 120-h flask cultivation and 26.32 g L⁻¹ after 60-h fed-batch fermentation. The conversion ratio of L-glutamate to GABA reached 0.60-0.74 mol mol⁻¹. By co-expressing gadB1 and gadB2 and optimising the urea addition method, C. glutamicum was genetically improved for de novo biosynthesis of GABA from its own accumulated L-glutamate.

  15. The influence of the cell free solution of lactic acid bacteria on tyramine production by food borne-pathogens in tyrosine decarboxylase broth.

    PubMed

    Toy, Nurten; Özogul, Fatih; Özogul, Yesim

    2015-04-15

    The function of cell-free solutions (CFSs) of lactic acid bacteria (LAB) on tyramine and other biogenic amine production by different food borne-pathogens (FBPs) was investigated in tyrosine decarboxylase broth (TDB) using HPLC. Cell free solutions were prepared from four LAB strains. Two different concentrations which were 50% (5 ml CFS+5 ml medium/1:1) and 25% (2.5 ml CFS+7.5 ml medium/1:3) CFS and the control without CFS were prepared. Both concentration of CFS of Streptococcus thermophilus and 50% CFS of Pediococcus acidophilus inhibited tyramine production up to 98% by Salmonella paratyphi A. Tyramine production by Escherichia coli was also inhibited by 50% CFS of Lactococcus lactis subsp. lactis and 25% CFS of Leuconostoc lactis. subsp. cremoris. The inhibitor effect of 50% CFS of P. acidophilus was the highest on tyramine production (55%) by Listeria monocytogenes, following Lc. lactis subsp. lactis and Leuconostoc mesenteroides subsp. cremoris (20%) whilst 25% CFS of Leu. mes. subsp. cremoris and Lc. lactis subsp. lactis showed stimulator effects (160%). The stimulation effects of 50% CFS of S. thermophilus and Lc. lactis subsp. lactis were more than 70% by Staphylococcus aureus comparing to the control. CFS of LAB strains showed statistically inhibitor effect since lactic acid inhibited microbial growth, decreased pH quickly and reduced the formation of AMN and BAs. Consequently, in order to avoid the formation of high concentrations of biogenic amines in fermented food by bacteria, it is advisable to use CFS for food and food products.

  16. Differential gene expression for glutamic acid decarboxylase and type II calcium-calmodulin-dependent protein kinase in basal ganglia, thalamus, and hypothalamus of the monkey

    SciTech Connect

    Benson, D.L.; Isackson, P.J.; Hendry, S.H.; Jones, E.G. )

    1991-06-01

    In situ hybridization histochemistry, using cRNA probes, revealed a complementarity in the distributions of cells in the basal ganglia, basal nucleus of Meynert, thalamus, hypothalamus, and rostral part of the midbrain that showed gene expression for glutamic acid decarboxylase (GAD) or the alpha-subunit of type II calcium-calmodulin-dependent protein kinase (CAM II kinase-alpha). Cells in certain nuclei such as the thalamic reticular nucleus, globus pallidus, and pars reticulata of the substantia nigra show GAD gene expression only; others in nuclei such as the basal nucleus of Meynert, medial mamillary nuclei, and ventromedial hypothalamic nuclei show CAM II kinase-alpha gene expression only. A few nuclei, for example, the pars compacta of the substantia nigra and the greater part of the subthalamic nucleus, display gene expression for neither GAD nor CAM II kinase-alpha. In other nuclei, notably those of the dorsal thalamus, and possibly in the striatum, GAD- and CAM II kinase-expressing cells appear to form two separate populations that, in most thalamic nuclei, together account for the total cell population. In situ hybridization reveals large amounts of CAM II kinase-alpha mRNA in the neuropil of most nuclei containing CAM II kinase-alpha-positive cells, suggesting its association with dendritic polyribosomes. The message may thus be translated at those sites, close to the synapses with which the protein is associated. The in situ hybridization results, coupled with those from immunocytochemical staining for CAM II kinase-alpha protein, indicate that CAM II kinase-alpha is commonly found in certain non-GABAergic afferent fiber systems but is not necessarily present in the postsynaptic cells on which they terminate. It appears to be absent from most GABAergic fiber systems but can be present in the cells on which they terminate.

  17. Tomato Glutamate Decarboxylase Genes SlGAD2 and SlGAD3 Play Key Roles in Regulating γ-Aminobutyric Acid Levels in Tomato (Solanum lycopersicum).

    PubMed

    Takayama, Mariko; Koike, Satoshi; Kusano, Miyako; Matsukura, Chiaki; Saito, Kazuki; Ariizumi, Tohru; Ezura, Hiroshi

    2015-08-01

    Tomato (Solanum lycopersicum) can accumulate relatively high levels of γ-aminobutyric acid (GABA) during fruit development. However, the molecular mechanism underlying GABA accumulation and its physiological function in tomato fruits remain elusive. We previously identified three tomato genes (SlGAD1, SlGAD2 and SlGAD3) encoding glutamate decarboxylase (GAD), likely the key enzyme for GABA biosynthesis in tomato fruits. In this study, we generated transgenic tomato plants in which each SlGAD was suppressed and those in which all three SlGADs were simultaneously suppressed. A significant decrease in GABA levels, i.e. 50-81% compared with wild-type (WT) levels, was observed in mature green (MG) fruits of the SlGAD2-suppressed lines, while a more drastic reduction (up to <10% of WT levels) was observed in the SlGAD3- and triple SlGAD-suppressed lines. These findings suggest that both SlGAD2 and SlGAD3 expression are crucial for GABA biosynthesis in tomato fruits. The importance of SlGAD3 expression was also confirmed by generating transgenic tomato plants that over-expressed SlGAD3. The MG and red fruits of the over-expressing transgenic lines contained higher levels of GABA (2.7- to 5.2-fold) than those of the WT. We also determined that strong down-regulation of the SlGADs had little effect on overall plant growth, fruit development or primary fruit metabolism under normal growth conditions.

  18. A distinct immunogenic region of glutamic acid decarboxylase 65 is naturally processed and presented by human islet cells to cytotoxic CD8 T cells.

    PubMed

    Knight, R R; Dolton, G; Kronenberg-Versteeg, D; Eichmann, M; Zhao, M; Huang, G C; Beck, K; Cole, D K; Sewell, A K; Skowera, A; Peakman, M

    2015-01-01

    CD8 T cells specific for islet autoantigens are major effectors of β cell damage in type 1 diabetes, and measurement of their number and functional characteristics in blood represent potentially important disease biomarkers. CD8 T cell reactivity against glutamic acid decarboxylase 65 (GAD65) in HLA-A*0201 subjects has been reported to focus on an immunogenic region 114-123 (VMNILLQYVV), with studies demonstrating both 114-123 and 114-122 epitopes being targeted. However, the fine specificity of this response is unclear and the key question as to which epitope(s) β cells naturally process and present and, therefore, the pathogenic potential of CD8 T cells with different specificities within this region has not been addressed. We generated human leucocyte antigen (HLA)-A*0201-restricted CD8 T cell clones recognizing either 114-122 alone or both 114-122 and 114-123. Both clone types show potent and comparable effector functions (cytokine and chemokine secretion) and killing of indicator target cells externally pulsed with cognate peptide. However, only clones recognizing 114-123 kill target cells transfected with HLA-A*0201 and GAD2 and HLA-A*0201(+) human islet cells. We conclude that the endogenous pathway of antigen processing by HLA-A*0201-expressing cells generates GAD65114-123 as the predominant epitope in this region. These studies highlight the importance of understanding β cell epitope presentation in the design of immune monitoring for potentially pathogenic CD8 T cells.

  19. Harmonization of Glutamic Acid Decarboxylase and Islet Antigen-2 Autoantibody Assays for National Institute of Diabetes and Digestive and Kidney Diseases Consortia

    PubMed Central

    Bonifacio, Ezio; Yu, Liping; Williams, Alastair K.; Eisenbarth, George S.; Bingley, Polly J.; Marcovina, Santica M.; Adler, Kerstin; Ziegler, Anette G.; Mueller, Patricia W.; Schatz, Desmond A.; Krischer, Jeffrey P.; Steffes, Michael W.; Akolkar, Beena

    2010-01-01

    Background/Rationale: Autoantibodies to islet antigen-2 (IA-2A) and glutamic acid decarboxylase (GADA) are markers for diagnosis, screening, and measuring outcomes in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) consortia studies. A harmonization program was established to increase comparability of results within and among these studies. Methods: Large volumes of six working calibrators were prepared from pooled sera with GADA 4.8–493 World Health Organization (WHO) units/ml and IA-2A 2–235 WHO units/ml. Harmonized assay protocols for IA-2A and GADA using 35S-methionine-labelled in vitro transcribed and translated antigens were developed based on methods in use in three NIDDK laboratories. Antibody thresholds were defined using sera from patients with recent onset type 1 diabetes and healthy controls. To evaluate the impact of the harmonized assay protocol on concordance of IA-2A and GADA results, two laboratories retested stored TEDDY study sera using the harmonized assays. Results: The harmonized assays gave comparable but not identical results in the three laboratories. For IA-2A, using a common threshold of 5 DK units/ml, 549 of 550 control and patient samples were concordantly scored as positive or negative, specificity was greater than 99% with sensitivity 64% in all laboratories. For GADA, using thresholds equivalent to the 97th percentile of 974 control samples in each laboratory, 1051 (97.9%) of 1074 samples were concordant. On the retested TEDDY samples, discordance decreased from 4 to 1.8% for IA-2A (n = 604 samples; P = 0.02) and from 15.4 to 2.7% for GADA (n = 515 samples; P < 0.0001). Conclusion: Harmonization of GADA and IA-2A is feasible using large volume working calibrators and common protocols and is an effective approach to ensure consistency in autoantibody measurements. PMID:20444913

  20. The novel R347g pathogenic mutation of aromatic amino acid decarboxylase provides additional molecular insights into enzyme catalysis and deficiency.

    PubMed

    Montioli, Riccardo; Paiardini, Alessandro; Kurian, Manju A; Dindo, Mirco; Rossignoli, Giada; Heales, Simon J R; Pope, Simon; Voltattorni, Carla Borri; Bertoldi, Mariarita

    2016-06-01

    We report here a clinical case of a patient with a novel mutation (Arg347→Gly) in the gene encoding aromatic amino acid decarboxylase (AADC) that is associated with AADC deficiency. The variant R347G in the purified recombinant form exhibits, similarly to the pathogenic mutation R347Q previously studied, a 475-fold drop of kcat compared to the wild-type enzyme. In attempting to unravel the reason(s) for this catalytic defect, we have carried out bioinformatics analyses of the crystal structure of AADC-carbidopa complex with the modelled catalytic loop (residues 328-339). Arg347 appears to interact with Phe103, as well as with both Leu333 and Asp345. We have then prepared and characterized the artificial F103L, R347K and D345A mutants. F103L, D345A and R347K exhibit about 13-, 97-, and 345-fold kcat decrease compared to the wild-type AADC, respectively. However, unlike F103L, the R347G, R347K and R347Q mutants as well as the D345A variant appear to be more defective in catalysis than in protein folding. Moreover, the latter mutants, unlike the wild-type protein and the F103L variant, share a peculiar binding mode of dopa methyl ester consisting of formation of a quinonoid intermediate. This finding strongly suggests that their catalytic defects are mainly due to a misplacement of the substrate at the active site. Taken together, our results highlight the importance of the Arg347-Leu333-Asp345 hydrogen-bonds network in the catalysis of AADC and reveal the molecular basis for the pathogenicity of the variants R347. Following the above results, a therapeutic treatment for patients bearing the mutation R347G is proposed.

  1. Structural perspective on the direct inhibition mechanism of EGCG on mammalian histidine decarboxylase and DOPA decarboxylase.

    PubMed

    Ruiz-Pérez, M Victoria; Pino-Ángeles, Almudena; Medina, Miguel A; Sánchez-Jiménez, Francisca; Moya-García, Aurelio A

    2012-01-23

    Histidine decarboxylase (HDC) and l-aromatic amino acid decarboxylase (DDC) are homologous enzymes that are responsible for the synthesis of important neuroactive amines related to inflammatory, neurodegenerative, and neoplastic diseases. Epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, has been shown to target histamine-producing cells and to promote anti-inflammatory, antitumor, and antiangiogenic effects. Previous experimental work has demonstrated that EGCG has a direct inhibitory effect on both HDC and DDC. In this study, we investigated the binding modes of EGCG to HDC and DDC as a first step for designing new polyphenol-based HDC/DDC-specific inhibitors. PMID:22107329

  2. Evolution of Substrate Specificity within a Diverse Family of [beta/alpha]-Barrel-fold Basic Amino Acid Decarboxylases X-ray Structure Determination of Enzymes with Specificity for L-Arginine and Carboxynorspermidine

    SciTech Connect

    Deng, Xiaoyi; Lee, Jeongmi; Michael, Anthony J.; Tomchick, Diana R.; Goldsmith, Elizabeth J.; Phillips, Margaret A.

    2010-08-26

    Pyridoxal 5{prime}-phosphate (PLP)-dependent basic amino acid decarboxylases from the {beta}/{alpha}-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-{angstrom} resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the {beta}/{alpha}-barrel domain of one subunit and the {beta}-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the {beta}-barrel domain we refer to as the 'specificity helix.' Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu{sup 314}, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5{prime}-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.

  3. T-cell reactivity to glutamic acid decarboxylase in stiff-man syndrome and cerebellar ataxia associated with polyendocrine autoimmunity

    PubMed Central

    Costa, M; Saiz, A; Casamitjana, R; Castañer, M FernÁndez; SanmartÍ, A; Graus, F; Jaraquemada, D

    2002-01-01

    Antibodies to glutamic acid decarboxilase (GAD-Abs) are present in the serum of 60–80% of newly diagnosed type 1 diabetes (DM1) patients and patients with autoimmune polyendocrine syndrome (APS) associated with DM1. Higher titre of GAD-Abs are also present in the serum of 60% of patients with stiff-man syndrome (SMS) and all reported patients with cerebellar ataxia associated with polyendocrine autoimmunity (CAPA). Several studies suggest that GAD-Abs may play a critical role in the pathogenesis of SMS and CAPA but little is known about T-cell responsiveness to GAD-65 in these neurological diseases. To analyse cell-mediated responses to GAD, we studied the peripheral blood lymphocyte proliferation and cytokine responses to recombinant human GAD-65 in 5 patients with SMS, 6 with CAPA, 9 with DM1, 8 with APS and 15 control subjects. GAD-65-specific cellular proliferation was significantly higher in SMS than in CAPA, DM1, APS or controls. In contrast, only T cells from CAPA patients showed a significantly high production of interferon-γ after GAD stimulation, compared to all other patients and controls. No differences were found for IL-4 production. These results suggest that, despite similar humoral autoreactivity, cellular responses to GAD are different between SMS and CAPA, with a greater inflammatory response in CAPA, and this difference may be relevant to the pathogenesis of these diseases. PMID:12197888

  4. 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. ...

  5. Uncovering the Lactobacillus plantarum WCFS1 gallate decarboxylase involved in tannin degradation.

    PubMed

    Jiménez, Natalia; Curiel, José Antonio; Reverón, Inés; de Las Rivas, Blanca; Muñoz, Rosario

    2013-07-01

    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.

  6. 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

  7. Glycine decarboxylase in Rhodopseudomonas spheroides and in rat liver mitochondria

    PubMed Central

    Tait, G. H.

    1970-01-01

    1. Glycine decarboxylase and glycine–bicarbonate exchange activities were detected in extracts of Rhodopseudomonas spheroides and in rat liver mitochondria and their properties were studied. 2. The glycine decarboxylase activity from both sources is stimulated when glyoxylate is added to the assay system. 3. Several proteins participate in these reactions and a heat-stable low-molecular-weight protein was purified from both sources. 4. These enzyme activities increase markedly when R. spheroides is grown in the presence of glycine, glyoxylate, glycollate, oxalate or serine. 5. All the enzymes required to catalyse the conversion of glycine into acetyl-CoA via serine and pyruvate were detected in extracts of R. spheroides; of these glycine decarboxylase has the lowest activity. 6. The increase in the activity of glycine decarboxylase on illumination of R. spheroides in a medium containing glycine, and the greater increase when ATP is also present in the medium, probably accounts for the increased incorporation of the methylene carbon atom of glycine into fatty acids found previously under these conditions (Gajdos, Gajdos-Török, Gorchein, Neuberger & Tait, 1968). 7. The results are compared with those obtained by other workers on the glycine decarboxylase and glycine–bicarbonate exchange activities in other systems. PMID:5476725

  8. Active site directed irreversible inactivation of brewers' yeast pyruvate decarboxylase by the conjugated substrate analogue (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid: development of a suicide substrate.

    PubMed

    Kuo, D J; Jordan, F

    1983-08-01

    (E)-4-(4-Chlorophenyl)-2-oxo-3-butenoic acid (CPB) was found to irreversibly inactivate brewers' yeast pyruvate decarboxylase (PDC, EC 4.1.1.1) in a biphasic, sigmoidal manner, as is found for the kinetic behavior of substrate. An expression was derived for two-site irreversible inhibition of allosteric enzymes, and the kinetic behavior of CPB fit the expression for two-site binding. The calculated Ki's of 0.7 mM and 0.3 mM for CPB were assigned to the catalytic site and the regulatory site, respectively. The presence of pyruvic acid at high concentrations protected PDC from inactivation, whereas low concentrations of pyruvic acid accelerated inactivation by CPB. Pyruvamide, a known allosteric activator of PDC, was found to enhance inactivation by CPB. The results can be explained if pyruvamide binds only to a regulatory site, but CPB and pyruvic acid compete for both the regulatory and the catalytic centers. [1-14C]CPB was found to lose 14CO2 concurrently with the inactivation of the enzyme. Therefore, CPB was being turned over by PDC, in addition to inactivating it. CPB can be labeled a suicide-type inactivator for PDC.

  9. Crenarchaeal Arginine Decarboxylase Evolved from an S-Adenosylmethionine Decarboxylase Enzyme*S⃞

    PubMed Central

    Giles, Teresa N.; Graham, David E.

    2008-01-01

    The crenarchaeon Sulfolobus solfataricus uses arginine to produce putrescine for polyamine biosynthesis. However, genome sequences from S. solfataricus and most crenarchaea have no known homologs of the previously characterized pyridoxal 5′-phosphate or pyruvoyl-dependent arginine decarboxylases that catalyze the first step in this pathway. Instead they have two paralogs of the S-adenosylmethionine decarboxylase (AdoMetDC). The gene at locus SSO0585 produces an AdoMetDC enzyme, whereas the gene at locus SSO0536 produces a novel arginine decarboxylase (ArgDC). Both thermostable enzymes self-cleave at conserved serine residues to form amino-terminal β-domains and carboxyl-terminal α-domains with reactive pyruvoyl cofactors. The ArgDC enzyme specifically catalyzed arginine decarboxylation more efficiently than previously studied pyruvoyl enzymes. α-Difluoromethylarginine significantly reduced the ArgDC activity of purified enzyme, and treating growing S. solfataricus cells with this inhibitor reduced the cells' ratio of spermidine to norspermine by decreasing the putrescine pool. The crenarchaeal ArgDC had no AdoMetDC activity, whereas its AdoMetDC paralog had no ArgDC activity. A chimeric protein containing the β-subunit of SSO0536 and the α-subunit of SSO0585 had ArgDC activity, implicating residues responsible for substrate specificity in the amino-terminal domain. This crenarchaeal ArgDC is the first example of alternative substrate specificity in the AdoMetDC family. ArgDC activity has evolved through convergent evolution at least five times, demonstrating the utility of this enzyme and the plasticity of amino acid decarboxylases. PMID:18650422

  10. An examination of aspartate decarboxylase and glutamate decarboxylase activity in mosquitoes

    PubMed Central

    Richardson, Graham; Ding, Haizhen; Rocheleau, Tom; Mayhew, George; Reddy, Erin; Han, Qian; Christensen, Bruce M.; Li, Jianyong

    2010-01-01

    A major pathway of beta-alanine synthesis in insects is through the alpha-decarboxylation of aspartate, but the enzyme involved in the decarboxylation of aspartate has not been clearly defined in mosquitoes and characterized in any insect species. In this study, we expressed two putative mosquito glutamate decarboxylase-like enzymes of mosquitoes and critically analyzed their substrate specificity and biochemical properties. Our results provide clear biochemical evidence establishing that one of them is an aspartate decarboxylase and the other is a glutamate decarboxylase. The mosquito aspartate decarboxylase functions exclusively on the production of beta-alanine with no activity with glutamate. Likewise the mosquito glutamate decarboxylase is highly specific to glutamate with essentially no activity with aspartate. Although insect aspartate decarboxylase shares high sequence identity with glutamate decarboxylase, we are able to closely predict aspartate decarboxylase from glutamate decarboxylase based on the difference of their active site residues. PMID:19842059

  11. 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

  12. Characterization of arginine decarboxylase from Dianthus caryophyllus.

    PubMed

    Ha, Byung Hak; Cho, Ki Joon; Choi, Yu Jin; Park, Ky Young; Kim, Kyung Hyun

    2004-04-01

    Arginine decarboxylase (ADC, EC 4.1.1.9) is a key enzyme in the biosynthesis of polyamines in higher plants, whereas ornithine decarboxylase represents the sole pathway of polyamine biosynthesis in animals. Previously, we characterized a genomic clone from Dianthus caryophyllus, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 78 kDa. In the present study, the ADC gene was subcloned into the pGEX4T1 expression vector in combination with glutathione S-transferase (GST). The fusion protein GST-ADC was water-soluble and thus was purified by sequential GSTrap-arginine affinity chromatography. A thrombin-mediated on-column cleavage reaction was employed to release free ADC from GST. Hiload superdex gel filtration FPLC was then used to obtain a highly purified ADC. The identity of the ADC was confirmed by immunoblot analysis, and its specific activity with respect to (14)C-arginine decarboxylation reaction was determined to be 0.9 CO(2) pkat mg(-1) protein. K(m) and V(max) of the reaction between ADC and the substrate were 0.077 +/- 0.001 mM and 6.0 +/- 0.6 pkat mg(-1) protein, respectively. ADC activity was reduced by 70% in the presence of 0.1 mM Cu(2+) or CO(2+), but was only marginally affected by Mg(2+), or Ca(2+) at the same concentration. Moreover, spermine at 1 mM significantly reduced its activity by 30%.

  13. 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...

  14. 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...

  15. Brain glutamate decarboxylase and pyrroloquinoline quinone.

    PubMed

    Choi, S Y; Khemlani, L S; Churchich, J E

    1992-01-01

    Porcine brain glutamate decarboxylase was examined for the presence of covalently bound pyrroloquinoline quinone (PQQ). HPLC analysis of pure glutamate decarboxylase subjected to the hexanol extraction procedure gave negative results when monitored at 320 nm, the maximum of absorbance of 4-hydroxy-5-hexoxy-PQQ. Resolved glutamate decarboxylase exhibits a structureless absorption band at wavelengths longer than 300 nm which cannot be attributed to PQQ. The holoenzyme is not a pyridoxal-quinoprotein; its catalytic mechanism involves the participation of only one cofactor, i.e. pyridoxal-5-P. Free PQQ is a strong inhibitor of the decarboxylase (Ki = 13 microM) and the reaction with the protein results in spectral changes resembling those of polylysine treated with PQQ. If the concentration of free PQQ in some regions of the brain reaches the micromolar level, then PQQ might play a role in the regulation of glutamate decarboxylase activity.

  16. Role of the NR2A/2B subunits of the N-methyl-D-aspartate receptor in glutamate-induced glutamic acid decarboxylase alteration in cortical GABAergic neurons in vitro.

    PubMed

    Monnerie, H; Hsu, F-C; Coulter, D A; Le Roux, P D

    2010-12-29

    The vulnerability of brain neuronal cell subpopulations to neurologic insults varies greatly. Among cells that survive a pathological insult, for example ischemia or brain trauma, some may undergo morphological and/or biochemical changes that may compromise brain function. The present study is a follow-up of our previous studies that investigated the effect of glutamate-induced excitotoxicity on the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67)'s expression in surviving DIV 11 cortical GABAergic neurons in vitro [Monnerie and Le Roux, (2007) Exp Neurol 205:367-382, (2008) Exp Neurol 213:145-153]. An N-methyl-D-aspartate receptor (NMDAR)-mediated decrease in GAD expression was found following glutamate exposure. Here we examined which NMDAR subtype(s) mediated the glutamate-induced change in GAD protein levels. Western blotting techniques on cortical neuron cultures showed that glutamate's effect on GAD proteins was not altered by NR2B-containing diheteromeric (NR1/NR2B) receptor blockade. By contrast, blockade of triheteromeric (NR1/NR2A/NR2B) receptors fully protected against a decrease in GAD protein levels following glutamate exposure. When receptor location on the postsynaptic membrane was examined, extrasynaptic NMDAR stimulation was observed to be sufficient to decrease GAD protein levels similar to that observed after glutamate bath application. Blocking diheteromeric receptors prevented glutamate's effect on GAD proteins after extrasynaptic NMDAR stimulation. Finally, NR2B subunit examination with site-specific antibodies demonstrated a glutamate-induced, calpain-mediated alteration in NR2B expression. These results suggest that glutamate-induced excitotoxic NMDAR stimulation in cultured GABAergic cortical neurons depends upon subunit composition and receptor location (synaptic vs. extrasynaptic) on the neuronal membrane. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered

  17. Locomotor response to L-DOPA in reserpine-treated rats following central inhibition of aromatic L-amino acid decarboxylase: further evidence for non-dopaminergic actions of L-DOPA and its metabolites.

    PubMed

    Alachkar, Amal; Brotchie, Jonathan M; Jones, Owen T

    2010-09-01

    L-DOPA is the most widely used treatment for Parkinson's disease. The anti-parkinsonian and pro-dyskinetic actions of L-DOPA are widely attributed to its conversion, by the enzyme aromatic L-amino acid decarboxylase (AADC), to dopamine. We investigated the hypothesis that exogenous L-DOPA can induce behavioural effects without being converted to dopamine in the reserpine-treated rat-model of Parkinson's disease. A parkinsonian state was induced with reserpine (3 mg/kg s.c.). Eighteen hours later, the rats were administered L-DOPA plus the peripherally acting AADC inhibitor benserazide (25 mg/kg), with or without the centrally acting AADC inhibitor NSD1015 (100 mg/kg). L-DOPA/benserazide alone reversed reserpine-induced akinesia (4158+/-1125 activity counts/6 h, cf vehicle 1327+/-227). Addition of NSD1015 elicited hyperactive behaviour that was approximately 7-fold higher than L-DOPA/benserazide (35755+/-5226, P<0.001). The hyperactivity induced by L-DOPA and NSD1015 was reduced by the alpha(2C) antagonist rauwolscine (1 mg/kg) and the 5-HT(2C) agonist MK212 (5 mg/kg), but not by the D2 dopamine receptor antagonist remoxipride (3 mg/kg) or the D1 dopamine receptor antagonist SCH23390 (1 mg/kg). These data suggest that L-DOPA, or metabolites produced via routes not involving AADC, might be responsible for the generation of at least some L-DOPA actions in reserpine-treated rats. PMID:20542064

  18. Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter.

    PubMed

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P; Geller, Alfred I

    2007-05-01

    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an approximately 9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported approximately 90% glutamatergic neuron-specific expression. The GAD67 promoter supported approximately 90% GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine

  19. 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.

  20. Glutamic Acid Decarboxylase 65 and Islet Cell Antigen 512/IA-2 Autoantibodies in Relation to Human Leukocyte Antigen Class II DR and DQ Alleles and Haplotypes in Type 1 Diabetes Mellitus ▿

    PubMed Central

    Stayoussef, Mouna; Benmansour, Jihen; Al-Jenaidi, Fayza A.; Said, Hichem B.; Rayana, Chiheb B.; Mahjoub, Touhami; Almawi, Wassim Y.

    2011-01-01

    The frequencies of autoantibodies against glutamic acid decarboxylase 65 (GAD65) and islet cell antigen (ICA) 512/IA-2 (512/IA-2) are functions of the specific human leukocyte antigen (HLA) in type 1 diabetes mellitus (T1D). We investigated the association of HLA class II (DR and DQ) alleles and haplotypes with the presence of GAD and IA-2 autoantibodies in T1D. Autoantibodies were tested in 88 Tunisian T1D patients and 112 age- and gender-matched normoglycemic control subjects by enzyme immunoassay. Among T1D patients, mean anti-GAD antibody titers were higher in the DRB1*030101 allele (P < 0.001), together with the DRB1*030101/DQB1*0201 (P < 0.001) and DRB1*040101/DQB1*0302 (P = 0.002) haplotypes, while lower anti-GAD titers were associated with the DRB1*070101 (P = 0.001) and DRB1*110101 (P < 0.001) alleles and DRB1*070101/DQB1*0201 (P = 0.001) and DRB1*110101/DQB1*030101 (P = 0.001) haplotypes. Mean anti-IA-2 antibody titers were higher in the DRB1*040101 allele (P = 0.007) and DRB1*040101/DQB1*0302 (P = 0.001) haplotypes but were lower in the DRB1*110101 allele (P = 0.010) and the DRB1*110101 (P < 0.001) and DRB1*110101/DQB1*030101 (P = 0.025) haplotypes. Multinomial regression analysis confirmed the positive association of DRB1*030101 and the negative association of DRB1*110101 and DQB1*030101, along with the DRB1*070101/DQB1*0201 and DRB1*110101/DQB1*030101 haplotypes, with anti-GAD levels. In contrast, only the DRB1*040101/DQB1*0302 haplotype was positively associated with altered anti-IA-2 titers. Increased GAD65 and IA-2 antibody positivity is differentially associated with select HLA class II alleles and haplotypes, confirming the heterogeneous nature of T1D. PMID:21490167

  1. STEREOLOGICAL ESTIMATES OF THE BASAL FOREBRAIN CELL POPULATION IN THE RAT, INCLUDING NEURONS CONTAINING CHOLINE ACETYLTRANSFERASE (ChAT), GLUTAMIC ACID DECARBOXYLASE (GAD) OR PHOSPHATE-ACTIVATED GLUTAMINASE (PAG) AND COLOCALIZING VESICULAR GLUTAMATE TRANSPORTERS (VGluTs)

    PubMed Central

    GRITTI, I.; HENNY, P.; GALLONI, F.; MAINVILLE, L.; MARIOTTI, M.; JONES, B. E.

    2006-01-01

    The basal forebrain (BF) plays an important role in modulating cortical activity and influencing attention, learning and memory. These activities are fulfilled importantly yet not entirely by cholinergic neurons. Noncholinergic neurons also contribute and are comprised by GABAergic neurons and other possibly glutamatergic neurons. The aim of the present study was to estimate the total number of cells in the BF of the rat and the proportions of that total represented by cholinergic, GABAergic and glutamatergic neurons. For this purpose, cells were counted using unbiased stereological methods within the medial septum, diagonal band, magnocellular preoptic nucleus, substantia innominata and globus pallidus in sections stained for Nissl substance and/or the neurotransmitter enzymes, choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or phosphate-activated glutaminase (PAG). In Nissl-stained sections, the total number of neurons in the BF was estimated as ~355,000 and the numbers of ChAT-immuno-positive (+) as ~22,000, GAD+ ~119,000 and PAG+ ~316,000, corresponding to ~5%, ~35% and ~90% of the total. Thus, of the large population of BF neurons, only a small proportion has the capacity to synthesize acetylcholine (ACh), one third to synthesize GABA and the vast majority to synthesize glutamate (Glu). Moreover, through the presence of PAG, a proportion of ACh- and GABA-synthesizing neurons also have the capacity to synthesize Glu. In sections dual fluorescent immunostained for vesicular transporters, VGluT3 and not VGluT2 was present in the cell bodies of most PAG+ and ChAT+ and half the GAD+ cells. Given previous results showing that VGluT2 and not VGluT3 was present in BF axon terminals and not colocalized with VAChT or VGAT, we conclude that the BF cell population influences cortical and subcortical regions through neurons which release ACh, GABA or Glu from their terminals but which in part can also synthesize and release Glu from their soma or

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

    PubMed

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

    1986-03-01

    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.

  3. Identification of the Enterococcus faecalis Tyrosine Decarboxylase Operon Involved in Tyramine Production

    PubMed Central

    Connil, Nathalie; Le Breton, Yoann; Dousset, Xavier; Auffray, Yanick; Rincé, Alain; Prévost, Hervé

    2002-01-01

    Screening of a library of Enterococcus faecalis insertional mutants allowed isolation of a mutant affected in tyramine production. The growth of this mutant was similar to that of the wild-type E. faecalis JH2-2 strain in Maijala broth, whereas high-performance liquid chromatography analyses showed that tyramine production, which reached 1,000 μg ml−1 for the wild-type strain, was completely abolished. Genetic analysis of the insertion locus revealed a gene encoding a decarboxylase with similarity to eukaryotic tyrosine decarboxylases. Sequence analysis revealed a pyridoxal phosphate binding site, indicating that this enzyme belongs to the family of amino acid decarboxylases using this cofactor. Reverse transcription-PCR analyses demonstrated that the gene (tdc) encoding the putative tyrosine decarboxylase of E. faecalis JH2-2 is cotranscribed with the downstream gene encoding a putative tyrosine-tyramine antiporter and with the upstream tyrosyl-tRNA synthetase gene. This study is the first description of a tyrosine decarboxylase gene in prokaryotes. PMID:12089039

  4. Retina maturation following administration of thyroxine in developing rats: effects on polyamine metabolism and glutamate decarboxylase.

    PubMed

    Macaione, S; Di Giorgio, R M; Nicotina, P A; Ientile, R

    1984-08-01

    The effects of subcutaneous daily treatment with thyroxine on cell proliferation, differentiation, polyamines, and gamma-aminobutyric acid metabolism in the rat retina were studied during the first 20 postnatal days. The retinal layers of the treated rats displayed an enhanced cell differentiation which reached its maximum 9-12 days from birth; but this effect stopped very quickly and was finished by the 20th postnatal day. Primarily there was an increase in ornithine decarboxylase activity which was accompanied by an increase in putrescine, spermidine, and spermine levels. S-Adenosylmethionine decarboxylase was induced later than ODC; corresponding with the enhanced synaptogenesis, glutamate decarboxylase increased 15-fold between the fourth and 15th days. Our data are consistent with the hypothesis that thyroxine may exert some of its effects by inducing the enzymes which regulate polyamine metabolism and synaptogenesis.

  5. Aerobically incubated medium for decarboxylase testing of Enterobacteriaceae by replica-plating methods.

    PubMed

    Maccani, J E

    1979-12-01

    An aerobically incubated, agar-based medium was developed for amino acid decarboxylase testing of Enterobacteriaceae family members by replica-plating methods. Results with the new medium agreed 97 to 99% with the reference broth method of Moeller, and no false-positive reactions were encountered.

  6. Expression of human arginine decarboxylase, the biosynthetic enzyme for agmatine

    PubMed Central

    Zhu, Meng-Yang; Iyo, Abiye; Piletz, John E.; Regunathan, Soundar

    2011-01-01

    Agmatine, an amine formed by decarboxylation of L-arginine by arginine decarboxylase (ADC), has been recently discovered in mammalian brain and other tissues. While the cloning and sequencing of ADC from plant and bacteria have been reported extensively, the structure of mammalian enzyme is not known. Using homology screening approach, we have identified a human cDNA clone that exhibits ADC activity when expressed in COS-7 cells. The cDNA and deduced amino acid sequence of this human ADC clone is distinct from ADC of other forms. Human ADC is a 460-amino acid protein that shows about 48% identity to mammalian ornithine decarboxylase (ODC) but has no ODC activity. While naive COS-7 cells do not make agmatine, these cells are able to produce agmatine, as measured by HPLC, when transfected with ADC cDNA. Northern blot analysis using the cDNA probe indicated the expression of ADC message in selective human brain regions and other human tissues. PMID:14738999

  7. Genetic manipulation of the γ-aminobutyric acid (GABA) shunt in rice: overexpression of truncated glutamate decarboxylase (GAD2) and knockdown of γ-aminobutyric acid transaminase (GABA-T) lead to sustained and high levels of GABA accumulation in rice kernels.

    PubMed

    Shimajiri, Yasuka; Oonishi, Takayuki; Ozaki, Kae; Kainou, Kumiko; Akama, Kazuhito

    2013-06-01

    Gamma-aminobutyric acid (GABA) is a non-protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA-transaminase, GABA-T), we attempted seed-specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB-1) or rice embryo globulin promoters (REG) and GABA-T-based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T₁ and T₂ generations of rice lines displayed high GABA concentrations (2-100 mg/100 g grain). In analyses of two selected lines from the T₃ generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA-T expression was relatively weak. In these two lines both with two T-DNA copies, their starch, amylose, and protein levels were slightly lower than non-transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75-350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts.

  8. Mammalian Dopa decarboxylase: structure, catalytic activity and inhibition.

    PubMed

    Bertoldi, Mariarita

    2014-03-15

    Mammalian Dopa decarboxylase catalyzes the conversion of L-Dopa and L-5-hydroxytryptophan to dopamine and serotonin, respectively. Both of them are biologically active neurotransmitters whose levels should be finely tuned. In fact, an altered concentration of dopamine is the cause of neurodegenerative diseases, such as Parkinson's disease. The chemistry of the enzyme is based on the features of its coenzyme pyridoxal 5'-phosphate (PLP). The cofactor is highly reactive and able to perform multiple reactions, besides decarboxylation, such as oxidative deamination, half-transamination and Pictet-Spengler cyclization. The structure resolution shows that the enzyme has a dimeric arrangement and provides a molecular basis to identify the residues involved in each catalytic activity. This information has been combined with kinetic studies under steady-state and pre-steady-state conditions as a function of pH to shed light on residues important for catalysis. A great effort in DDC research is devoted to design efficient and specific inhibitors in addition to those already used in therapy that are not highly specific and are responsible for the side effects exerted by clinical approach to either Parkinson's disease or aromatic amino acid decarboxylase deficiency. PMID:24407024

  9. A novel approach to inhibit intracellular vitamin B6-dependent enzymes: proof of principle with human and plasmodium ornithine decarboxylase and human histidine decarboxylase.

    PubMed

    Wu, Fang; Christen, Philipp; Gehring, Heinz

    2011-07-01

    Pyridoxal-5'-phosphate (vitamin B(6))-dependent enzymes play central roles in the metabolism of amino acids. Moreover, the synthesis of polyamines, which are essential for cell growth, and of biogenic amines, such as histamine and other signal transmitters, relies on these enzymes. Certain B(6) enzymes thus are prime targets for pharmacotherapeutic intervention. We have devised a novel, in principle generally applicable strategy for obtaining small-molecule cell-permeant inhibitors of specific B(6) enzymes. The imine adduct of pyridoxal-5'-phosphate and the specific amino acid substrate, the first intermediate in all pyridoxal-5'-phosphate-dependent reactions of amino acids, was reduced to a stable secondary amine. This coenzyme-substrate-conjugate was modified further to make it membrane-permeant and, guided by structure-based modeling, to boost its affinity to the apoform of the target enzyme. Inhibitors of this type effectively decreased the respective intracellular enzymatic activity (IC(50) in low micromolar range), providing lead compounds for inhibitors of human ornithine decarboxylase (hODC), plasmodium ornithine decarboxylase, and human histidine decarboxylase. The inhibitors of hODC interfere with the metabolism of polyamines and efficiently prevent the proliferation of tumor cell lines (IC(50)∼ 25 μM). This approach to specific inhibition of intracellular B(6) enzymes might be applied in a straightforward manner to other B(6) enzymes of emerging medicinal interest. PMID:21454364

  10. 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.

  11. 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.

  12. The Ornithine Decarboxylase Gene of Caenorhabditis Elegans: Cloning, Mapping and Mutagenesis

    PubMed Central

    Macrae, M.; Plasterk, RHA.; Coffino, P.

    1995-01-01

    The gene (odc-1) encoding ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was cloned and characterized. Two introns interrupt the coding sequence of the gene. The deduced protein contains 422 amino acids and is homologous to ornithine decarboxylases of other eukaryotic species. In vitro translation of a transcript of the cDNA yielded an enzymatically active product. The mRNA is 1.5 kb in size and is formed by trans-splicing to SL1, a common 5' RNA segment. odc-1 maps to the middle of LG V, between dpy-11 and unc-42 and near a breakpoint of the nDf32 deficiency strain. Enzymatic activity is low in starved stage 1 (L1) larva and, after feeding, rises progressively as the worms develop. Targeted gene disruption was used to create a null allele. Homozygous mutants are normally viable and show no apparent defects, with the exception of a somewhat reduced brood size. In vitro assays for ornithine decarboxylase activity, however, show no detectable enzymatic activity, suggesting that ornithine decarboxylase is dispensible for nematode growth in the laboratory. PMID:7498733

  13. The ornithine decarboxylase gene of Caenorhabditis elegans: Cloning, mapping and mutagenesis

    SciTech Connect

    Macrae, M.; Coffino, P.; Plasterk, R.H.A.

    1995-06-01

    The gene (odc-1) encoding ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was cloned and characterized. Two introns interrupt the coding sequence of the gene. The deduced protein contains 442 amino acids and is homologous to ornithine decarboxylases of other eukaryotic species. In vitro translation of a transcript of the cDNA yielded an enzymatically active product. The mRNA is 1.5 kb in size and is formed by trans-splicing to SL1, a common 5{prime} RNA segment. odc-1 maps to the middle of LG V, between dpy-11 and unc-42 and near a breakpoint of the nDf32 deficiency strain. Enzymatic activity is low in starved 1 (L1) larva and, after feeding, rises progressively as the worms develop. Targeted gene disruption was used to create a null allele. Homozygous mutants are normally viable and show no apparent defects, with the exception of a somewhat reduced brood size. In vitro assays for ornithine decarboxylase activity, however, show no detectable enzymatic activity, suggesting that ornithine decarboxylase is dispensible for nematode growth in the laboratory. 37 refs., 6 figs., 1 tab.

  14. A defect in pyruvate decarboxylase in a child with an intermittent movement disorder

    PubMed Central

    Blass, John P.; Avigan, Joel; Uhlendorf, B. William

    1970-01-01

    A patient with an intermittent movement disorder has been found to have an inherited defect in pyruvate decarboxylase ((2-oxo-acid carboxy-lyase, E.C. 4.1.1.1.). The patient is a 9 yr old boy who since infancy has had repeated episodes of a combined cerebellar and choreoathetoid movement disorder. He has an elevated level of pyruvic acid in his blood, an elevated urinary alanine content, and less marked elevations in blood alanine and lactate. Methods were developed to study his metabolic abnormality in dilute suspensions of white blood cells and cultured skin fibroblasts, as well as in cell-free sonicates of fibroblasts. Oxidation of pyruvic acid-1-14C and pyruvic acid-2-14C by his cells and pyruvate decarboxylase activity in sonicates of his cells were less than 20% of those in cells from control subjects. Oxidation of glutamic acid-U-14C, acetate-1-14C, and palmitate-1-14C was normal, as was incorporation of alanine-U-14C into protein. The rate of oxidation of pyruvic acid by the father's cells and the activity of pyruvate decarboxylase in the father's sonicated fibroblasts were intermediate between those of the patient and those of controls. Values for the mother were at or just below the lower limits of the ranges in controls. Kinetic data suggested the posibility of several forms of pyruvate decarboxylase in this family. Possible mechanisms relating the chemical abnormality and the clinical symptoms in this patient are discussed. PMID:4313434

  15. Glycine decarboxylase controls photosynthesis and plant growth.

    PubMed

    Timm, Stefan; Florian, Alexandra; Arrivault, Stephanie; Stitt, Mark; Fernie, Alisdair R; Bauwe, Hermann

    2012-10-19

    Photorespiration makes oxygenic photosynthesis possible by scavenging 2-phosphoglycolate. Hence, compromising photorespiration impairs photosynthesis. We examined whether facilitating photorespiratory carbon flow in turn accelerates photosynthesis and found that overexpression of the H-protein of glycine decarboxylase indeed considerably enhanced net-photosynthesis and growth of Arabidopsis thaliana. At the molecular level, lower glycine levels confirmed elevated GDC activity in vivo, and lower levels of the CO(2) acceptor ribulose 1,5-bisphosphate indicated higher drain from CO(2) fixation. Thus, the photorespiratory enzyme glycine decarboxylase appears as an important feed-back signaller that contributes to the control of the Calvin-Benson cycle and hence carbon flow through both photosynthesis and photorespiration.

  16. Biosynthetic arginine decarboxylase in phytopathogenic fungi.

    PubMed

    Khan, A J; Minocha, S C

    1989-01-01

    It has been reported that while bacteria and higher plants possess two different pathways for the biosynthesis of putrescine, via ornithine decarboxylase (ODC) and arginine decarboxylase (ADC); the fungi, like animals, only use the former pathway. We found that contrary to the earlier reports, two of the phytopathogenic fungi (Ceratocystis minor and Verticillium dahliae) contain significant levels of ADC activity with very little ODC. The ADC in these fungi has high pH optimum (8.4) and low Km (0.237 mM for C. minor, 0.103 mM for V. dahliae), and is strongly inhibited by alpha-difluoromethylarginine (DFMA), putrescine and spermidine, further showing that this enzyme is probably involved in the biosynthesis of polyamines and not in the catabolism of arginine as in Escherichia coli. The growth of these fungi is strongly inhibited by DFMA while alpha-difluoromethylornithine (DFMO) has little effect.

  17. Additivity and associative effects of metabolizable energy and amino acid digestibility in barley and canola meal for White Pekin ducks.

    PubMed

    Hong, D; Ragland, D; Adeola, O

    2001-11-01

    An experiment was conducted using the TMEn bioassay method to investigate the additivity and associative effects of metabolizable energy and amino acid digestibility in barley and canola meal for White Pekin ducks. Additivity was tested by comparing the difference between observed values determined in a complete diet and predicted values from measurements determined with individual ingredients (barley and canola meal). Six ducks each were assigned to diets of barley, canola meal, the complete diet, and dextrose. Dextrose-fed ducks were used for estimation of endogenous losses for calculation of true amino acid digestibility. The observed AME, TME, AMEn, and TMEn values in the complete diet were 0.065, 0.083, 0.016, and 0.023 (kcal/g), respectively, numerically higher than predicted values. Differences between observed and predicted values were not significant (P > 0.05), indicating that the AME, AMEn, TME, and TMEn in barley and canola meal were all additive. In general, observed values for apparent amino acid digestibility (AAAD) and true amino acid digestibility (TAAD) in the complete diet were higher than those predicted from individual ingredients. Observed AAAD for lysine, histidine, tryptophan, alanine, and aspartate were higher (P < 0.05) than predicted values, indicating that digestibilities of these amino acids were not additive. The mean of AAAD in canola meal (77.29%) was higher (P < 0.05) than the observed values of barley (52.2%) and complete diet (64.55%). For TAAD values, differences between observed and predicted values were significant for lysine, histidine, and tryptophan (P < 0.05). The mean of TAAD in canola meal, barley, and complete diet were 85.88, 80.87, and 81.33%, respectively. The average difference between observed and predicted values for TAAD (1.18 %) was smaller than that of AAAD (5.41%). These results indicated that ME values for barley and canola meal were additive in the complete diet but that digestibilities of some amino acids

  18. Structures of Bacterial Biosynthetic Arginine Decarboxylases

    SciTech Connect

    F Forouhar; S Lew; J Seetharaman; R Xiao; T Acton; G Montelione; L Tong

    2011-12-31

    Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. The TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.

  19. In vitro inhibition of lysine decarboxylase activity by organophosphate esters.

    PubMed

    Wang, Sufang; Wan, Bin; Zhang, Lianying; Yang, Yu; Guo, Liang-Hong

    2014-12-01

    Organophosphate esters (OPEs), a major group of organophosphorus flame retardants, are regarded as emerging environmental contaminants of health concern. Amino acid decarboxylases catalyze the conversion of amino acids into polyamines that are essential for cell proliferation, hypertrophy and tissue growth. In this paper, inhibitory effect of twelve OPEs with aromatic, alkyl or chlorinated alkyl substituents on the activity of lysine decarboxylase (LDC) was assessed quantitatively with an economic and label-free fluorescence sensor and cell assay. The sensor comprises a macrocyclic host (cucurbit[7]uril) and a fluorescent dye (acridine orange) reporter. The twelve OPEs were found to vary in their capacity to inhibit LDC activity. Alkyl group substituted OPEs had no inhibitory effect. By contrast, six OPEs substituted with aromatic or chlorinated alkyl groups inhibited LDC activity significantly with IC50 ranging from 1.32 μM to 9.07 μM. Among them, the inhibitory effect of tri-m-cresyl phosphate (TCrP) was even more effective as an inhibitor than guanosine 5'-diphosphate-3'-diphosphate (ppGpp) (1.60 μM), an LDC natural inhibitor in vivo. Moreover, at non-cytotoxic concentrations, these six OPEs showed perceptible inhibitory effects on LDC activity in PC12 living cells, and led to a marked loss in the cadaverine content. Molecular docking analysis of the LDC/OPE complexes revealed that different binding modes contribute to the difference in their inhibitory effect. Our finding suggested that LDC, as a new potential biological target of OPEs, might be implicated in toxicological and pathogenic mechanism of OPEs. PMID:25264276

  20. Nitrogen isotope effects on glutamate decarboxylase from Escherichia coli

    SciTech Connect

    Abell, L.M.; O'Leary, M.H.

    1988-05-03

    The nitrogen isotope effect on the decarboxylation of glutamic acid by glutamate decarboxylase from Escherichia coli has been measured by comparison of the isotopic composition of the amino nitrogen of the product ..gamma..-aminobutyric acid isolated after 10-20% reaction with that of the starting glutamic acid. At pH 4.7, 37 /sup 0/C, the isotope effect is k/sup 14//k/sup 15/ = 0.9855 +/- 0.0006 when compared to unprotonated glutamic acid. Interpretation of this result requires knowledge of the equilibrium nitrogen isotope effect for Schiff base formation. This equilibrium isotope effect is K/sup 14//K/sup 15/ - 0.9824 for the formation of the unprotonated Schiff base between unprotonated valine and salicylaldehyde. Analysis of the nitrogen isotope effect on decarboxylation of glutamic acid and of the previously measured carbon isotope effect on this same reaction shows that decarboxylation and Schiff base formation are jointly rate limiting. The enzyme-bound Schiff base between glutamate and pyridoxal 5'-phosphate partitions approximately 2:1 between decarboxylation and return to the starting state. The nitrogen isotope effect also reveals that the Schiff base nitrogen is protonated in this intermediate.

  1. 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.

  2. 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...

  3. Evidence for PQQ as cofactor in 3,4-dihydroxyphenylalanine (dopa) decarboxylase of pig kidney.

    PubMed

    Groen, B W; van der Meer, R A; Duine, J A

    1988-09-12

    Pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase (EC 4.1.1.28) was purified to homogeneity. Treatment of the enzyme with phenylhydrazine (PH) according to a procedure developed for analysis of quinoproteins gave products which were identified as the hydrazone of pyridoxal phosphate (PLP) and the C(5)-hydrazone of pyrroloquinoline quinone (PQQ). This method failed, however, in quantifying the amounts of cofactor. Direct hydrolysis of the enzyme by refluxing with hexanol and concentrated HCl led to detachment of PQQ from the protein in a quantity of 1 PQQ per enzyme molecule. In view of the reactivity of PQQ towards amines and amino acids, we postulate that it participates as a covalently bound cofactor in the catalytic cycle of the enzyme, in interplay with PLP. Since several other enzymes have been reported to show the atypical behaviour of dopa decarboxylase, it seems that the PLP-containing group of enzymes can be subdivided into pyridoxoproteins and pyridoxo-quinoproteins.

  4. Observation of superoxide production during catalysis of Bacillus subtilis oxalate decarboxylase at pH 4.

    PubMed

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

    2015-03-01

    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 are 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

  5. 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

  6. Sequencing, characterization, and gene expression analysis of the histidine decarboxylase gene cluster of Morganella morganii.

    PubMed

    Ferrario, Chiara; Borgo, Francesca; de Las Rivas, Blanca; Muñoz, Rosario; Ricci, Giovanni; Fortina, Maria Grazia

    2014-03-01

    The histidine decarboxylase gene cluster of Morganella morganii DSM30146(T) was sequenced, and four open reading frames, named hdcT1, hdc, hdcT2, and hisRS were identified. Two putative histidine/histamine antiporters (hdcT1 and hdcT2) were located upstream and downstream the hdc gene, codifying a pyridoxal-P dependent histidine decarboxylase, and followed by hisRS gene encoding a histidyl-tRNA synthetase. This organization was comparable with the gene cluster of other known Gram negative bacteria, particularly with that of Klebsiella oxytoca. Recombinant Escherichia coli strains harboring plasmids carrying the M. morganii hdc gene were shown to overproduce histidine decarboxylase, after IPTG induction at 37 °C for 4 h. Quantitative RT-PCR experiments revealed the hdc and hisRS genes were highly induced under acidic and histidine-rich conditions. This work represents the first description and identification of the hdc-related genes in M. morganii. Results support the hypothesis that the histidine decarboxylation reaction in this prolific histamine producing species may play a role in acid survival. The knowledge of the role and the regulation of genes involved in histidine decarboxylation should improve the design of rational strategies to avoid toxic histamine production in foods.

  7. 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

  8. The ornithine decarboxylase gene odc is required for alcaligin siderophore biosynthesis in Bordetella spp.: putrescine is a precursor of alcaligin.

    PubMed Central

    Brickman, T J; Armstrong, S K

    1996-01-01

    Chromosomal insertions defining Bordetella bronchiseptica siderophore phenotypic complementation group III mutants BRM3 and BRM5 were found to reside approximately 200 to 300 bp apart by restriction mapping of cloned genomic regions associated with the insertion markers. DNA hybridization analysis using B. bronchiseptica genomic DNA sequences flanking the cloned BRM3 insertion marker identified homologous Bordetella pertussis UT25 cosmids that complemented the siderophore biosynthesis defect of the group III B. bronchiseptica mutants. Subcloning and complementation analysis localized the complementing activity to a 2.8-kb B. pertussis genomic DNA region. Nucleotide sequencing identified an open reading frame predicted to encode a polypeptide exhibiting strong similarity at the primary amino acid level with several pyridoxal phosphate-dependent amino acid decarboxylases. Alcaligin production was fully restored to group III mutants by supplementation of iron-depleted culture media with putrescine (1,4-diaminobutane), consistent with defects in an ornithine decarboxylase activity required for alcaligin siderophore biosynthesis. Concordantly, the alcaligin biosynthesis defect of BRM3 was functionally complemented by the heterologous Escherichia coli speC gene encoding an ornithine decarboxylase activity. Enzyme assays confirmed that group III B. bronchiseptica siderophore-deficient mutants lack an ornithine decarboxylase activity required for the biosynthesis of alcaligin. Siderophore production by an analogous mutant of B. pertussis constructed by allelic exchange was undetectable. We propose the designation odc for the gene defined by these mutations that abrogate alcaligin siderophore production. Putrescine is an essential precursor of alcaligin in Bordetella spp. PMID:8550442

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

    PubMed Central

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

    1996-01-01

    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. PMID:8660289

  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. 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.

  12. A role for glutamate decarboxylase during tomato ripening: the characterisation of a cDNA encoding a putative glutamate decarboxylase with a calmodulin-binding site.

    PubMed

    Gallego, P P; Whotton, L; Picton, S; Grierson, D; Gray, J E

    1995-03-01

    A tomato fruit cDNA library was differentially screened to identify mRNAs present at higher levels in fruit of the tomato ripening mutant rin (ripening inhibitor). Complete sequencing of a unique clone ERT D1 revealed an open reading frame with homology to several glutamate decarboxylases. The deduced polypeptide sequence has 80% overall amino acid sequence similarity to a Petunia hybrida glutamate decarboxylase (petGAD) which carries a calmodulin-binding site at its carboxyl terminus and ERT D1 appears to have a similar domain. ERT D1 mRNA levels peaked at the first visible sign of fruit colour change during normal tomato ripening and then declined, whereas in fruit of the ripening impaired mutant, rin, accumulation of this mRNA continued until at least 14 days after the onset of ripening. This mRNA was present at much lower levels in other tissues, such as leaves, roots and stem, and was not increased by wounding. Possible roles for GAD, and its product gamma-aminobutyric acid (GABA) in fruit, are discussed.

  13. Characterization of a second ornithine decarboxylase isolated from Morganella morganii.

    PubMed

    De Las Rivas, Blanca; González, Ramón; Landete, José María; Muñoz, Rosario

    2008-03-01

    The genes involved in the putrescine formation by Morganella morganii were investigated because putrescine is an indicator of food process deterioration. We report here on the existence of a new gene for ornithine decarboxylase (ODC) in M. morganii. The sequenced 5,311-bp DNA region showed the presence of four complete and one partial open reading frame. Putative functions have been assigned to several gene products by sequence comparison with the proteins included in the databases. The third open reading frame (speC) encoded a 722-amino acid protein showing 70.9% identity to the M. morganii ODC previously characterized (SpeF). The speC gene has been expressed in Escherichia coli, resulting in ODC activity. The presence of a functional promoter (PspeC) located upstream of speC has been demonstrated. Quantitative real-time reverse transcription PCR assay was used to quantify expression of both M. morganii ODC-encoding genes, speC and speF, under different growth conditions. This assay allows us to identify SpeF as the inducible M. morganii ODC, since it was highly expressed in the presence of ornithine.

  14. Ornithine decarboxylase and S-adenosyl methionine decarboxylase in skin fibroblasts of normal and cystic fibrosis patients.

    PubMed

    Buehler, B; Wright, R; Schott, S; Darby, B; Rennert, O M

    1977-03-01

    The key enzymes in the synthesis of the naturally occurring polyamines, ornithine decarboxylase (ODC) and S-adenosyl methionine (SAM) decarboxylase, were investigated during cell growth and aging in fibroblast cultures from normal patients and patients with cystic fibrosis. A linear correlation between increased S-adenosyl methionine activity and putrescine concentration was apparent in all cell lines. A putrescine concentration of 0.8 mM was optimal for enhancement of SAM decarboxylase activity. The passage number of the cell line correlated inversely with maximal putrescine-stimulated SAM decarboxylase activity, earlier passage numbers having the highest specific activity (Fig. 1). No significant differences in basal or putrescine-stimulated SAM decarboxylase activity were noted between normal fibroblast cultures and cells from patients with cystic fibrosis (Fig. 2). SAM decarboxylase activity increased as the cell lines approached confluence. Activity was lowest during exponential growth (Fig. 3). ODC activity was increased during early exponential growth and fell as cells reached confluence (Fig. 4). No differences in ODC activity and putrescine inhibition between the normal and cystic fibrosis cell cultures at equivalent points of exponential growth were noted.

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

    PubMed

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

    2002-11-01

    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.

  16. 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

  17. 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

  18. Cysteinesulfinate decarboxylase: Characterization, inhibition, and metabolic role in taurine formation

    SciTech Connect

    Weinstein, C.L.

    1988-01-01

    Cysteinesulfinate decarboxylase, an enzyme that plays a major role in the formation of taurine from cysteine, has been purified from rat liver to homogeneity and characterized. The physical properties of the enzyme were studied, along with its substrate specificity. Multiple forms of the enzyme were found in rat liver, kidney, and brain with isoelectric points ranging from pH 5.6 to 4.9. These multiple forms did not differ in their substrate specificity. It was found by using gel electrofocusing and polyclonal antibodies raised to the liver enzyme that the different forms of cysteinesulfinate decarboxylase are identical in the various rat tissues studied. Various inhibitors of the enzyme were tested both in vitro and in vivo in order to evaluate the role of cysteinesulfinate decarboxylase in taurine formation in mammalian tissues. In in vitro studies, cysteinesulfinate decarboxylase was irreversibly inhibited by {beta}-ethylidene-DL-aspartate (Ki = 10 mM), and competitive inhibition was found using mercaptomethylsuccinate (Ki = 0.1 mM) and D-cysteinesulfinate (Ki = 0.32 mM) when L-cysteinesulfinate was used as a substrate. In order to be able to test these inhibitors in vivo, L-(1-{sup 14}C)cysteinesulfonate was evaluated as a probe for the in vivo measurement of cysteinesulfinate decarboxylase activity. The metabolism of cysteinesulfonate and the product of its transamination, {beta}-sulfopyruvate, was studied, and it was found that L-(1-{sup 14}C)cysteinesulfonate is an accurate and convenient probe for cysteinesulfinate decarboxylase activity. Using L-(1-{sup 14}C)cysteinesulfonate, it was found that D-cysteinesulfinate inhibits cysteinesulfinate decarboxylase activity by greater than 90% in the intact mouse and that inhibition lasts for up to fifteen hours.

  19. 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. PMID

  20. 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

  1. 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.

  2. Ornithine Decarboxylase, Polyamines, and Pyrrolizidine Alkaloids in Senecio and Crotalaria

    PubMed Central

    Birecka, Helena; Birecki, Mieczyslaw; Cohen, Eric J.; Bitonti, Alan J.; McCann, Peter P.

    1988-01-01

    When tested for ornithine and arginine decarboxylases, pyrrolizidine alkaloid-bearing Senecio riddellii, S. longilobus (Compositae), and Crotalaria retusa (Leguminosae) plants exhibited only ornithine decarboxylase activity. This contrasts with previous studies of four species of pyrrolizidine alkaloid-bearing Heliotropium (Boraginaceae) in which arginine decarboxylase activity was very high relative to that of ornithine decarboxylase. Unlike Heliotropium angiospermum and Heliotropium indicum, in which endogenous arginine was the only detectable precursor of putrescine channeled into pyrrolizidines, in the species studied here—using difluoromethylornithine and difluoromethylarginine as the enzyme inhibitors—endogenous ornithine was the main if not the only precursor of putrescine converted into the alkaloid aminoalcohol moiety. In S. riddellii and C. retusa at flowering, ornithine decarboxylase activity was present mainly in leaves, especially the young ones. However, other very young organs such as inflorescence and growing roots exhibited much lower or very low activities; the enzyme activity in stems was negligible. There was no correlation between the enzyme activity and polyamine or alkaloid content in either species. In both species only free polyamines were detected except for C. retusa roots and inflorescence—with relatively very high levels of these compounds—in which conjugated putrescine, spermidine, and spermine were also found; agmatine was not identified by HPLC in any plant organ except for C. retusa roots with rhizobial nodules. Organ- or age-dependent differences in the polyamine levels were small or insignificant. The highest alkaloid contents were found in young leaves and inflorescence. PMID:16665870

  3. Frequent coexpression of the vesicular glutamate transporter 1 and 2 genes, as well as coexpression with genes for choline acetyltransferase or glutamic acid decarboxylase in neurons of rat brain.

    PubMed

    Danik, Marc; Cassoly, Estelle; Manseau, Frédéric; Sotty, Florence; Mouginot, Didier; Williams, Sylvain

    2005-08-15

    It is widely believed that expression of the vesicular glutamate transporter genes VGLUT1 and VGLUT2 is restricted to glutamatergic neurons and that the two transporters segregate in different sets of neurons. Using single-cell multiplex RT-PCR (sc-RT-mPCR), we show that VGLUT1 and VGLUT2 mRNAs were coexpressed in most of the sampled neurons from the rat hippocampus, cortex, and cerebellum at postnatal Day (P)14 but not P60. In accordance, changes in VGLUT1 and VGLUT2 mRNA concentrations were found to occur in these and other brain areas between P14 and P60, as revealed by semiquantitative RT-PCR and quantitated by ribonuclease protection assay. VGLUT1 and -2 coexpression in the hippocampal formation is supported further by in situ hybridization data showing that virtually all cells in the CA1-CA3 pyramidal and granule cell layers were highly positive for both transcripts until P14. It was revealed using sc-RT-mPCR that transcripts for VGLUT1 and VGLUT2 were also present in neurons of the cerebellum, striatum, and septum that expressed markers for gamma-aminobutyric acid (GABA)ergic or cholinergic phenotypes, as well as in hippocampal cells containing transcripts for the glial fibrillary acidic protein. Our study suggests that VGLUT1 and VGLUT2 proteins may often transport glutamate into vesicles within the same neuron, especially during early postnatal development, and that they are expressed widely in presumed glutamatergic, GABAergic, and cholinergic neurons, as well as in astrocytes. Furthermore, our study shows that such coexpressing neurons remain in the adult brain and identifies several areas that contain them in both young and adult rats. PMID:15983996

  4. 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+).

  5. 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.

  6. Expression of arginine decarboxylase and ornithine decarboxylase genes in apple cells and stressed shoots.

    PubMed

    Hao, Yu-Jin; Kitashiba, Hiroyasu; Honda, Chikako; Nada, Kazuyoshi; Moriguchi, Takaya

    2005-04-01

    Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) are two important enzymes responsible for putrescine biosynthesis. In this study, a full-length ADC cDNA (MdADC) was isolated from apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. Meanwhile, a partial ODC (pMdODC) could be amplified only by a second RCR from the RT-PCR products, whereas a full-length ODC could not be obtained by either cDNA library screening or 5'- and 3'-RACEs, suggesting quite low expression. Moreover, D-arginine, an ADC inhibitor, caused a decrease in ADC activity and severely inhibited the growth of apple callus, which could be partially resumed by exogenous addition of putrescine, whereas alpha-difluoromethylornithine (DFMO), an inhibitor for ODC, caused the incomplete repression of callus growth without changing ODC activity. RNA gel blot showed that the expression level of MdADC was high in young tissues/organs with rapid cell division and was positively induced by chilling, salt, and dehydration, implying its involvement in both cell growth and these stress responses. By contrast, the transcript of ODC could not be detected by RNA gel blot analysis. Based on the present study, it is possible to conclude that (i) the ODC pathway is active in apple, although the expression level of the pMdODC gene homologous with its counterparts found in other plant species is quite low; and (ii) MdADC expression correlates with cell growth and stress responses to chilling, salt, and dehydration, suggesting that ADC is a primary biosynthetic pathway for putrescine biosynthesis in apple.

  7. Crystal structures of the wild-type, P1A mutant, and inactivated malonate semialdehyde decarboxylase: a structural basis for the decarboxylase and hydratase activities.

    PubMed

    Almrud, Jeffrey J; Poelarends, Gerrit J; Johnson, William H; Serrano, Hector; Hackert, Marvin L; Whitman, Christian P

    2005-11-15

    Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 is a tautomerase superfamily member that converts malonate semialdehyde to acetaldehyde by a mechanism utilizing Pro-1 and Arg-75. Pro-1 and Arg-75 have also been implicated in the hydratase activity of MSAD in which 2-oxo-3-pentynoate is processed to acetopyruvate. Crystal structures of MSAD (1.8 A resolution), the P1A mutant of MSAD (2.7 A resolution), and MSAD inactivated by 3-chloropropiolate (1.6 A resolution), a mechanism-based inhibitor activated by the hydratase activity of MSAD, have been determined. A comparison of the P1A-MSAD and MSAD structures reveals little geometric alteration, indicating that Pro-1 plays an important catalytic role but not a critical structural role. The structures of wild-type MSAD and MSAD covalently modified at Pro-1 by 3-oxopropanoate, the adduct resulting from the incubation of MSAD and 3-chloropropiolate, implicate Asp-37 as the residue that activates a water molecule for attack at C-3 of 3-chloropropiolate to initiate a Michael addition of water. The interactions of Arg-73 and Arg-75 with the C-1 carboxylate group of the adduct suggest these residues polarize the alpha,beta-unsaturated acid and facilitate the addition of water. On the basis of these structures, a mechanism for the inactivation of MSAD by 3-chloropropiolate can be formulated along with mechanisms for the decarboxylase and hydratase activities. The results also provide additional evidence supporting the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, a tautomerase superfamily member preceding MSAD in the trans-1,3-dichloropropene degradation pathway, diverged from a common ancestor but retained the key elements for the conjugate addition of water.

  8. Assaying Ornithine and Arginine Decarboxylases in Some Plant Species 1

    PubMed Central

    Birecka, Helena; Bitonti, Alan J.; McCann, Peter P.

    1985-01-01

    A release of 14CO2 not related to ornithine decarboxylase activity was found in crude leaf extracts from Lycopersicon esculentum, Avena sativa, and especially from the pyrrolizidine alkaloid-bearing Heliotropium angiospermum when incubated with [1-14C]- or [U-14C]ornithine. The total 14CO2 produced was about 5- to 100-fold higher than that due to ornithine decarboxylase activities calculated from labeled putrescine (Put) found by thin-layer electrophoresis in the incubation mixtures. Partial purification with (NH4)2SO4 did not eliminate completely the interfering decarboxylation. When incubated with labeled arginine, a very significant 14CO2 release not related to arginine decarboxylase activity was observed only in extracts from H. angiospermum leaves, especially in Tris·HCl buffer. Under the assay conditions, these extracts exhibited oxidative degradation of added Put and agmatine (Agm) and also revealed a high arginase activity. Amino-guanidine at 0.1 to 0.2 millimolar prevented Put degradation and greatly decreased oxidative degradation of Agm; ornithine at 15 to 20 millimolar significantly inhibited arginase activity. A verification of the reliability of the standard 14CO2-based method by assessing labeled Put and/or Agm—formed in the presence of added aminoguanidine and/or ornithine when needed—is recommended especially when crude or semicrude plant extracts are assayed. When based on Put and/or Agm formed at 1.0 to 2.5 millimolar of substrate, the activities of ornithine decarboxylase and arginine decarboxylase in the youngest leaves of the tested species ranged between 1.1 and 3.6 and 1 and 1600 nanomoles per hour per gram fresh weight, respectively. The enzyme activities are discussed in relation to the biosynthesis of pyrrolizidine alkaloids. PMID:16664441

  9. Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes.

    PubMed

    Lee, Won-Heong; Seo, Seung-Oh; Bae, Yi-Hyun; Nan, Hong; Jin, Yong-Su; Seo, Jin-Ho

    2012-11-01

    Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93 mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151 mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.

  10. The hydratase activity of malonate semialdehyde decarboxylase: mechanistic and evolutionary implications.

    PubMed

    Poelarends, Gerrit J; Serrano, Hector; Johnson, William H; Hoffman, David W; Whitman, Christian P

    2004-12-01

    Malonate semialdehyde decarboxylase (MSAD) is a member of the tautomerase superfamily, a group of structurally homologous proteins that have a characteristic beta-alpha-beta-fold and a catalytic amino-terminal proline. In addition to its physiological decarboxylase activity, the conversion of malonate semialdehyde to acetaldehyde and carbon dioxide, the enzyme has now been found to display a promiscuous hydratase activity, converting 2-oxo-3-pentynoate to acetopyruvate, with a kcat/Km value of 6.0 x 102 M-1 s-1. Pro-1 and Arg-75 are critical for both activities, and the pKa of Pro-1 was determined to be approximately 9.2 by a direct 15N NMR titration. These observations implicate a decarboxylation mechanism in which Pro-1 polarizes the carbonyl oxygen of substrate by hydrogen bonding and/or an electrostatic interaction. Arg-75 may position the carboxylate group into a favorable orientation for decarboxylation. Both the hydratase activity and the pKa value of Pro-1 are shared with trans-3-chloroacrylic acid dehalogenase, another tautomerase superfamily member that precedes MSAD in a bacterial degradation pathway for trans-1,3-dichloropropene. Hence, MSAD and CaaD could have evolved by divergent evolution from a common ancestral protein, retaining the necessary catalytic components for the conjugate addition of water.

  11. 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

  12. A second 5-carboxyvanillate decarboxylase gene, ligW2, is important for lignin-related biphenyl catabolism in Sphingomonas paucimobilis SYK-6.

    PubMed

    Peng, Xue; Masai, Eiji; Kasai, Daisuke; Miyauchi, Keisuke; Katayama, Yoshihiro; Fukuda, Masao

    2005-09-01

    A lignin-related biphenyl compound, 5,5'-dehydrodivanillate (DDVA), is degraded to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by DDVA O-demethylase (LigX), meta-cleavage oxygenase (LigZ), and meta-cleavage compound hydrolase (LigY) in Sphingomonas paucimobilis SYK-6. 5CVA is then transformed to vanillate by a nonoxidative 5CVA decarboxylase and is further degraded through the protocatechuate 4,5-cleavage pathway. A 5CVA decarboxylase gene, ligW, was isolated from SYK-6 (X. Peng, E. Masai, H. Kitayama, K. Harada, Y, Katayama, and M. Fukuda, Appl. Environ. Microbiol. 68:4407-4415, 2002). However, disruption of ligW slightly affected the 5CVA decarboxylase activity and the growth rate on DDVA of the mutant, suggesting the presence of an alternative 5CVA decarboxylase gene. Here we isolated a second 5CVA decarboxylase gene, ligW2, which consists of a 1,050-bp open reading frame encoding a polypeptide with a molecular mass of 39,379 Da. The deduced amino acid sequence encoded by ligW2 exhibits 37% identity with the sequence encoded by ligW. Based on a gas chromatography-mass spectrometry analysis of the reaction product from 5CVA catalyzed by LigW2 in the presence of deuterium oxide, LigW2 was indicated to be a nonoxidative decarboxylase of 5CVA, like LigW. After disruption of ligW2, both the growth rate on DDVA and the 5CVA decarboxylase activity of the mutant were decreased to approximately 30% of the wild-type levels. The ligW ligW2 double mutant lost both the ability to grow on DDVA and the 5CVA decarboxylase activity. These results indicate that both ligW and ligW2 contribute to 5CVA degradation, although ligW2 plays the more important role in the growth of SYK-6 cells on DDVA.

  13. Analysis of Mammalian Histidine Decarboxylase Dimerization Interface Reveals an Electrostatic Hotspot Important for Catalytic Site Topology and Function.

    PubMed

    Moya-García, Aurelio A; Rodríguez-Agudo, Daniel; Hayashi, Hideyuki; Medina, Miguel Angel; Urdiales, José Luis; Sánchez-Jiménez, Francisca

    2011-06-14

    Selective intervention of mammalian histidine decarboxylase (EC 4.1.1.22) could provide a useful antihistaminic strategy against many different pathologies. It is known that global conformational changes must occur during reaction that involves the monomer-monomer interface of the enzyme. Thus, the dimerization surface is a promising target for histidine decarboxylase inhibition. In this work, a rat apoenzyme structural model is used to analyze the interface of the dimeric active HDC. The dimerization surface mainly involves the fragments 1-213 and 308-371 from both subunits. Part of the overlapping surfaces conforms each catalytic site entrance and the substrate-binding sites. In addition, a cluster of charged residues is located in each overlapping surface, so that both electrostatic hotspots mediate in the interaction between the catalytic sites of the dimeric enzyme. It is experimentally demonstrated that the carboxyl group of aspartate 315 is critical for the proper conformation of the holoenzyme and the progression of the reaction. Comparison to the available information on other evolutionary related enzymes also provides new insights for characterization and intervention of homologous l-amino acid decarboxylases. PMID:26596454

  14. Increased Putrescine Biosynthesis through Transfer of Mouse Ornithine Decarboxylase cDNA in Carrot Promotes Somatic Embryogenesis.

    PubMed Central

    Bastola, D. R.; Minocha, S. C.

    1995-01-01

    Carrot (Daucus carota L.) cells were transformed with Agrobacterium tumefaciens strains containing 3[prime]-truncated mouse ornithine decarboxylase (ODC) cDNA under the control of a cauliflower mosaic virus 35S promoter. A neomycin phosphotransferase gene linked with a nopaline synthase promoter was used to select transformed cell lines on kanamycin. Although the nontransformed cells contained no ODC, high amounts of mouse-specific ODC activity were observed in the transformed cells. Transgenic cells showed a significant increase in the cellular content of putrescine compared to control cells. Spermidine, however, remained unaffected. Not only did the transformed cells exhibit improved somatic embryogenesis in the auxin-free medium, they also regenerated some embryos in the presence of inhibitory concentrations of 2,4-dichlorophenoxyacetic acid. These cells acquired tolerance to [alpha]-difluoromethylarginine (a potent inhibitor of arginine decarboxylase) at concentrations that inhibit growth as well as embryogenesis in nontransformed carrot cells, showing that the mouse ODC can replace the carrot arginine decarboxylase for putrescine biosynthesis in the transgenic cells. PMID:12228581

  15. Bacterial Lysine Decarboxylase Influences Human Dental Biofilm Lysine Content, Biofilm Accumulation and Sub-Clinical Gingival Inflammation

    PubMed Central

    Lohinai, Z.; Keremi, B.; Szoko, E.; Tabi, T.; Szabo, C.; Tulassay, Z.; Levine, M.

    2012-01-01

    Background Dental biofilms contain a protein that inhibits mammalian cell growth, possibly lysine decarboxylase from Eikenella corrodens. This enzyme decarboxylates lysine, an essential amino acid for dentally attached cell turnover in gingival sulci. Lysine depletion may stop this turnover, impairing the barrier to bacterial compounds. The aims of this study were to determine biofilm lysine and cadaverine contents before oral hygiene restriction (OHR), and their association with plaque index (PI) and gingival crevicular fluid (GCF) after OHR for a week. Methods Laser-induced fluorescence after capillary electrophoresis was used to determine lysine and cadaverine contents in dental biofilm, tongue biofilm and saliva before OHR and in dental biofilm after OHR. Results Before OHR, lysine and cadaverine contents of dental biofilm were similar and 10-fold greater than in saliva or tongue biofilm. After a week of OHR, the biofilm content of cadaverine increased and that of lysine decreased, consistent with greater biofilm lysine decarboxylase activity. Regression indicated that PI and GCF exudation were positively related to biofilm lysine post-OHR, unless biofilm lysine exceeded the minimal blood plasma content in which case PI was further increased but GCF exudation was reduced. Conclusions After OHR, lysine decarboxylase activity seems to determine biofilm lysine content and biofilm accumulation. When biofilm lysine exceeds minimal blood plasma content after OHR, less GCF appeared despite more biofilm. Lysine appears important for biofilm accumulation and the epithelial barrier to bacterial proinflammatory agents. Clinical Relevance Inhibiting lysine decarboxylase may retard the increased GCF exudation required for microbial development and gingivitis. PMID:22141361

  16. 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.

  17. [Simultaneous demonstration of glutamate decarboxylase and synaptophysin in paraffin sections of rat cerebellum].

    PubMed

    Korzhevskiy, D E; Gilerovich, Ye G; Kirik, O V; Alekseyeva, O S; Grigoriyev, I P

    2015-01-01

    The article presents highly reproducible and inexpensive protocol for simultaneous demonstration of glutamate decarboxylase (GAD67), the key enzyme of gamma-aminobutyric acid (GABA) synthesis and synaptophysin (SYP), a marker protein of synaptic vesicles using confocal laser microscopy. In the cerebellar cortex, GAD labels Purkinje cells and pinceaux in their basal parts and is unevenly distributed in the neuropil of molecular and granular layers. SYP clearly marks the contours of large dendrites of Purkinje cells in molecular layer, while in the granular layers it labels parts of cerebellar glomeruli--the terminals of the mossy fibers. GAD-immunopositive structures (GABA-ergic axons of stellate cells--Golgi cells) are often located at periphery of the glomeruli. In the peripheral zone of the glomeruli, colocalization of GAD- and SYP-immunopositive structures was observed, suggesting the presence of GABA-ergic synapses in this zone.

  18. Molecular cloning and sequence analysis of the cDNA encoding rat liver cysteine sulfinate decarboxylase (CSD).

    PubMed

    Reymond, I; Sergeant, A; Tappaz, M

    1996-06-01

    The taurine biosynthesis enzyme, cysteine sulfinate decarboxylase (CSD), was purified to homogeneity from rat liver. Three CSD peptides generated by tryptic cleavage were isolated and partially sequenced. Two of them showed a marked homology with glutamate decarboxylase and their respective position on the CSD amino acid sequence was postulated accordingly. Using appropriate degenerated primers derived from these two peptides, a PCR amplified DNA fragment was generated from liver poly(A)+ mRNA, cloned and used as a probe to screen a rat liver cDNA library. Three cDNAs, length around 1800 bp, were isolated which all contained an open reading frame (ORF) encoding a 493 amino acid protein with a calculated molecular mass of 55.2 kDa close to the experimental values for CSD. The encoded protein contained the sequence of the three peptides isolated from homogenous liver CSD. Our data confirm and significantly extend those recently published (Kaisaki et al. (1995) Biochim. Biophys. Acta 1262, 79-82). Indeed, an additional base pair found 1371 bp downstream from the initiation codon led to a shift in the open reading frame which extended the carboxy-terminal end by 15 amino acid residues and altogether modified 36 amino acids. The validity of this correction is supported by the finding that the corrected reading frame encoded a peptide issued from CSD tryptic cleavage that was not encoded anywhere in the CSD sequence previously reported. PMID:8679699

  19. 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

  20. Pantothenic acid biosynthesis in zymomonas

    SciTech Connect

    Tao, Luan; Tomb, Jean-Francois; Viitanen, Paul V.

    2014-07-01

    Zymomonas is unable to synthesize pantothenic acid and requires this essential vitamin in growth medium. Zymomonas strains transformed with an operon for expression of 2-dehydropantoate reductase and aspartate 1-decarboxylase were able to grow in medium lacking pantothenic acid. These strains may be used for ethanol production without pantothenic acid supplementation in seed culture and fermentation media.

  1. Inactivation of malonate semialdehyde decarboxylase by 3-halopropiolates: evidence for hydratase activity.

    PubMed

    Poelarends, Gerrit J; Serrano, Hector; Johnson, William H; Whitman, Christian P

    2005-07-01

    Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 catalyzes the metal ion-independent decarboxylation of malonate semialdehyde and represents one of three known enzymatic activities in the tautomerase superfamily. The characterized members of this superfamily are structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. Sequence analysis, chemical labeling studies, site-directed mutagenesis, and NMR studies of MSAD identified Pro-1 as a key active site residue in which the amino group has a pKa value of 9.2. The available evidence suggests a mechanism involving polarization of the C-3 carbonyl group of malonate semialdehyde by the cationic Pro-1. A second critical active site residue, Arg-75, could assist in the reaction by placing the substrate's carboxylate group in a favorable conformation for decarboxylation. In addition to the decarboxylase activity, MSAD has a hydratase activity as demonstrated by the MSAD-catalyzed conversion of 2-oxo-3-pentynoate to acetopyruvate. In view of this activity, MSAD was incubated with 3-bromo- and 3-chloropropiolate, and the subsequent reactions were characterized. Both compounds result in the irreversible inactivation of MSAD, making them the first identified inhibitors of MSAD. Inactivation by 3-chloropropiolate occurs in a time- and concentration-dependent manner and is due to the covalent modification of Pro-1. The proposed mechanism for inactivation involves the initial hydration of the 3-halopropiolate followed by a rearrangement to an alkylating agent, either an acyl halide or a ketene. The results provide additional evidence for the hydratase activity of MSAD and further support for the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, the preceding enzyme in the trans-1,3-dichloropropene catabolic pathway, diverged from a common ancestor but conserved the necessary catalytic machinery for the conjugate addition of water.

  2. Decarboxylases involved in polyamine biosynthesis and their inactivation by nitric oxide.

    PubMed

    Hillary, Rebecca A; Pegg, Anthony E

    2003-04-11

    Polyamines are ubiquitous cellular components that are involved in normal and neoplastic growth. Polyamine biosynthesis is very highly regulated in mammalian cells by the activities of two key decarboxylases acting on ornithine and S-adenosylmethionine. Recent studies, which include crystallographic analysis of the recombinant human proteins, have provided a detailed knowledge of their structure and function. Ornithine decarboxylase is a PLP-requiring decarboxylase, whereas S-adenosylmethionine decarboxylase (AdoMetDC) contains a covalently bound pyruvate prosthetic group. Both enzymes have a key cysteine residue, which is involved in protonation of the Schiff base intermediate C(alpha) to form the product. These residues, Cys360 in ornithine decarboxylase (ODC) and Cys82 in AdoMetDC, react readily with nitric oxide (NO), which is therefore a potent inactivator of polyamine synthesis. The inactivation of these enzymes may mediate some of the antiproliferative actions of NO.

  3. 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. PMID:2760634

  4. 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.

  5. Two isoforms of glutamate decarboxylase in Arabidopsis are regulated by calcium/calmodulin and differ in organ distribution.

    PubMed

    Zik, M; Arazi, T; Snedden, W A; Fromm, H

    1998-08-01

    The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [35S] calmodulin (CaM) in the presence of calcium, and a region of 30-32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflorescence stems and flowers. Our study provides the first evidence for the occurrence of multiple functional Ca2+/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis GAD activity involves modulation of isoform-specific gene expression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.

  6. Crystal structure of pyruvate decarboxylase from Zymobacter palmae.

    PubMed

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

    2016-09-01

    Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg(2+) 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 Rr.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 Rwork = 0.186 (0.271 in the highest resolution bin) and Rfree = 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

  7. 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

  8. Activities of Arginine and Ornithine Decarboxylases in Various Plant Species 1

    PubMed Central

    Birecka, Helena; Bitonti, Alan J.; McCann, Peter P.

    1985-01-01

    In extracts from the youngest leaves of Avena sativa, Hordeum vulgare, Zea Mays, Pisum sativum, Phaseolus vulgaris, Lactuca sativa, and four pyrrolizidine alkaloid-bearing species of Heliotropium, the activities of ornithine decarboxylase, close to Vmax, ranged between traces and 1.5 nanomoles per hour per gram fresh weight when based on putrescine formed during incubation with labeled ornithine. The arginine decarboxylase activities in the same extracts ranged between 8 and 8000 nanomoles per hour per gram fresh weight being lowest in the borages and highest in oat and barley. α-Difluoromethylornithine and α-difluoromethylarginine inhibited ornithine and arginine decarboxylases, respectively, in all species. Agmatine, putrescine, spermidine, and spermine were found in all, diaminopropane in eight, and cadaverine in three species. No correlation was observed between arginine or ornithine decarboxylase level and the levels of total polyamines. The in vitro decarboxylase activities found in the borages cannot explain the high accumulation of putrescine-derived pyrrolizidines in their youngest leaves if the pyrrolizidines are produced in situ from arginine and/or ornithine as precursors; other possibilities are discussed. In assays of ornithine decarboxylase, an interference of decarboxylation not due to this enzyme was observed in extracts from all species. In arginine decarboxylase assays, the interfering decarboxylation as well as the interference of arginase were apparent in two species. Addition of aminoguanidine was needed to suppress oxidative degradation of putrescine and agmatine during incubation of extracts from pea, bean, lettuce, Heliotropium angiospermum, and Heliotropium indicum. PMID:16664442

  9. Expression and localization of cysteine sulfinate decarboxylase in major salivary glands of male mice.

    PubMed

    Liu, Shengnan; Liu, Ying; Ma, Qiwang; Cui, Sheng; Liu, Jiali

    2015-04-01

    Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in mammalian cells. It plays a significant role in cell development, nutrition, and survival, such as in the regulation of ion transport and osmoregulation. Cysteine sulfinate decarboxylase (CSD) is the rate-limiting biosynthetic enzyme of taurine. Recently, the synthesis of taurine has been observed in the central nervous system, kidney, liver, and muscle. However, the synthesis of taurine in the salivary glands has still not been described in detail. We have detected CSD expression in the major salivary glands of adult male mice by real-time polymerase chain reaction (RT-PCR), Western blot, and immunofluorescence. In addition, we determined the content of taurine by high-performance liquid chromatography (HPLC). The results show that taurine is present in high concentrations in the major salivary glands of male mice. CSD messenger RNA (mRNA) and protein are expressed in the major salivary glands of male mice. The relative levels of CSD mRNA increase from the submandibular gland (SMG) to the sublingual gland (SLG) and parotid gland (PG), but the levels of the CSD protein are the opposite. The immunofluorescence results indicate that CSD is mainly located in the excretory ducts (EDs) and interlobular duct (IL) of SMG and ED in SLG, respectively. These results suggest that the major salivary glands of male mice produce taurine through the CSD pathway, and the synthesis of taurine might be related to sodium reabsorption in the salivary glands. PMID:25645459

  10. 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.

  11. The Bifunctional Pyruvate Decarboxylase/Pyruvate Ferredoxin Oxidoreductase from Thermococcus guaymasensis

    PubMed Central

    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. PMID:24982594

  12. 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.

  13. Antiinflammatory drug effects on ultraviolet light-induced epidermal ornithine decarboxylase and DNA synthesis

    SciTech Connect

    Lowe, N.J.; Breeding, J.

    1980-06-01

    Epidermal ornithine decarboxylase activity is greatly elevated in response to tumor promoting agents and ultraviolet light. The purpose of this paper is to report modification of ultraviolet-induced epidermal ornithine decarboxylase activity by antiinflammatory agents. Topical triamoinolone acetonide and indomethacin were found to significantly inhibit the UV-B induction of epidermal ornithine decarboxylase in hairless mice when applied following ultraviolet light irradiation. The corticosteroid also showed inhibition of ultraviolet light increased epidermal DNA synthesis. Indomethacin failed to show any inhibition of DNA synthesis.

  14. Structural features of mammalian histidine decarboxylase reveal the basis for specific inhibition

    PubMed Central

    Moya-García, AA; Pino-Ángeles, A; Gil-Redondo, R; Morreale, A; Sánchez-Jiménez, F

    2009-01-01

    For a long time the structural and molecular features of mammalian histidine decarboxylase (EC 4.1.1.22), the enzyme that produces histamine, have evaded characterization. We overcome the experimental problems for the study of this enzyme by using a computer-based modelling and simulation approach, and have now the conditions to use histidine decarboxylase as a target in histamine pharmacology. In this review, we present the recent (last 5 years) advances in the structure–function relationship of histidine decarboxylase and the strategy for the discovery of new drugs. PMID:19413567

  15. Structure of the Homodimeric Glycine Decarboxylase P-protein from Synechocystis sp. PCC 6803 Suggests a Mechanism for Redox Regulation*

    PubMed Central

    Hasse, Dirk; Andersson, Evalena; Carlsson, Gunilla; Masloboy, Axel; Hagemann, Martin; Bauwe, Hermann; Andersson, Inger

    2013-01-01

    Glycine decarboxylase, or P-protein, is a pyridoxal 5′-phosphate (PLP)-dependent enzyme in one-carbon metabolism of all organisms, in the glycine and serine catabolism of vertebrates, and in the photorespiratory pathway of oxygenic phototrophs. P-protein from the cyanobacterium Synechocystis sp. PCC 6803 is an α2 homodimer with high homology to eukaryotic P-proteins. The crystal structure of the apoenzyme shows the C terminus locked in a closed conformation by a disulfide bond between Cys972 in the C terminus and Cys353 located in the active site. The presence of the disulfide bridge isolates the active site from solvent and hinders the binding of PLP and glycine in the active site. Variants produced by substitution of Cys972 and Cys353 by Ser using site-directed mutagenesis have distinctly lower specific activities, supporting the crucial role of these highly conserved redox-sensitive amino acid residues for P-protein activity. Reduction of the 353–972 disulfide releases the C terminus and allows access to the active site. PLP and the substrate glycine bind in the active site of this reduced enzyme and appear to cause further conformational changes involving a flexible surface loop. The observation of the disulfide bond that acts to stabilize the closed form suggests a molecular mechanism for the redox-dependent activation of glycine decarboxylase observed earlier. PMID:24121504

  16. Cloning and expression of pig kidney dopa decarboxylase: comparison of the naturally occurring and recombinant enzymes.

    PubMed Central

    Moore, P S; Dominici, P; Borri Voltattorni, C

    1996-01-01

    L-Aromatic amino acid decarboxylase (dopa decarboxylase; DDC) is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme that catalyses the decarboxylation of L-dopa and other L-aromatic amino acids. To advance structure-function studies with the enzyme, a cDNA that codes for the protein from pig kidney has been cloned by joining a partial cDNA obtained by library screening with a synthetic portion constructed by the annealing and extension of long oligonucleotides. The hybrid cDNA was then expressed in Escherichia coli to produce recombinant protein. During characterization of the recombinant enzyme it was unexpectedly observed that it possesses certain differences from the enzyme purified from pig kidney. Whereas the later protein binds 1 molecule of PLP per dimer, the recombinant enzyme was found to bind two molecules of coenzyme per dimer. Moreover, the Vmax was twice that of the protein purified from tissue. On addition of substrate, the absorbance changes accompanying transaldimination were likewise 2-fold greater in the recombinant enzyme. Examination of the respective apoenzymes by absorbance, CD and fluorescence spectroscopy revealed distinct differences. The recombinant apoprotein has no significant absorbance at 335 nm, unlike the pig kidney apoenzyme; in the latter case this residual absorbance is associated with a positive dichroic signal. When excited at 335 nm the pig kidney apoenzyme has a pronounced emission maximum at 385 nm, in contrast with its recombinant counterpart, which shows a weak broad emission at about 400 nm. However, the holoenzyme-apoenzyme transition did not markedly alter the respective fluorescence properties of either recombinant or pig kidney DDC when excited at 335 nm. Taken together, these findings indicate that recombinant pig kidney DDC has two active-site PLP molecules and therefore displays structural characteristics typical of PLP-dependent homodimeric enzymes. The natural enzyme contains one active-site PLP molecule

  17. The Saccharomyces cerevisiae mevalonate diphosphate decarboxylase is essential for viability, and a single Leu-to-Pro mutation in a conserved sequence leads to thermosensitivity.

    PubMed Central

    Bergès, T; Guyonnet, D; Karst, F

    1997-01-01

    The mevalonate diphosphate decarboxylase is an enzyme which converts mevalonate diphosphate to isopentenyl diphosphate, the building block of isoprenoids. We used the Saccharomyces cerevisiae temperature-sensitive mutant defective for mevalonate diphosphate decarboxylase previously described (C. Chambon, V. Ladeveve, M. Servouse, L. Blanchard, C. Javelot, B. Vladescu, and F. Karst, Lipids 26:633-636, 1991) to characterize the mutated allele. We showed that a single change in a conserved amino acid accounts for the temperature-sensitive phenotype of the mutant. Complementation experiments were done both in the erg19-mutated background and in a strain in which the ERG19 gene, which was shown to be an essential gene for yeast, was disrupted. Epitope tagging of the wild-type mevalonate diphosphate decarboxylase allowed us to isolate the enzyme in an active form by a versatile one-step immunoprecipitation procedure. Furthermore, during the course of this study, we observed that a high level of expression of the wild-type ERG19 gene led to a lower sterol steady-state accumulation compared to that of a wild-type strain, suggesting that this enzyme may be a key enzyme in mevalonate pathway regulation. PMID:9244250

  18. 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.

  19. 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.

  20. The electrochemical investigation of the catalytic power of pyruvate decarboxylase and its coenzyme.

    PubMed

    Bell, Patrick; Hoyt, Kathryn; Shabangi, Masangu

    2006-05-01

    The change in the energy barriers for the heterogeneous reduction of pyruvate decarboxylase (PDC) relative to its coenzyme, thiamin pyrophosphate (ThPP), was determined experimentally using square wave voltammetry (SWV) to be 5.3 kcal/mol. These results are in agreement with those of reaction rate acceleration provided by thiamin-dependent decarboxylases relative to their coenzyme as determined kinetically based on the pK(a) suppression by the enzyme environment.

  1. Characterization of Glutamate Decarboxylase (GAD) from Lactobacillus sakei A156 Isolated from Jeot-gal.

    PubMed

    Sa, Hyun Deok; Park, Ji Yeong; Jeong, Seon-Ju; Lee, Kang Wook; Kim, Jeong Hwan

    2015-05-01

    A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from jeot-gal (anchovy), a Korean fermented seafood. The isolate, A156, produced GABA profusely when incubated in MRS broth with monosodium glutamate (3% (w/v)) at 37°C for 48 h. A156 was identified as Lactobacillus sakei by 16S rRNA gene sequencing. The GABA conversion yield was 86% as determined by GABase enzyme assay. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB. The operon structure of gadCB was confirmed by RT-PCR. gadB was overexpressed in Escherichia coli BL21(DE3) and recombinant GAD was purified. The purified GAD was 54.4 kDa in size by SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 55°C and the activity was dependent on pyridoxal 5'-phosphate. The Km and Vmax of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.

  2. Mutational Analysis of Substrate Interactions with the Active Site of Dialkylglycine Decarboxylase

    PubMed Central

    Fogle, Emily J.; Toney, Michael D.

    2010-01-01

    Pyridoxal phosphate (PLP) dependent enzymes catalyze many different types of reactions at the α-, β-, and γ-carbons of amine and amino acid substrates. Dialkylglycine decarboxylase (DGD) is an unusual PLP dependent enzyme that catalyzes two reaction types, decarboxylation and transamination, in the same active site. A structurally-based, functional model has been proposed for the DGD active site, which maintains that R406 is important in determining substrate specificity through interactions with the substrate carboxylate while W138 provides specificity for short-chain alkyl groups. The mechanistic roles of R406 and W138 were investigated using site directed mutagenesis, alternate substrates, and analysis of steady-state and half-reaction kinetics. Experiments on the R406M and R406K mutants confirm the importance of R406 in substrate binding. Surprisingly, this work also shows that the positive charge of R406 facilitates catalysis of decarboxylation. The W138F mutant demonstrates that W138 indeed acts to limit the size of the subsite C binding pocket, determining specificity for 2,2-dialkylglycines with small side chains as predicted by the model. Finally, work with the double mutant W138F/M141R shows that these mutations expand substrate specificity to include L-glutamate and lead to an increase in specificity for L-glutamate over 2-aminoisobutyrate of approximately eight orders of magnitude compared to WT DGD. PMID:20540501

  3. Deletion of pyruvate decarboxylase by a new method for efficient markerless gene deletions in Gluconobacter oxydans.

    PubMed

    Peters, Björn; Junker, Anja; Brauer, Katharina; Mühlthaler, Bernadette; Kostner, David; Mientus, Markus; Liebl, Wolfgang; Ehrenreich, Armin

    2013-03-01

    Gluconobacter oxydans, a biotechnologically relevant species which incompletely oxidizes a large variety of carbohydrates, alcohols, and related compounds, contains a gene for pyruvate decarboxylase (PDC). This enzyme is found only in very few species of bacteria where it is normally involved in anaerobic ethanol formation via acetaldehyde. In order to clarify the role of PDC in the strictly oxidative metabolism of acetic acid bacteria, we developed a markerless in-frame deletion system for strain G. oxydans 621H which uses 5-fluorouracil together with a plasmid-encoded uracil phosphoribosyltransferase as counter selection method and used this technique to delete the PDC gene (GOX1081) of G. oxydans 621H. The PDC deletion mutant accumulated large amounts of pyruvate but almost no acetate during growth on D-mannitol, D-fructose or in the presence of L-lactate. This suggested that in G. oxydans acetate formation occurs by decarboxylation of pyruvate and subsequent oxidation of acetaldehyde to acetate. This observation and the efficiency of the markerless deletion system were confirmed by constructing deletion mutants of two acetaldehyde dehydrogenases (GOX1122 and GOX2018) and of the acetyl-CoA-synthetase (GOX0412). Acetate formation during growth of these mutants on mannitol did not differ significantly from the wild-type strain.

  4. Design of inhibitors of orotidine monophosphate decarboxylase using bioisosteric replacement and determination of inhibition kinetics.

    PubMed

    Poduch, Ewa; Bello, Angelica M; Tang, Sishi; Fujihashi, Masahiro; Pai, Emil F; Kotra, Lakshmi P

    2006-08-10

    Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (k(cat) = 21 s(-1) and KM = 5 microM) and the molar enthalpy (DeltaH(app) = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (Ki) were determined to be 12.4 microM and 29 microM for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.

  5. Molecular characterization of Mtb-OMP decarboxylase by modeling, docking and dynamic studies.

    PubMed

    Madhusudana, P; Babajan, B; Chaitanya, M; Anuradha, C M; Shobharani, C; Chikati, Rajasekar; Kumar, Chitta Suresh; Rao, K R S Sambasiva; Poda, Sudhakar

    2012-06-01

    Tuberculosis (TB), the second most deadly disease in the world is caused by Mycobacterium tuberculosis (Mtb). In the present work a unique enzyme of Mtb orotidine 5' monophosphate decarboxylase (Mtb-OMP Decase) is selected as drug target due to its indispensible role in biosynthesis of pyrimidines. The present work is focused on understanding the structural and functional aspects of Mtb-OMP Decase at molecular level. Due to absence of crystal structure, the 3D structure of Mtb-OMP Decase was predicted by MODELLER9V7 using a known structural template 3L52. Energy minimization and refinement of the developed 3D model was carried out with Gromacs 3.2.1 and the optimized homology model was validated by PROCHECK,WHAT-IF and PROSA2003. Further, the surface active site amino acids were quantified by WHAT-IF pocket. The exact binding interactions of the ligands, 6-idiouridine 5' monophosphate and its designed analogues with the receptor Mtb-OMP Decase were predicted by docking analysis with AUTODOCK 4.0. This would be helpful in understanding the blockade mechanism of OMP Decase and provide a candidate lead for the discovery of Mtb-OMP Decase inhibitors, which may bring insights into outcome new therapy to treat drug resistant Mtb.

  6. 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.

  7. Identification of malic and soluble oxaloacetate decarboxylase enzymes in Enterococcus faecalis.

    PubMed

    Espariz, Martín; Repizo, Guillermo; Blancato, Víctor; Mortera, Pablo; Alarcón, Sergio; Magni, Christian

    2011-06-01

    Two paralogous genes, maeE and citM, that encode putative malic enzyme family members were identified in the Enterococcus faecalis genome. MaeE (41 kDa) and CitM (42 kDa) share a high degree of homology between them (47% identities and 68% conservative substitutions). However, the genetic context of each gene suggested that maeE is associated with malate utilization whereas citM is linked to the citrate fermentation pathway. In the present work, we focus on the biochemical characterization and physiological contribution of these enzymes in E. faecalis. With this aim, the recombinant versions of the two proteins were expressed in Escherichia coli, affinity purified and finally their kinetic parameters were determined. This approach allowed us to establish that MaeE is a malate oxidative decarboxylating enzyme and CitM is a soluble oxaloacetate decarboxylase. Moreover, our genetic studies in E. faecalis showed that the citrate fermentation phenotype is not affected by citM deletion. On the other hand, maeE gene disruption resulted in a malate fermentation deficient strain indicating that MaeE is responsible for malate metabolism in E. faecalis. Lastly, it was demonstrated that malate fermentation in E. faecalis is associated with cytoplasmic and extracellular alkalinization which clearly contributes to pH homeostasis in neutral or mild acidic conditions. PMID:21518252

  8. 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

  9. 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.

  10. 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. PMID:27191596

  11. 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.

  12. Mechanistic characterization of a bacterial malonate semialdehyde decarboxylase: identification of a new activity on the tautomerase superfamily.

    PubMed

    Poelarends, Gerrit J; Johnson, William H; Murzin, Alexey G; Whitman, Christian P

    2003-12-01

    Malonate semialdehyde decarboxylase (MSAD) has been identified as the protein encoded by the orf130 gene from Pseudomonas pavonaceae 170 on the basis of the genomic context of the gene as well as its ability to catalyze the decarboxylation of malonate semialdehyde to generate acetaldehyde. The enzyme is found in a degradative pathway for the xenobiotic nematocide trans-1,3-dichloropropene. MSAD has no sequence homology to previously characterized decarboxylases, but the presence of a conserved motif (Pro1-(X)8 -Gly-Arg11-X-Asp-X-Gln) in its N-terminal region suggested a relationship to the tautomerase superfamily. Sequence analysis identified Pro1 and Arg75 as potential active site residues that might be involved in the MSAD activity. The results of site-directed mutagenesis experiments confirmed the importance of these residues to activity and provided further evidence to implicate MSAD as a new member of the tautomerase superfamily. MSAD is the first identified decarboxylase in the superfamily and is possibly the first characterized member of a new and distinct family within this superfamily. Malonate semialdehyde is analogous to a beta-keto acid, and enzymes that catalyze the decarboxylation of these acids generally utilize metal ion catalysis, a Schiff base intermediate, or polarization of the carbonyl group by hydrogen bonding and/or electrostatic interactions. A mechanistic analysis shows that the rate of the reaction is not affected by the presence of a metal ion or EDTA while the incubation of MSAD with the substrate in the presence of sodium cyanoborohydride results in the irreversible inactivation of the enzyme. The site of modification is Pro1. These observations are consistent with the latter two mechanisms, but do not exclude the first mechanism. Based on the sequence analysis, the outcome of the mutagenesis and mechanistic experiments, and the roles determined for Pro1 and the conserved arginine in all tautomerase superfamily members characterized

  13. Cloning and expression of the gene encoding alpha-acetolactate decarboxylase from Acetobacter aceti ssp. xylinum in brewer's yeast.

    PubMed

    Yamano, S; Tanaka, J; Inoue, T

    1994-02-14

    Acetobacter aceti ssp. xylinum genomic library was constructed using cosmid pJB8 in Escherichia coli. The gene encoding alpha-acetolactate decarboxylase (ALDC) was isolated from the library by direct measurement of ALDC activity. The ALDC gene was expressed by its own promoter in E. coli. The nucleotide sequence was determined, and an open reading frame which may encode a protein composed of 304 amino acids with a molecular weight of 33,747 was found. A brewer's yeast was transformed with the YEp-type plasmid containing the ALDC gene placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The laboratory-scale growth test confirmed that the total diacetyl concentration was considerably reduced by the transformant. The analysis of the wort indicates that the Acetobacter ALDC reduces the concentration of diacetyl more effectively than that of 2,3-pentanedione.

  14. The DOPA decarboxylase (DDC) gene is associated with alerting attention.

    PubMed

    Zhu, Bi; Chen, Chuansheng; Moyzis, Robert K; Dong, Qi; Chen, Chunhui; He, Qinghua; Li, Jin; Li, Jun; Lei, Xuemei; Lin, Chongde

    2013-06-01

    DOPA decarboxylase (DDC) is involved in the synthesis of dopamine, norepinephrine and serotonin. It has been suggested that genes involved in the dopamine, norepinephrine, and cholinergic systems play an essential role in the efficiency of human attention networks. Attention refers to the cognitive process of obtaining and maintaining the alert state, orienting to sensory events, and regulating the conflicts of thoughts and behavior. The present study tested seven single nucleotide polymorphisms (SNPs) within the DDC gene for association with attention, which was assessed by the Attention Network Test to detect three networks of attention, including alerting, orienting, and executive attention, in a healthy Han Chinese sample (N=451). Association analysis for individual SNPs indicated that four of the seven SNPs (rs3887825, rs7786398, rs10499695, and rs6969081) were significantly associated with alerting attention. Haplotype-based association analysis revealed that alerting was associated with the haplotype G-A-T for SNPs rs7786398-rs10499695-rs6969081. These associations remained significant after correcting for multiple testing by max(T) permutation. No association was found for orienting and executive attention. This study provides the first evidence for the involvement of the DDC gene in alerting attention. A better understanding of the genetic basis of distinct attention networks would allow us to develop more effective diagnosis, treatment, and prevention of deficient or underdeveloped alerting attention as well as its related prevalent neuropsychiatric disorders.

  15. 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.

  16. 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

  17. Amiloride inhibits rat mucosal ornithine decarboxylase activity and DNA synthesis

    SciTech Connect

    Ulrich-Baker, M.G.; Wang, P.; Fitzpatrick, L.; Johnson, L.R. )

    1988-03-01

    Refeeding fasted rats induces a dramatic trophic response in gastrointestinal mucosa and is associated with elevations in both rate of DNA synthesis and ornithine decarboxylase (ODC) activity. The signal for these increases is unknown. Amiloride prevents cell alkalinization by blocking Na{sup +}-H{sup +} exchange at apical epithelial cell membranes. In study 1, rats were fasted 48 h, treated with amiloride (0.5 to 500 mg/kg), and refed for 4 h. Refeeding increased ODC activities in the jejunal mucosa (X8) and liver (X19) but not in the oxyntic gland mucosa. In the jejunum, but not the liver, the activation of ODC was completely abolished by 100 mg/kg amiloride. In study 2, the rate of DNA synthesis was determine by measuring the rate of ({sup 3}H)thymidine incorporation 16 h after refeeding. Refeeding resulted in significantly increased rates of DNA synthesis over fasted levels, and amiloride at 100 mg/kg significantly reduced the elevations in the jejenum and liver. In conclusion, amiloride inhibits the postprandial increases in jejunal ODC activity and DNA synthesis in the jejunum and liver. The results indicate that (1) the Na{sup +}-H{sup +} antiport is essential to the increased ODC activity in the jejunum and liver after a meal and (2) increases in DNA synthesis and their suppression by amiloride are not necessary linked to ODC activity.

  18. 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.

  19. Localization of histidine decarboxylase mRNA in rat brain.

    PubMed

    Bayliss, D A; Wang, Y M; Zahnow, C A; Joseph, D R; Millhorn, D E

    1990-08-01

    The recent cloning of a cDNA encoding fetal rat liver histidine decarboxylase (HDC), the synthesizing enzyme for histamine, allows the study of the central histaminergic system at the molecular level. To this end, Northern blot and in situ hybridization analyses were used to determine the regional and cellular distribution of neurons which express HDC mRNA in rat brain. Three hybridizing species which migrate as 1.6-, 2.6-, and 3.5-kb RNA were identified with Northern blots. The major (2.6 kb) and minor (3.5 kb) species, characteristic of HDC mRNA in fetal liver, were expressed at high levels in diencephalon and at just detectable levels in hippocampus, but not in other brain regions. In contrast, the 1.6-kb species was present in all brain regions examined except the olfactory bulb. Cells which contain HDC mRNA were found by in situ hybridization in the hypothalamus; HDC mRNA-containing cells were not detected in other areas, including the hippocampus. Hypothalamic neurons which express HDC mRNA were localized to all aspects of the tuberomammillary nucleus, a result consistent with previous immunohistochemical findings. PMID:19912749

  20. Chloroform induction of ornithine decarboxylase activity in rats.

    PubMed Central

    Savage, R E; Westrich, C; Guion, C; Pereira, M A

    1982-01-01

    Chloroform is a drinking water contaminant that has been demonstrated to be carcinogenic to mice and rats resulting in an increased incidence of liver and kidney tumors, respectively. The mechanism of chloroform carcinogenicity might be by tumor initiation and/or promotion. Since induction of ornithine decarboxylase (ODC) activity has been proposed as a molecular marker for tumor promoters, we have investigated the effect of chloroform on ODC activity in rats. Chloroform induced a dose-dependent increase of hepatic ODC with an apparent threshold at 100 mg/kg body weight. Female rats were two to four times more susceptible to to chloroform. Upon daily dosing of chloroform for 7 days the liver became less susceptible, with the last dose of chloroform resulting in only 10% of the activity observed after a single dose. Nuclear RNA polymerase I activity was also induced by chloroform. Chloroform, rather than increasing the activity of renal ODC, resulted in a 35% reduction. The induction by chloroform of hepatic ODC activity might be associated with regenerative hyperplasia while the renal carcinogenicity of chloroform could not be demonstrated to be associated with ODC induction. PMID:7151757

  1. 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

  2. Characterization and translational regulation of the arginine decarboxylase gene in carnation (Dianthus caryophyllus L.).

    PubMed

    Chang, K S; Lee, S H; Hwang, S B; Park, K Y

    2000-10-01

    Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We characterized a carnation genomic clone, gDcADC8, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 77.7 kDa. The unusually long 5'-UTR that contained a short upstream open reading frame (uORF) of seven amino acids (MQKSLHI) was predicted to form an extensive secondary structure (free energy of approximately -117 kcal mol-1) using the Zuker m-fold algorithm. The result that an ADC antibody detected two bands of 45 and 33 kDa in a petal extract suggested the full length of the 78 kDa polypeptide precursor converted into two polypeptides in the processing reaction. To investigate the role of the transcript leader in translation, in vitro transcription/translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The ADC transcript leader affected positively downstream translation in both wheatgerm extract and primary transformant overexpressing ADC gene. It was demonstrated that heptapeptide (8.6 kDa) encoded by the ADC uORF was synthesized in vitro. Both uORF peptide, and the synthetic heptapeptide MQKSLHI of the uORF, repressed the translation of downstream ORF. Mutation of the uORF ATG codon alleviated the inhibitory effect. ORF translation was not affected by either a frame-shift mutation in uORF or a random peptide. To our knowledge, this is the first report to provide evidence that a uORF may inhibit the translation of a downstream ORF, not only in cis but also in trans, and that the leader sequence of the ADC gene is important for efficient translation.

  3. Insect ornithine decarboxylase (ODC) complements SPE1 knock-out of yeast Saccharomyces cerevisiae.

    PubMed

    Choi, Soon-Yong; Park, Hee Yun; Paek, Aron; Kim, Gil Seob; Jeong, Seong Eun

    2009-12-31

    Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines, which are essential for cell growth, differentiation, and proliferation. This report presents the characterization of an ODC-encoding cDNA (SlitODC) isolated from a moth species, the tobacco cutworm, Spodoptera litura (Lepidoptera); its expression in a polyamine-deficient strain of yeast, S. cerevisiae; and the recovery in polyamine levels and proliferation rate with the introduction of the insect enzyme. SlitODC encodes 448 amino acid residues, 4 amino acids longer than B. Mori ODC that has 71% identity, and has a longer C-terminus, consistent with B. mori ODC, than the reported dipteran enzymes. The null mutant yeast strain in the ODC gene, SPE1, showed remarkably depleted polyamine levels; in putrescine, spermidine, and spermine, the levels were > 7, > 1, and > 4%, respectively, of the levels in the wild-type strain. This consequently caused a significant arrest in cell proliferation of > 4% of the wild-type strain in polyaminefree media. The transformed strain, with the substituted SlitODC for the deleted endogenous ODC, grew and proliferated rapidly at even a higher rate than the wild-type strain. Furthermore, its polyamine content was significantly higher than even that in the wild-type strain as well as the spe1-null mutant, particularly with a very continuously enhanced putrescine level, reflecting no inhibition mechanism operating in the putrescine synthesis step by any corresponding insect ODC antizymes to SlitODC in this yeast system. PMID:19937472

  4. Structural Basis for Putrescine Activation of Human S-Adenosylmethionine Decarboxylase

    SciTech Connect

    Bale, Shridhar; Lopez, Maria M.; Makhatadze, George I.; Fang, Qingming; Pegg, Anthony E.; Ealick, Steven E.

    2009-01-23

    Putrescine (1,4-diaminobutane) activates the autoprocessing and decarboxylation reactions of human S-adenosylmethionine decarboxylase (AdoMetDC), a critical enzyme in the polyamine biosynthetic pathway. In human AdoMetDC, putrescine binds in a buried pocket containing acidic residues Asp174, Glu178, and Glu256. The pocket is away from the active site but near the dimer interface; however, a series of hydrophilic residues connect the putrescine binding site and the active site. Mutation of these acidic residues modulates the effects of putrescine. D174N, E178Q, and E256Q mutants were expressed and dialyzed to remove putrescine and studied biochemically using X-ray crystallography, UV-CD spectroscopy, analytical ultracentrifugation, and ITC binding studies. The results show that the binding of putrescine to the wild type dimeric protein is cooperative. The D174N mutant does not bind putrescine, and the E178Q and E256Q mutants bind putrescine weakly with no cooperativity. The crystal structure of the mutants with and without putrescine and their complexes with S-adenosylmethionine methyl ester were obtained. Binding of putrescine results in a reorganization of four aromatic residues (Phe285, Phe315, Tyr318, and Phe320) and a conformational change in the loop 312-320. The loop shields putrescine from the external solvent, enhancing its electrostatic and hydrogen bonding effects. The E256Q mutant with putrescine added shows an alternate conformation of His243, Glu11, Lys80, and Ser229, the residues that link the active site and the putrescine binding site, suggesting that putrescine activates the enzyme through electrostatic effects and acts as a switch to correctly orient key catalytic residues.

  5. Arginine Decarboxylase and Putrescine Oxidase in Ovaries of Pisum sativum L. (Changes during Ovary Senescence and Early Stages of Fruit Development).

    PubMed Central

    Perez-Amador, M. A.; Carbonell, J.

    1995-01-01

    Enzymatic activities involved in putrescine metabolism in ovaries of Pisum sativum L. during ovary senescence and fruit set were investigated. Accumulation of putrescine was observed during incubation of extracts from gibberellic acid-treated unpollinated ovaries (young developing fruits) but not in extracts from untreated ovaries (senescent ovaries). Extracts from pea ovaries showed arginine decarboxylase (ADC) activity, but ornithine decarboxylase and arginase activity were not detected. ADC activity decreased in presenescent ovaries and increased markedly after induction of fruit set with gibberellic acid. Increases in ADC activity were also observed with application of other plant growth substances (benzy-ladenine and 2,4-dichlorophenoxyacetic acid), after pollination, and in the slender (la crys) pea mutant. By contrast, putrescine oxidase activity increased in presenescent ovaries but did not increase during early fruit development. All of these results suggest that ADC and putrescine oxidase are involved in the control of putrescine metabolism. Ovary senescence is characterized by the absence of putrescine biosynthesis enzymes and increased levels of putrescine oxidase and fruit development by an increase in ADC and a constant level of putrescine oxidase. PMID:12228409

  6. DL-a-Monofluoromethylputrescine is a potent irreversible inhibitor of Escherichia coli ornithine decarboxylase.

    PubMed Central

    Kallio, A; McCann, P P; Bey, P

    1982-01-01

    DL-alpha-Monofluoromethylputrescine (compound R.M.I. 71864) is an enzyme-activated irreversible inhibitor of the biosynthetic enzyme ornithine decarboxylase from Escherichia coli. This compound, however, has much less effect in vitro on ornithine decarboxylase obtained from Pseudomonas aeruginosa. These findings are in contrast with those previously found with the substrate analogue DL-alpha-difluoromethylornithine (compound R.M.I. 71782). The K1 of the DL-alpha-monofluoromethylputrescine for the E. coli ornithine decarboxylase is 110 microM, and the half-life (t1/2) calculated for an infinite concentration of inhibitor is 2.1 min. When DL-alpha-monofluoromethylputrescine is used in combination with DL-alpha-difluoromethylarginine (R.M.I. 71897), an irreversible inhibitor of arginine decarboxylase, in vivo in E. coli, both decarboxylase activities are inhibited (greater than 95%) but putrescine levels are only decreased to about one-third of control values and spermidine levels are slightly increased. PMID:6812566

  7. Ultraviolet radiation induction of ornithine decarboxylase in rat keratinocytes

    SciTech Connect

    Rosen, C.F.; Gajic, D.; Drucker, D.J. )

    1990-05-01

    UV radiation plays an important role in the induction of cutaneous malignancy, including basal cell and squamous cell carcinomas and malignant melanoma. In addition to its effects on DNA damage and repair mechanisms, UV radiation has been shown to modulate the expression of specific genes, altering the levels of their mRNAs and the synthesis of their corresponding proteins. In order to gain further information about the molecular effects of UV radiation, we have studied the regulation of ornithine decarboxylase (ODC) gene expression in response to UVB radiation. ODC is the rate-limiting enzyme in polyamine biosynthesis, is involved in growth and differentiation, and has been implicated in carcinogenesis. Keratinocytes grown in culture were either sham-irradiated or exposed to increasing doses of UVB (1-5 mJ/cm2). Northern blot analysis of keratinocyte RNA under basal conditions demonstrated the presence of two ODC mRNA transcripts. Increasing exposure to UVB resulted in a dose-dependent increase in the levels of both ODC mRNA transcripts. The induction of ODC gene expression following UVB was noted 2 h after UVB exposure, and ODC mRNA levels continued to increase up to 24 h after UVB exposure. The UVB-induced increase in ODC gene expression was not serum dependent, despite the ability of serum alone to induce ODC gene expression. The mRNA transcripts for actin and hexosaminidase A were not induced after UVB exposure. These studies show that the UVB-induced increase in ODC activity is due, at least in part, to an increase in ODC gene expression and they provide a useful model for the analysis of the molecular effects of UVB radiation.

  8. Catalysis of acetoin formation by brewers' yeast pyruvate decarboxylase isozymes.

    PubMed

    Stivers, J T; Washabaugh, M W

    1993-12-14

    Catalysis of C(alpha)-proton transfer from 2-(1-hydroxyethyl)thiamin diphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determining the steady-state kinetics of the reaction of [1-L]acetaldehyde (L = H, D, or T) to form acetoin and the primary kinetic isotope effects on the reaction. The PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) have different steady-state kinetic parameters and isotope effects for acetoin formation in the presence and absence of the nonsubstrate allosteric effector pyruvamide: pyruvamide activation occurs by stabilization of the acetaldehyde/PDC ternary complex. The magnitudes of primary L(V/K)-type (L = D or T) isotope effects on C(alpha)-proton transfer from alpha 4-PDC-bound HETDP provide no evidence for significant breakdown of the Swain-Schaad relationship that would indicate partitioning of the putative C(alpha)-carbanion/enamine intermediate between HETDP and products. The substrate concentration dependence of the deuterium primary kinetic isotope effects provides evidence for an intrinsic isotope effect of 4.1 for C(alpha)-proton transfer from alpha 4-PDC-bound HETDP. A 1.10 +/- 0.02-fold 14C isotope discrimination against [1,2-14C]acetaldehyde in acetoin formation is inconsistent with a stepwise mechanism, in which the addition step occurs after rate-limiting formation of the C(alpha)-carbanion/enamine as a discrete enzyme-bound intermediate, and provides evidence for a concerted reaction mechanism with an important component of carbon-carbon bond formation in the transition state.

  9. Inhibition of Ornithine Decarboxylase and Growth of the Fungus Helminthosporium maydis1

    PubMed Central

    Birecka, Helena; Garraway, Michael O.; Baumann, Russell J.; McCann, Peter P.

    1986-01-01

    α-dl-Difluoromethylornithine (DFMO), a specific enzyme-activated inhibitor of ornithine decarboxylase, at 0.5 to 2.0 millimolar significantly inhibited mycelial growth and especially sporulation of Helminthosporium maydis in the dark; its inhibitory effect on sporulation was greatly increased under light conditions. Putrescine at 0.25 millimolar fully prevented the inhibitory effects of DFMO; the inhibition caused by the latter could not be prevented by cadaverine or CaCl2. α-dl-Difluoromethylarginine, a specific enzyme-activated inhibitor of arginine decarboxylase, at 0.1 to 2.0 millimolar had a weak inhibitory effect on the fungus. The effect was not dependent on the inhibitor concentration and there was no detectable arginine decarboxylase activity in the fungus. PMID:16664707

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

    PubMed

    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

  11. 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

  12. 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

  13. Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis.

    PubMed

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

    2016-08-01

    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

  14. 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.

  15. Epigenetic signature of panic disorder: a role of glutamate decarboxylase 1 (GAD1) DNA hypomethylation?

    PubMed

    Domschke, Katharina; Tidow, Nicola; Schrempf, Marie; Schwarte, Kathrin; Klauke, Benedikt; Reif, Andreas; Kersting, Anette; Arolt, Volker; Zwanzger, Peter; Deckert, Jürgen

    2013-10-01

    Glutamate decarboxylases (GAD67/65; GAD1/GAD2) are crucially involved in gamma-aminobutyric acid (GABA) synthesis and thus were repeatedly suggested to play an important role in the pathogenesis of anxiety disorders. In the present study, DNA methylation patterns in the GAD1 and GAD2 promoter and GAD1 intron 2 regions were investigated for association with panic disorder, with particular attention to possible effects of environmental factors. Sixty-five patients with panic disorder (f=44, m=21) and 65 matched healthy controls were analyzed for DNA methylation status at 38 GAD1 promoter/intron2 and 10 GAD2 promoter CpG sites via direct sequencing of sodium bisulfate treated DNA extracted from blood cells. Recent positive and negative life events were ascertained. Patients and controls were genotyped for GAD1 rs3762556, rs3791878 and rs3762555, all of which are located in the analyzed promoter region. Patients with panic disorder exhibited significantly lower average GAD1 methylation than healthy controls (p<0.001), particularly at three CpG sites in the promoter as well as in intron 2. The occurrence of negative life events was correlated with relatively decreased average methylation mainly in the female subsample (p=0.01). GAD1 SNP rs3762555 conferred a significantly lower methylation at three GAD1 intron 2 CpG sites (p<0.001). No differential methylation was observed in the GAD2 gene. The present pilot data suggest a potentially compensatory role of GAD1 gene hypomethylation in panic disorder possibly mediating the influence of negative life events and depending on genetic variation. Future studies are warranted to replicate the present finding in independent samples, preferably in a longitudinal design.

  16. 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

  17. 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.

  18. Reduced Glutamate Decarboxylase 65 Protein Within Primary Auditory Cortex Inhibitory Boutons in Schizophrenia

    PubMed Central

    Moyer, Caitlin E.; Delevich, Kristen M.; Fish, Kenneth N.; Asafu-Adjei, Josephine K.; Sampson, Allan R.; Dorph-Petersen, Karl-Anton; Lewis, David A.; Sweet, Robert A.

    2012-01-01

    Background Schizophrenia is associated with perceptual and physiological auditory processing impairments that may result from primary auditory cortex excitatory and inhibitory circuit pathology. High-frequency oscillations are important for auditory function and are often reported to be disrupted in schizophrenia. These oscillations may, in part, depend on upregulation of gamma-aminobutyric acid synthesis by glutamate decarboxylase 65 (GAD65) in response to high interneuron firing rates. It is not known whether levels of GAD65 protein or GAD65-expressing boutons are altered in schizophrenia. Methods We studied two cohorts of subjects with schizophrenia and matched control subjects, comprising 27 pairs of subjects. Relative fluorescence intensity, density, volume, and number of GAD65-immunoreactive boutons in primary auditory cortex were measured using quantitative confocal microscopy and stereologic sampling methods. Bouton fluorescence intensities were used to compare the relative expression of GAD65 protein within boutons between diagnostic groups. Additionally, we assessed the correlation between previously measured dendritic spine densities and GAD65-immunoreactive bouton fluorescence intensities. Results GAD65-immunoreactive bouton fluorescence intensity was reduced by 40% in subjects with schizophrenia and was correlated with previously measured reduced spine density. The reduction was greater in subjects who were not living independently at time of death. In contrast, GAD65-immunoreactive bouton density and number were not altered in deep layer 3 of primary auditory cortex of subjects with schizophrenia. Conclusions Decreased expression of GAD65 protein within inhibitory boutons could contribute to auditory impairments in schizophrenia. The correlated reductions in dendritic spines and GAD65 protein suggest a relationship between inhibitory and excitatory synapse pathology in primary auditory cortex. PMID:22624794

  19. Enzyme architecture: deconstruction of the enzyme-activating phosphodianion interactions of orotidine 5'-monophosphate decarboxylase.

    PubMed

    Goldman, Lawrence M; Amyes, Tina L; Goryanova, Bogdana; Gerlt, John A; Richard, John P

    2014-07-16

    The mechanism for activation of orotidine 5'-monophosphate decarboxylase (OMPDC) by interactions of side chains from Gln215 and Try217 at a gripper loop and R235, adjacent to this loop, with the phosphodianion of OMP was probed by determining the kinetic parameters k(cat) and K(m) for all combinations of single, double, and triple Q215A, Y217F, and R235A mutations. The 12 kcal/mol intrinsic binding energy of the phosphodianion is shown to be equal to the sum of the binding energies of the side chains of R235 (6 kcal/mol), Q215 (2 kcal/mol), Y217 (2 kcal/mol), and hydrogen bonds to the G234 and R235 backbone amides (2 kcal/mol). Analysis of a triple mutant cube shows small (ca. 1 kcal/mol) interactions between phosphodianion gripper side chains, which are consistent with steric crowding of the side chains around the phosphodianion at wild-type OMPDC. These mutations result in the same change in the activation barrier to the OMPDC-catalyzed reactions of the whole substrate OMP and the substrate pieces (1-β-D-erythrofuranosyl)orotic acid (EO) and phosphite dianion. This shows that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The 12 kcal/mol intrinsic phosphodianion binding energy of OMP is divided between the 8 kcal/mol of binding energy, which is utilized to drive a thermodynamically unfavorable conformational change of the free enzyme, resulting in an increase in (k(cat))(obs) for OMPDC-catalyzed decarboxylation of OMP, and the 4 kcal/mol of binding energy, which is utilized to stabilize the Michaelis complex, resulting in a decrease in (K(m))(obs).

  20. Plant ornithine decarboxylase is not post-transcriptionally feedback regulated by polyamines but can interact with a cytosolic ribosomal protein S15 polypeptide.

    PubMed

    Illingworth, Crista; Michael, Anthony J

    2012-02-01

    The formation of putrescine by ornithine decarboxylase (ODC) is a key regulatory step in polyamine biosynthesis in metazoa and fungi. Excess polyamines post-transcriptionally induce the synthesis of a unique non-competitive protein inhibitor of ODC, termed antizyme. Binding of antizyme to an ODC monomer subunit results in enzymatic inhibition, rapid ubiquitin-independent degradation of ODC by the 26S proteasome and recycling of antizyme. Plants possess an additional route for synthesizing putrescine via arginine decarboxylase (ADC). No homologue of ODC antizyme has been detected in plant genomes but several biochemical studies have reported plant ODC antizyme proteins of 9 and 16 kDa. Here we show that plant cells grown in liquid culture do not exhibit any substantial post-transcriptional, polyamine-responsive feedback regulation of ODC or ADC. However, using the yeast two hybrid system, a plant ODC-binding polypeptide was detected: the C-terminal 84-87 amino acids of cytosolic ribosomal protein (rp) S15. The Arabidopsis rpS15 polypeptide interacted specifically with plant ODC but not with human or Saccharomyces cerevisiae ODCs. Co-expression of either the full length or C-terminal rpS15 polypeptides with a plant ODC in yeast did not reduce ODC enzymatic activity. Only the full length mRNA encoding rpS15 was detected in Arabidopsis cells, suggesting that the C-terminal rpS15 polypeptide is encoded by a low abundance mRNA or the polypeptide is not physiologically relevant in plants. These results confirm the primacy of S-adenosylmethionine decarboxylase as the key regulatory enzyme in plant polyamine biosynthesis. PMID:21814791

  1. 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.

  2. Subunit gamma of the oxaloacetate decarboxylase Na(+) pump: interaction with other subunits/domains of the complex and binding site for the Zn(2+) metal ion.

    PubMed

    Schmid, Markus; Wild, Markus R; Dahinden, Pius; Dimroth, Peter

    2002-01-29

    The oxaloacetate decarboxylase Na(+) pump of Klebsiella pneumoniae is an enzyme complex composed of the peripheral alpha subunit and the two integral membrane-bound subunits beta and gamma. The alpha subunit consists of the N-terminal carboxyltransferase domain and the C-terminal biotin domain, which are connected by a flexible proline/alanine-rich linker peptide. To probe interactions between the two domains of the alpha subunit and between alpha-subunit domains and the gamma subunit, the relevant polypeptides were synthesized in Escherichia coli and subjected to copurification studies. The two alpha-subunit domains had no distinct affinity toward each other and could, therefore, not be purified as a unit on avidin-sepharose. The two domains reacted together catalytically, however, performing the carboxyl transfer from oxaloacetate to protein-bound biotin. This reaction was enhanced up to 6-fold in the presence of the Zn(2+)-containing gamma subunit. On the basis of copurification with different tagged proteins, the C-terminal biotin domain but not the N-terminal carboxyltransferase domain of the alpha subunit formed a strong complex with the gamma subunit. Upon the mutation of gamma H78 to alanine, the binding affinity to subunit alpha was lost, indicating that this amino acid may be essential for formation of the oxaloacetate decarboxylase enzyme complex. The binding residues for the Zn(2+) metal ion were identified by site-directed and deletion mutagenesis. In the gamma D62A or gamma H77A mutant, the Zn(2+) content of the decarboxylase decreased to 35% or 10% of the wild-type enzyme, respectively. Less than 5% of the Zn(2+) present in the wild-type enzyme was found if the two C-terminal gamma-subunit residues H82 and P83 were deleted. Corresponding with the reduced Zn(2+) contents in these mutants, the oxaloacetate decarboxylase activities were diminished. These results indicate that aspartate 62, histidine 77, and histidine 82 of the gamma subunit are ligands

  3. Putrescine and spermidine control degradation and synthesis of ornithine decarboxylase in Neurospora crassa

    SciTech Connect

    Barnett, G.R.; Seyfzadeh, M.; Davis, R.H.

    1988-07-15

    Neurospora crassa mycelia, when starved for polyamines, have 50-70-fold more ornithine decarboxylase activity and enzyme protein than unstarved mycelia. Using isotopic labeling and immunoprecipitation, we determined the half-life and the synthetic rate of the enzyme in mycelia differing in the rates of synthesis of putrescine, the product of ornithine decarboxylase, and spermidine, the main end-product of the polyamine pathway. When the pathway was blocked between putrescine and spermidine, ornithine decarboxylase synthesis rose 4-5-fold, regardless of the accumulation of putrescine. This indicates that spermidine is a specific signal for the repression of enzyme synthesis. When both putrescine and spermidine synthesis were reduced, the half-life of the enzyme rapidly increased 10-fold. The presence of either putrescine or spermidine restored the normal enzyme half-life of 55 min. Tests for an ornithine decarboxylase inhibitory protein (antizyme) were negative. The regulatory mechanisms activated by putrescine and spermidine account for most or all of the regulatory amplitude of this enzyme in N. crassa.

  4. Draft Genome Sequence of Bordetella bronchiseptica KU1201, the First Isolation Source of Arylmalonate Decarboxylase.

    PubMed

    Yoshida, Shosuke; Enoki, Junichi; Hemmi, Risa; Kourist, Robert; Kawakami, Norifumi; Miyamoto, Kenji

    2015-01-01

    The analysis of the 6.8-Mbp draft genome sequence of the phenylmalonate-assimilating bacterium Bordetella bronchiseptica KU1201 identified 6,358 protein-coding sequences. This will give us an insight into the catabolic variability of this strain for aromatic compounds, along with the roles of arylmalonate decarboxylases in nature. PMID:25953178

  5. Draft Genome Sequence of Bordetella bronchiseptica KU1201, the First Isolation Source of Arylmalonate Decarboxylase.

    PubMed

    Yoshida, Shosuke; Enoki, Junichi; Hemmi, Risa; Kourist, Robert; Kawakami, Norifumi; Miyamoto, Kenji

    2015-01-01

    The analysis of the 6.8-Mbp draft genome sequence of the phenylmalonate-assimilating bacterium Bordetella bronchiseptica KU1201 identified 6,358 protein-coding sequences. This will give us an insight into the catabolic variability of this strain for aromatic compounds, along with the roles of arylmalonate decarboxylases in nature.

  6. Polyamine and differentiation: induction of ornithine decarboxylase by parathyroid hormone is a good marker of differentiated chondrocytes.

    PubMed Central

    Takigawa, M; Ishida, H; Takano, T; Suzuki, F

    1980-01-01

    The activity of ornithine decarboxylase (OD-Case:L-ornithine carboxy-lyase, EC 4.1.1.17) in rabbit costal chondrocytes in culture increased markedly after addition of parathyroid hormone (PTH), reaching a maximum 4 to 5 hr after PTH addition. The increase in ODCase activity was followed by increase in the intracellular concentrations of polyamines, especially putrescine, which increased in 6 hr to about 3-fold that of untreated cultures. The induction of ODCase by PTH was not observed in L, 3T3, HeLa, buffalo rat liver, or BHK cells. Retinyl acetate and retinoic acid both inhibited expression of the differentiated phenotype of chondrocytes by rabbit costal chondrocytes in culture within 3 days after their addition, as judged by morphological change and decrease in sulfate incorporation into glycosaminoglycans but did not inhibit cell proliferation. PTH could not induce an increase in ODCase in de-differentiated cells that had been pretreated with retinyl acetate or retinoic acid for 3 days. but 4 days after removal of the retinoids, these de-differentiated cells regained the ability to synthesize ODCase in response to PTH. These facts suggest that the induction of ODCase and the formation of putrescine by PTH are good markers of the differentiated phenotype of cultured chondrocytes. Images PMID:6929498

  7. Studies on polyamine and ornithine metabolism in rat colon: effects of two synergistically. Acting inducers of ornithine decarboxylase activity

    SciTech Connect

    Stanley, B.A.

    1987-01-01

    Ornithine decarboxylase (ODC) activity in rat colon mucosa was determined by the release of /sup 14/CO/sub 2/ from radiolabeled ornithine in the presence (total enzyme) or absence (holoenzyme) of added pyridoxal-5'-phosphate (PLP). Total leucine incorporation into acid-precipitable protein over 30 minute was calculated by dividing the /sup 3/H-leucine in protein by the specific activity of the intracellular leucine. Amino acids, polyamines, and PLP-semicarbazide were quantified by high pressure liquid chromatography. Ornithine aminotransaminase activity (OAT) was measured as the quantity of pyrolline (5-carboxy) produced from alpha-ketoglutarate and ornithine. After 10 weeks on a high or no vitamin B/sub 6/ diet, no change in basal ODC activity was seen; however, sodium deoxycholate instillation in vitamin B/sub 6/ deficient rats led to a large increase in total but not holo-ODC activity. In rats fed normal chow diet, no increases in mucosal PLP levels were seen after either treatment. Increases in general protein synthesis rate could not account for the peaks in ODC activity after either stimulus. Putrescine increases were proportional to peaks of ODC activity after either stimulus, while spermine levels remained depressed for 18 hours after starvation/refeeding. Ornithine levels were increased after either stimulus, and this increase was linked to decreases in OAT activity, indicating short-term coordination of overall ornithine metabolism to favor polyamine biosynthesis.

  8. Rapid detection and quantification of tyrosine decarboxylase gene (tdc) and its expression in gram-positive bacteria associated with fermented foods using PCR-based methods.

    PubMed

    Torriani, Sandra; Gatto, Veronica; Sembeni, Silvia; Tofalo, Rosanna; Suzzi, Giovanna; Belletti, Nicoletta; Gardini, Fausto; Bover-Cid, Sara

    2008-01-01

    In this study, PCR-based procedures were developed to detect the occurrence and quantify the expression of the tyrosine decarboxylase gene (tdc) in gram-positive bacteria associated with fermented foods. Consensus primers were used in conventional and reverse transcription PCR to analyze a collection of 87 pure cultures of lactic acid bacteria and staphylococci. All enterococci, Staphylococcus epidermidis, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus fermentum strains and 1 of 10 Staphylococcus xylosus strains produced amplification products with the primers DEC5 and DEC3 in accordance with results of the screening plate method and with previously reported result obtained with high-performance liquid chromatography. No amplicons were obtained for tyramine-negative strains, confirming the high specificity of these new primers. A novel quantitative real-time PCR assay was successfully applied to quantify tdc and its transcript in pure cultures and in meat and meat products. This assay allowed estimation of the influence of different variables (pH, temperature, and NaCl concentration) on the tdc expression of the tyraminogenic strain Enterococcus faecalis EF37 after 72 h of growth in M17 medium. Data obtained suggest that stressful conditions could induce greater tyrosine decarboxylase activity. The culture-independent PCR procedures developed here may be used for reliable and fast detection and quantification of bacterial tyraminogenic activity without the limitations of conventional techniques.

  9. Rapid detection and quantification of tyrosine decarboxylase gene (tdc) and its expression in gram-positive bacteria associated with fermented foods using PCR-based methods.

    PubMed

    Torriani, Sandra; Gatto, Veronica; Sembeni, Silvia; Tofalo, Rosanna; Suzzi, Giovanna; Belletti, Nicoletta; Gardini, Fausto; Bover-Cid, Sara

    2008-01-01

    In this study, PCR-based procedures were developed to detect the occurrence and quantify the expression of the tyrosine decarboxylase gene (tdc) in gram-positive bacteria associated with fermented foods. Consensus primers were used in conventional and reverse transcription PCR to analyze a collection of 87 pure cultures of lactic acid bacteria and staphylococci. All enterococci, Staphylococcus epidermidis, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus fermentum strains and 1 of 10 Staphylococcus xylosus strains produced amplification products with the primers DEC5 and DEC3 in accordance with results of the screening plate method and with previously reported result obtained with high-performance liquid chromatography. No amplicons were obtained for tyramine-negative strains, confirming the high specificity of these new primers. A novel quantitative real-time PCR assay was successfully applied to quantify tdc and its transcript in pure cultures and in meat and meat products. This assay allowed estimation of the influence of different variables (pH, temperature, and NaCl concentration) on the tdc expression of the tyraminogenic strain Enterococcus faecalis EF37 after 72 h of growth in M17 medium. Data obtained suggest that stressful conditions could induce greater tyrosine decarboxylase activity. The culture-independent PCR procedures developed here may be used for reliable and fast detection and quantification of bacterial tyraminogenic activity without the limitations of conventional techniques. PMID:18236668

  10. Fern L-methionine decarboxylase: Kinetics and mechanism of decarboxylation and abortive transamination

    SciTech Connect

    Akhtar, M.; Stevenson, D.E.; Gani, D. )

    1990-08-21

    L-Methionine decarboxylase from Dryopteris filix-mas catalyzes the decarboxylation of L-methionine and a range of straight- and branched-chain L-amino acids to give the corresponding amine products. The deuterium solvent isotope effects for the decarboxylation of (2S)-methionine are {sup D}(V/K) = 6.5 and {sup D}V = 2.3, for (2S)-valine are {sup D}(V/K) = 1.9 and {sup D}V = 2.6, and for (2S)-lecuine are {sup D}(V/K) = 2.5 and {sup D}V = 1.0 at pL 5.5. At pL 6.0 and above, where the value of k{sub cat} for all of the substrates is low, the solvent isotope effects on V{sub max} for methionine are 1.1-1.2 whereas the effects on V/K remain unchanged, indicating that the solvent-sensitive transition state occurs before the first irreversible step, carbon dioxide desorption. At very high concentration, the product amine can promote transamination of the coenzyme. However, the reaction occurs infrequently and does not influence the partitioning between decarboxylation and substrate-mediated abortive transamination under steady-state turnover conditions. The partition ratio, normal catalytic versus abortive events, can be determined from the amount of substrate consumed by a known amount of enzyme at infinite time, and the rate of inactivation can be determined by measuring the decrease in enzyme activity with respect to time. Experiments conducted in deuterium oxide allowed the solvent isotope effects for the partition ratio and the abortive reaction to be determined. {sup 1}H NMR spectroscopic analysis of 3-(methylthio)-1-aminopropane isolated from incubations conducted in 50 molar % deuterium oxide at pL 4.8 and at pL 6.5 indicated that the proton donor was monoprotic and, therefore, is probably the imidazolium side chain of a histidine residue.

  11. Minimal antizyme peptide fully functioning in the binding and inhibition of ornithine decarboxylase and antizyme inhibitor.

    PubMed

    Hsieh, Ju-Yi; Yang, Jung-Yen; Lin, Chih-Li; Liu, Guang-Yaw; Hung, Hui-Chih

    2011-01-01

    Antizyme (AZ) is a protein with 228 amino acid residues that regulates ornithine decarboxylase (ODC) by binding to ODC and dissociating its homodimer, thus inhibiting its enzyme activity. Antizyme inhibitor (AZI) is homologous to ODC, but has a higher affinity than ODC for AZ. In this study, we quantified the biomolecular interactions between AZ and ODC as well as AZ and AZI to identify functional AZ peptides that could bind to ODC and AZI and inhibit their function as efficiently as the full-length AZ protein. For these AZ peptides, the inhibitory ability of AZ_95-228 was similar to that of AZ_WT. Furthermore, AZ_95-176 displayed an inhibition (IC(50): 0.20 µM) similar to that of AZ-95-228 (IC(50): 0.16 µM), even though a large segment spanning residues 177-228 was deleted. However, further deletion of AZ_95-176 from either the N-terminus or the C-terminus decreased its ability to inhibit ODC. The AZ_100-176 and AZ_95-169 peptides displayed a noteworthy decrease in ability to inhibit ODC, with IC(50) values of 0.43 and 0.37 µM, respectively. The AZ_95-228, AZ_100-228 and AZ_95-176 peptides had IC(50) values comparable to that of AZ_WT and formed AZ-ODC complexes with K(d,AZ-ODC) values of 1.5, 5.3 and 5.6 µM, respectively. Importantly, our data also indicate that AZI can rescue AZ peptide-inhibited ODC enzyme activity and that it can bind to AZ peptides with a higher affinity than ODC. Together, these data suggest that these truncated AZ proteins retain their AZI-binding ability. Thus, we suggest that AZ_95-176 is the minimal AZ peptide that is fully functioning in the binding of ODC and AZI and inhibition of their function. PMID:21931692

  12. Increased gene expression of catecholamine-synthesizing enzymes in adrenal glands contributes to high circulating catecholamines in pigs with tachycardia-induced cardiomyopathy.

    PubMed

    Tomaszek, A; Kiczak, L; Bania, J; Paslawska, U; Zacharski, M; Janiszewski, A; Noszczyk-Nowak, A; Dziegiel, P; Kuropka, P; Ponikowski, P; Jankowska, E A

    2015-04-01

    High levels of circulating catecholamines have been established as fundamental pathophysiological elements of heart failure (HF). However, it is unclear whether the increased gene expression of catecholamine-synthesis enzymes in the adrenal glands contributes to these hormone abnormalities in large animal HF models. We analyzed the mRNA levels of catecholamine-synthesizing enzymes: tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AAAD), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in adrenal glands of 18 pigs with chronic systolic non-ischaemic HF (tachycardia-induced cardiomyopathy due to right ventricle pacing) and 6 sham-operated controls. Pigs with severe HF demonstrated an increased expression of TH and DBH (but neither AAAD nor PNMT) as compared to animals with milder HF and controls (P<0.05 in all cases). The increased adrenal mRNA expression of TH and DBH was accompanied by a reduced left ventricle ejection fraction (LVEF) (P<0.001) and an elevated plasma B-type natriuretic peptide (BNP) (P<0.01), the other indices reflecting HF severity. There was a positive relationship between the increased adrenal mRNA expression of TH and DBH, and the high levels of circulating adrenaline and noradrenaline (all P<0.05). The association with noradrenaline remained significant also when adjusted for LVEF and plasma BNP, suggesting a significant contribution of adrenals to the circulating pool of catecholamines in subjects with systolic HF. PMID:25903953

  13. Increased gene expression of catecholamine-synthesizing enzymes in adrenal glands contributes to high circulating catecholamines in pigs with tachycardia-induced cardiomyopathy.

    PubMed

    Tomaszek, A; Kiczak, L; Bania, J; Paslawska, U; Zacharski, M; Janiszewski, A; Noszczyk-Nowak, A; Dziegiel, P; Kuropka, P; Ponikowski, P; Jankowska, E A

    2015-04-01

    High levels of circulating catecholamines have been established as fundamental pathophysiological elements of heart failure (HF). However, it is unclear whether the increased gene expression of catecholamine-synthesis enzymes in the adrenal glands contributes to these hormone abnormalities in large animal HF models. We analyzed the mRNA levels of catecholamine-synthesizing enzymes: tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AAAD), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in adrenal glands of 18 pigs with chronic systolic non-ischaemic HF (tachycardia-induced cardiomyopathy due to right ventricle pacing) and 6 sham-operated controls. Pigs with severe HF demonstrated an increased expression of TH and DBH (but neither AAAD nor PNMT) as compared to animals with milder HF and controls (P<0.05 in all cases). The increased adrenal mRNA expression of TH and DBH was accompanied by a reduced left ventricle ejection fraction (LVEF) (P<0.001) and an elevated plasma B-type natriuretic peptide (BNP) (P<0.01), the other indices reflecting HF severity. There was a positive relationship between the increased adrenal mRNA expression of TH and DBH, and the high levels of circulating adrenaline and noradrenaline (all P<0.05). The association with noradrenaline remained significant also when adjusted for LVEF and plasma BNP, suggesting a significant contribution of adrenals to the circulating pool of catecholamines in subjects with systolic HF.

  14. 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.

  15. 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

  16. Functional analysis of cis-aconitate decarboxylase and trans-aconitate metabolism in riboflavin-producing filamentous Ashbya gossypii.

    PubMed

    Sugimoto, Takashi; Kato, Tatsuya; Park, Enoch Y

    2014-05-01

    In Ashbya gossypii, isocitrate lyase (ICL1) is a very crucial enzyme for riboflavin production. Itaconate, the inhibitor of ICL1, has been used as an antimetabolite for mutagenic studies in A. gossypii. It has been reported that itaconate is produced from cis-aconitate by cis-aconitate decarboxylase (CAD1) in Aspergillus terreus. In this study, identification of CAD1 gene and determination of the presence of itaconate in the riboflavin biosynthetic pathway in A. gossypii were carried out to confirm itaconate metabolism. Although no CAD1 candidate gene was found and no itaconate production was observed, cis- and trans-aconitate were detected in the riboflavin production phase. It is known that trans-aconitate inhibits aconitase (ACO1) in the tricarboxylic acid cycle. In A. gossypii, the transcription level of AGR110Wp, the homolog of trans-aconitate 3-methyltransferase (TMT1), was enhanced by almost threefold during riboflavin production than that during its growth phase. TMT1 catalyzes the methylation reaction of trans-aconitate in Saccharomyces cerevisiae. Thus, these results suggest that the enhancement of the transcription level of this TMT1 homolog decreases the trans-aconitate level, which may mitigate the inhibition of ACO1 by oxidative stress in the riboflavin biosynthetic pathway in A. gossypii. This is a novel finding in A. gossypii, which may open new metabolic engineering ideas for improving riboflavin productivity. PMID:24315530

  17. 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.

  18. 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.

  19. Cloning and molecular characterization of an ornithine decarboxylase gene and its expression during embryonic development of the housefly, Musca domestica.

    PubMed

    Toutges, Michelle J; Santoso, Adi

    2011-10-01

    We are interested in identifying targets that may be used to develop new control products for the common housefly, Musca domestica, a vector of disease for many vertebrates. One such target, ornithine decarboxylase (ODC), is an embryonic enzyme involved in the regulation of polyamines and is a critical enzyme during M. domestica development. In this study, the cDNA for ODC from M. domestica was cloned, sequenced, and characterized. The full-length cDNA was 1,337-bp, consistent with a single band of approximately 1.35 kb obtained by northern analysis. The open-reading frame contains 1,191 bp, yielding a deduced polypeptide of 396 amino acid residues with a predicted mass of 44,618 Da. The deduced M. domestica ODC protein was homologous to other ODC proteins. mRNA expression profiles analyzed by real-time PCR indicated that the ODC transcript is temporally regulated throughout embryogenesis. Sequence data and Southern blot analysis suggests that there were likely only one or two closely linked copies of the M. domestica ODC gene.

  20. Cloning and molecular characterization of an ornithine decarboxylase gene and its expression during embryonic development of the housefly, Musca domestica.

    PubMed

    Toutges, Michelle J; Santoso, Adi

    2011-10-01

    We are interested in identifying targets that may be used to develop new control products for the common housefly, Musca domestica, a vector of disease for many vertebrates. One such target, ornithine decarboxylase (ODC), is an embryonic enzyme involved in the regulation of polyamines and is a critical enzyme during M. domestica development. In this study, the cDNA for ODC from M. domestica was cloned, sequenced, and characterized. The full-length cDNA was 1,337-bp, consistent with a single band of approximately 1.35 kb obtained by northern analysis. The open-reading frame contains 1,191 bp, yielding a deduced polypeptide of 396 amino acid residues with a predicted mass of 44,618 Da. The deduced M. domestica ODC protein was homologous to other ODC proteins. mRNA expression profiles analyzed by real-time PCR indicated that the ODC transcript is temporally regulated throughout embryogenesis. Sequence data and Southern blot analysis suggests that there were likely only one or two closely linked copies of the M. domestica ODC gene. PMID:21928394

  1. Histidine decarboxylase deficiency causes tourette syndrome: parallel findings in humans and mice.

    PubMed

    Castellan Baldan, Lissandra; Williams, Kyle A; Gallezot, Jean-Dominique; Pogorelov, Vladimir; 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

    2014-01-01

    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 (DA) 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 DA levels; in Hdc knockout mice, striatal DA was increased and the DA-regulated immediate early gene Fos was upregulated. DA D2/D3 receptor binding was altered both in mice and in humans carrying the Hdc mutation. These data confirm histidine decarboxylase deficiency as a rare cause of TS and identify HA-DA interactions in the basal ganglia as an important locus of pathology. PMID:24411733

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. Distribution of the thiamin diphosphate C(2)-proton during catalysis of acetaldehyde formation by brewers' yeast pyruvate decarboxylase.

    PubMed

    Harris, T K; Washabaugh, M W

    1995-10-31

    The distribution of tritium derived from enzyme-bound [thiazole-2-T]thiamin diphosphate (TDP) during the reaction of pyruvate to form acetaldehyde catalyzed by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was determined under single-turnover conditions ([E] > [S]) in the presence of the nonsubstrate allosteric effector pyruvamide. The specific radioactivity of the [1-L]acetaldehyde product and solvent ([L]H2O) was 43 +/- 4% and 54 +/- 2%, respectively, of the initial specific radioactivity of PDC-bound [thiazole-2-T]TDP and was independent of the extent of the single-turnover reaction. There is little (< or = 3%) or no return of the abstracted C(2)-hydron to the C(2) position of PDC-bound TDP. This provides evidence that the abstracted C(2)-hydron is involved in the specific protonation of the C(alpha) position of the PDC-bound intermediate 2-(1-hydroxyethyl)thiamin diphosphate (HETDP), which is cleaved to form [1-L]acetaldehyde and PDC-bound [thiazole-2-H]TDP. The partial exchange of C(2)-derived tritium into solvent requires that (1) hydron transfer from C(2) occurs to a catalytic-base in which the conjugate catalytic acid is partially shielded from hydron exchange with the solvent, (2) the conjugate catalytic acid transfers the C(2)-derived hydron to the C(alpha) position of HETDP, and (3) hydron transfer to C(2) to regenerate the coenzyme occurs either from solvent directly or from a second catalytic acid of the enzyme that undergoes rapid hydron exchange with the solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. 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.

  11. The root-specific glutamate decarboxylase (GAD1) is essential for sustaining GABA levels in Arabidopsis.

    PubMed

    Bouché, Nicolas; Fait, Aaron; Zik, Moriyah; Fromm, Hillel

    2004-05-01

    In plants, as in most eukaryotes, glutamate decarboxylase catalyses the synthesis of GABA. The Arabidopsis genome contains five glutamate decarboxylase genes and one of these genes (glutamate decarboxylase1; i.e. GAD1 ) is expressed specifically in roots. By isolating and analyzing three gad1 T-DNA insertion alleles, derived from two ecotypes, we investigated the potential role of GAD1 in GABA production. We also analyzed a promoter region of the GAD1 gene and show that it confers root-specific expression when fused to reporter genes. Phenotypic analysis of the gad1 insertion mutants revealed that GABA levels in roots were drastically reduced compared with those in the wild type. The roots of the wild type contained about sevenfold more GABA than roots of the mutants. Disruption of the GAD1 gene also prevented the accumulation of GABA in roots in response to heat stress. Our results show that the root-specific calcium/calmodulin-regulated GAD1 plays a major role in GABA synthesis in plants under normal growth conditions and in response to stress.

  12. Identification, cloning, and nucleotide sequencing of the ornithine decarboxylase antizyme gene of Escherichia coli.

    PubMed Central

    Canellakis, E S; Paterakis, A A; Huang, S C; Panagiotidis, C A; Kyriakidis, D A

    1993-01-01

    The ornithine decarboxylase antizyme gene of Escherichia coli was identified by immunological screening of an E. coli genomic library. A 6.4-kilobase fragment containing the antizyme gene was subcloned and sequenced. The open reading frame encoding the antizyme was identified on the basis of its ability to direct the synthesis of immunoreactive antizyme. Antizyme shares significant homology with bacterial transcriptional activators of the two-component regulatory system family; these systems consist of a "sensor" kinase and a transcriptional regulator. The open reading frame next to antizyme is homologous to sensor kinases. Antizyme overproduction inhibits the activities of both ornithine and arginine decarboxylases without affecting their protein levels. Extracts from E. coli bearing an antizyme gene-containing plasmid exhibit increased antizyme activity. These data strongly suggest that (i) the cloned gene encodes the ornithine decarboxylase antizyme and (ii) antizyme is a bifunctional protein serving as both an inhibitor of polyamine biosynthesis as well as a transcriptional regulator of an as yet unknown set of genes. Images Fig. 2 Fig. 4 Fig. 6 PMID:8346225

  13. 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.

  14. 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.

  15. 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.

  16. Kinetic, mutational, and structural analysis of malonate semialdehyde decarboxylase from Coryneform bacterium strain FG41: mechanistic implications for the decarboxylase and hydratase activities.

    PubMed

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

    2013-07-16

    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 and 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. In terms of pairwise sequence, MSAD from Coryneform bacterium strain FG41 (designated FG41 MSAD) is 38% identical 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. 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 the 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 Pp 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.

  17. 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

  18. Development of a Novel Cysteine Sulfinic Acid Decarboxylase Knockout Mouse: Dietary Taurine Reduces Neonatal Mortality

    PubMed Central

    Park, Eunkyue; Park, Seung Yong; Schuller-Levis, Georgia

    2014-01-01

    We engineered a CSAD KO mouse to investigate the physiological roles of taurine. The disruption of the CSAD gene was verified by Southern, Northern, and Western blotting. HPLC indicated an 83% decrease of taurine concentration in the plasma of CSAD−/−. Although CSAD−/− generation (G)1 and G2 survived, offspring from G2 CSAD−/− had low brain and liver taurine concentrations and most died within 24 hrs of birth. Taurine concentrations in G3 CSAD−/− born from G2 CSAD−/− treated with taurine in the drinking water were restored and survival rates of G3 CSAD−/− increased from 15% to 92%. The mRNA expression of CDO, ADO, and TauT was not different in CSAD−/− compared to WT and CSAD mRNA was not expressed in CSAD−/−. Expression of Gpx 1 and 3 was increased significantly in CSAD−/− and restored to normal levels with taurine supplementation. Lactoferrin and the prolactin receptor were significantly decreased in CSAD−/−. The prolactin receptor was restored with taurine supplementation. These data indicated that CSAD KO is a good model for studying the effects of taurine deficiency and its treatment with taurine supplementation. PMID:24639894

  19. A calmodulin like EF hand protein positively regulates oxalate decarboxylase expression by interacting with E-box elements of the promoter

    PubMed Central

    Kamthan, Ayushi; Kamthan, Mohan; Kumar, Avinash; Sharma, Pratima; Ansari, Sekhu; Thakur, Sarjeet Singh; Chaudhuri, Abira; Datta, Asis

    2015-01-01

    Oxalate decarboxylase (OXDC) enzyme has immense biotechnological applications due to its ability to decompose anti-nutrient oxalic acid. Flammulina velutipes, an edible wood rotting fungus responds to oxalic acid by induction of OXDC to maintain steady levels of pH and oxalate anions outside the fungal hyphae. Here, we report that upon oxalic acid induction, a calmodulin (CaM) like protein-FvCaMLP, interacts with the OXDC promoter to regulate its expression. Electrophoretic mobility shift assay showed that FvCamlp specifically binds to two non-canonical E-box elements (AACGTG) in the OXDC promoter. Moreover, substitutions of amino acids in the EF hand motifs resulted in loss of DNA binding ability of FvCamlp. F. velutipes mycelia treated with synthetic siRNAs designed against FvCaMLP showed significant reduction in FvCaMLP as well as OXDC transcript pointing towards positive nature of the regulation. FvCaMLP is different from other known EF hand proteins. It shows sequence similarity to both CaMs and myosin regulatory light chain (Cdc4), but has properties typical of a calmodulin, like binding of 45Ca2+, heat stability and Ca2+ dependent electrophoretic shift. Hence, FvCaMLP can be considered a new addition to the category of unconventional Ca2+ binding transcriptional regulators. PMID:26455820

  20. Combination treatment for allergic conjunctivitis - Plant derived histidine decarboxylase inhibitor and H1 antihistaminic drug.

    PubMed

    Bakrania, Anita K; Patel, Snehal S

    2015-08-01

    Aim of present investigation was to study the effect of catechin and the combination of catechin and cetirizine in ovalbumin induced animal model of allergic conjunctivitis. Guinea pigs were divided into 5 groups: normal control, disease control, disease treated with catechin 100 mg/kg, disease treated with cetirizine 10 mg/kg, disease treated with combination of catechin and cetirizine, 50 mg/kg & 5 mg/kg respectively. Sensitization was carried out by intraperitoneal injection of ovalbumin for the period of 14 day. Simultaneously, catechin was administered orally for 14 days while, cetirizine was administered at the day of experiment. Determination of clinical scoring, mast cell and blood histamine content, histidine decarboxylase activity from stomach was carried out. Vascular permeability was measured by dye leakage after secondary challenge of allergen and conjunctival tissues were subjected for histopathological examinations. Treatment with catechin, cetirizine and combination showed significant (P < 0.05) decrease in clinical scoring and vascular permeability. While, catechin 100 mg/kg and catechin 50 mg/kg showed significant (P < 0.05) decrease in histamine content in mast and blood. The treatment also showed significant (P < 0.05) decrease in the histidine decarboxylase enzyme activity. However, cetirizine group did not show any difference in enzyme activity as well as histamine content. Histopathological examination also showed improvement in ulceration and decrease in edema and inflammation in all treatment groups. From the present study, we can conclude that catechin exhibits potent anti-allergic activity by histidine decarboxylase enzyme inhibition and combination shown significant anti-allergic activity at reduced dose by both enzyme inhibition as well as inhibition of histamine receptors.

  1. Mechanism of citrate metabolism by an oxaloacetate decarboxylase-deficient mutant of Lactococcus lactis IL1403.

    PubMed

    Pudlik, Agata M; Lolkema, Juke S

    2011-08-01

    Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706-714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of L. lactis when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of L-lactate, indicating exchange between oxaloacetate and L-lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of L. lactis is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate.

  2. Cloning, expression and characterization of the ornithine decarboxylase gene from Dictyostelium discoideum.

    PubMed

    Kumar, Rishikesh; Rafia, Sheikh; Saran, Shweta

    2014-01-01

    Ornithine decarboxylase (ODC) is a rate limiting enzyme in polyamine synthesis that decarboxylates ornithine to form the diamine putrescine. We report here the isolation, expression and characterization of a homolog of ODC from Dictyostelium discoideum. DdODC is conserved and shows sequence and structural homology with that from human. Both ODC transcript and protein are expressed at all stages of development and show high expression in prestalk/stalk cells. It is cytosolic and predominantly perinuclear in localization. Both overexpression of DdODC and putrescine treatment resulted in inhibition of cell proliferation. PMID:25896203

  3. Fusion of pyruvate decarboxylase and alcohol dehydrogenase increases ethanol production in Escherichia coli.

    PubMed

    Lewicka, Aleksandra J; Lyczakowski, Jan J; Blackhurst, Gavin; Pashkuleva, Christiana; Rothschild-Mancinelli, Kyle; Tautvaišas, Dainius; Thornton, Harry; Villanueva, Hugo; Xiao, Weike; Slikas, Justinas; Horsfall, Louise; Elfick, Alistair; French, Christopher

    2014-12-19

    Ethanol is an important biofuel. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) increases ethanol production in Escherichia coli. A fusion of PDC and ADH was generated and expressed in E. coli. The fusion enzyme was demonstrated to possess both activities. AdhB activity was significantly lower when fused to PDC than when the two enzymes were expressed separately. However, cells expressing the fusion protein generated ethanol more rapidly and to higher levels than cells coexpressing Pdc and AdhB, suggesting a specific rate enhancement due to the fusion of the two enzymes.

  4. Association between a polymorphism of the 65K-glutamate decarboxylase gene and insulin-dependent diabetes mellitus

    SciTech Connect

    Kure, S.; Aoki, Y.; Narisawa, K.

    1994-09-01

    Autoimmunity against 65K-glutamate decarboxylase (GAD65), one of two forms of the {gamma}-aminobutyric acid-synthesizing enzyme, is commonly associated with insulin-dependent diabetes mellitus (IDDM). To study the predisposing effect of the GAD65 genotype on IDDM, we performed a case-control study screening an association between a newly-identified GAD65 polymorphism and IDDM in the Japanese population. The identified polymorphism was a microsatellite that was located in an intron near the 3{prime} end of the GAD65 gene consisting of variable numbers of a (CA)-dinucleotide repeat. We amplified the polymorphic region by polymerase chain reaction, and, for each individual in the control group (n=254) and the IDDM group (n=108), determined a pair of (CA)-repeat numbers, each number derived from one or the other of their alleles. In both groups we found 13 allelic variants with different repeat numbers, ranging from 19 to 31 repeats of the (CA) dinucleotide. The most frequent allelic variant in the IDDM group was 20 repeats; (CA){sub 20}. A higher frequency of a genotype containing two (CA){sub 20} alleles (p=0.005) was observed in the IDDM group (41.7%) compared with the control group (26.8%). Odds ratio (a 95% confidence interval) for a heterozygote or a homozygote of (CA){sub 20} versus a subject without (CA){sub 20} was 1.2 (0.66-2.25) and 2.23 (1.18-4.21), respectively. No significant association was observed between the (CA)-repeat genotype and the appearance of anti-GAD antibodies in the patients whose duration of the diabetes was less than 4 years (n=35). Therefore, genetic variations in GAD65 appears to be associated with IDDM susceptibility.

  5. 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

  6. Identification of the active site of human mitochondrial malonyl-coenzyme a decarboxylase: A combined computational study.

    PubMed

    Ling, Baoping; Liu, Yuxia; Li, Xiaoping; Wang, Zhiguo; Bi, Siwei

    2016-06-01

    Malonyl-CoA decarboxylase (MCD) can control the level of malonyl-CoA in cell through the decarboxylation of malonyl-CoA to acetyl-CoA, and plays an essential role in regulating fatty acid metabolism, thus it is a potential target for drug discovery. However, the interactions of MCD with CoA derivatives are not well understood owing to unavailable crystal structure with a complete occupancy in the active site. To identify the active site of MCD, molecular docking and molecular dynamics simulations were performed to explore the interactions of human mitochondrial MCD (HmMCD) and CoA derivatives. The findings reveal that the active site of HmMCD indeed resides in the prominent groove which resembles that of CurA. However, the binding modes are slightly different from the one observed in CurA due to the occupancy of the side chain of Lys183 from the N-terminal helical domain instead of the adenine ring of CoA. The residues 300 - 305 play an essential role in maintaining the stability of complex mainly through hydrogen bond interactions with the pyrophosphate moiety of acetyl-CoA. Principle component analysis elucidates the conformational distribution and dominant concerted motions of HmMCD. MM_PBSA calculations present the crucial residues and the major driving force responsible for the binding of acetyl-CoA. These results provide useful information for understanding the interactions of HmMCD with CoA derivatives. Proteins 2016; 84:792-802. © 2016 Wiley Periodicals, Inc. PMID:26948533

  7. [Molecular cloning and characterization of S-adenosyl-L-methionine decarboxylase gene (DoSAMDC1) in Dendrobium officinale].

    PubMed

    Zhao, Ming-Ming; Zhang, Gang; Zhang, Da-Wei; Guo, Shun-Xing

    2013-06-01

    S-Adenosyl-L-methionine decarboxylase (SAMDC) is a key enzyme in the polyamines biosynthesis, thus is essential for basic physiological and biochemical processes in plant. In the present study, a full length cDNA of DoSAMDC1 gene was obtained from symbiotic germinated seeds of an endangered medicinal orchid species Dendrobium officinale, using the rapid amplification of cDNA ends (RACE)-PCR technique for the first time. The full length cDNA was 1 979 bp, with three open reading frames, i.e. tiny-uORF, small-uORF and main ORF (mORF). The mORF was deduced to encode a 368 amino acid (aa) protein with a molecular mass of 40.7 kD and a theoretical isoelectric point of 5.2. The deduced DoSAMDC1 protein, without signal peptide, had two highly conserved function domains (proenzyme cleavage site and PEST domain) and a 22-aa transmembrane domain (89-110). Multiple sequence alignments and phylogenetic relationship analyses revealed DoSAMDC1 had a higher level of sequence similarity to monocot SAMDCs than those of dicot. Expression patterns using qRT-PCR analyses showed that DoSAMDC1 transcripts were expressed constitutively without significant change in the five tissues (not infected with fungi). While in the symbiotic germinated seeds, the expression level was enhanced by 2.74 fold over that in the none-germinated seeds, indicating possible involvement of the gene in symbiotic seed germination of D. officinale.

  8. Secondary. beta. -deuterium isotope effects in decarboxylation and elimination reactions of. cap alpha. -lactylthiamin: intrinsic isotope effects of pyruvate decarboxylase

    SciTech Connect

    Kluger, R.; Brandl, M.

    1986-11-26

    The reactions of the adduct of pyruvate and thiamine, lactylthiamin (2-(lact-2-yl)thiamine), are accurate nonenzymic models for reactions of intermediates formed during catalysis by pyruvate decarboxylase. The enzymatic reaction generates lactylthiamin diphosphate from pyruvate and thiamine diphosphate. ..beta..-Deuterium isotope effects were determined for the nonenzymic reactions, and the results were related to isotope effects on the enzymic reaction. 2-(Lact-2-yl-..beta..-d/sub 3/) thiamine was prepared by condensation of methyl pyruvate-d/sub 3/ with thiamine followed by hydrolysis. The isotope effect for decarboxylation of lactylthiamin in acidic solution at 25/sup 0/C (k/sub H3//k/sub D3/) is 1.09 (standard deviation (SD) 0.015) in pH 3.8, 0.5 M sodium acetate: isotope effect = 1.095 (SD 0.014) in 0.001 M HCl. The reaction was also studied using 38% ethanolic aqueous sodium acetate (pH 3.8 before mixing with ethanol) since the enzymic sites are less polar than water and the reaction is significantly accelerated by the cosolvent. The isotope effect is within statistical range of that for the reaction in water, 1.105 (SD 0.016), indicating that acceleration by the solvent does not change the extent of hyperconjugative stabilization of the transition state relative to the ground state. The isotope effect for the base-catalyzed elimination of pyruvate from lactylthiamin was determined from kinetic studies by using multiwavelength analysis for reactions in pH 11 sodium carbonate solution. The isotope effect (k/sub H3//k/sub D3/) is 1.12 (SD 0.01), which is slightly higher than the effect on decarboxylation.

  9. Heterologous expression and characterization of tyrosine decarboxylase from Enterococcus faecalis R612Z1 and Enterococcus faecium R615Z1.

    PubMed

    Liu, Fang; Xu, Wenjuan; Du, Lihui; Wang, Daoying; Zhu, Yongzhi; Geng, Zhiming; Zhang, Muhan; Xu, Weimin

    2014-04-01

    Tyrosine decarboxylase (TDC) is responsible for tyramine production and can catalyze phenylalanine to produce β-phenylethylamine. Enterococcus strains are a group of bacteria predominantly producing tyramine and β-phenylethylamine in water-boiled salted duck. In this study, the heterologous expression and characterization of two TDCs from Enterococcus faecalis R612Z1 (612TDC) and Enterococcus faecium R615Z1 (615TDC) were studied. The recombinant putative proteins of 612TDC and 615TDC were heterologously expressed in Escherichia coli. 612TDC is a 620-amino-acid protein with a molecular mass of 70.0 kDa, whereas 615TDC is a 625-amino-acid protein with a molecular mass of 70.3 kDa. Both 612TDC and 615TDC showed an optimum temperature of 25 °C for the tyrosine and phenylalanine substrates. However, 612TDC revealed maximal activity at pH 5.5, whereas 615TDC revealed maximal activity at pH 6.0. Kinetic studies showed that 612TDC and 615TDC exhibited higher specificity for tyrosine than for phenylalanine. The catalysis abilities of both 612TDC and 615TDC for phenylalanine were restrained significantly with the increase in NaCl concentration, but this was not the case for tyrosine. This study revealed that the enzyme properties of the purified recombinant 612TDC and 615TDC were similar, although their amino acid sequences had 84% identity. PMID:24680070

  10. Isotope effect studies of the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii

    SciTech Connect

    Abell, L.M.; O'Leary, M.H.

    1988-08-09

    The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k/sup 14//k/sup 15/ = 0.9770 +/- 0.0021, a carbon isotope effect k/sup 12//k/sup 13/ = 1.0308 +/- 0.0006, and a carbon isotope effect for L-(..cap alpha..-/sup 2/H)histidine of 1.0333 +/- 0.0001 at pH 6.3, 37/sup 0/C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli, the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken.

  11. The effective molarity of the substrate phosphoryl group in the transition state for yeast OMP decarboxylase.

    PubMed

    Sievers, Annette; Wolfenden, Richard

    2005-02-01

    The second order rate constant (k(cat)/K(m)) for decarboxylation of orotidine by yeast OMP decarboxylase (ODCase), measured by trapping (14)CO(2) released during the reaction, is 2 x 10(-4)M(-1)s(-1). This very low activity may be compared with a value of 3 x 10(7)M(-1)s(-1) for the action of yeast OMP decarboxylase on the normal substrate OMP. Both activities are strongly inhibited by 6-hydroxy UMP (BMP), and abrogated by mutation of Asp-96 to alanine. These results, in conjunction with the binding affinity of inorganic phosphate as a competitive inhibitor (K(i)=7 x 10(-4)M), imply an effective concentration of 1.1 x 10(9)M for the substrate phosphoryl group in stabilizing the transition state for enzymatic decarboxylation of OMP. The observed difference in rate (1.5 x 10(11)-fold) is the largest effect of a simple substituent that appears to have been reported for an enzyme reaction.

  12. 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. PMID:26588105

  13. Structural analysis of mevalonate-3-kinase provides insight into the mechanisms of isoprenoid pathway decarboxylases

    PubMed Central

    Vinokur, Jeffrey M; Korman, Tyler P; Sawaya, Michael R; Collazo, Michael; Cascio, Duillio; Bowie, James U

    2015-01-01

    In animals, cholesterol is made from 5-carbon building blocks produced by the mevalonate pathway. Drugs that inhibit the mevalonate pathway such as atorvastatin (lipitor) have led to successful treatments for high cholesterol in humans. Another potential target for the inhibition of cholesterol synthesis is mevalonate diphosphate decarboxylase (MDD), which catalyzes the phosphorylation of (R)-mevalonate diphosphate, followed by decarboxylation to yield isopentenyl pyrophosphate. We recently discovered an MDD homolog, mevalonate-3-kinase (M3K) from Thermoplasma acidophilum, which catalyzes the identical phosphorylation of (R)-mevalonate, but without concomitant decarboxylation. Thus, M3K catalyzes half the reaction of the decarboxylase, allowing us to separate features of the active site that are required for decarboxylation from features required for phosphorylation. Here we determine the crystal structure of M3K in the apo form, and with bound substrates, and compare it to MDD structures. Structural and mutagenic analysis reveals modifications that allow M3K to bind mevalonate rather than mevalonate diphosphate. Comparison to homologous MDD structures show that both enzymes employ analogous Arg or Lys residues to catalyze phosphate transfer. However, an invariant active site Asp/Lys pair of MDD previously thought to play a role in phosphorylation is missing in M3K with no functional replacement. Thus, we suggest that the invariant Asp/Lys pair in MDD may be critical for decarboxylation rather than phosphorylation. PMID:25422158

  14. Structural analysis of mevalonate-3-kinase provides insight into the mechanisms of isoprenoid pathway decarboxylases.

    PubMed

    Vinokur, Jeffrey M; Korman, Tyler P; Sawaya, Michael R; Collazo, Michael; Cascio, Duillio; Bowie, James U

    2015-02-01

    In animals, cholesterol is made from 5-carbon building blocks produced by the mevalonate pathway. Drugs that inhibit the mevalonate pathway such as atorvastatin (lipitor) have led to successful treatments for high cholesterol in humans. Another potential target for the inhibition of cholesterol synthesis is mevalonate diphosphate decarboxylase (MDD), which catalyzes the phosphorylation of (R)-mevalonate diphosphate, followed by decarboxylation to yield isopentenyl pyrophosphate. We recently discovered an MDD homolog, mevalonate-3-kinase (M3K) from Thermoplasma acidophilum, which catalyzes the identical phosphorylation of (R)-mevalonate, but without concomitant decarboxylation. Thus, M3K catalyzes half the reaction of the decarboxylase, allowing us to separate features of the active site that are required for decarboxylation from features required for phosphorylation. Here we determine the crystal structure of M3K in the apo form, and with bound substrates, and compare it to MDD structures. Structural and mutagenic analysis reveals modifications that allow M3K to bind mevalonate rather than mevalonate diphosphate. Comparison to homologous MDD structures show that both enzymes employ analogous Arg or Lys residues to catalyze phosphate transfer. However, an invariant active site Asp/Lys pair of MDD previously thought to play a role in phosphorylation is missing in M3K with no functional replacement. Thus, we suggest that the invariant Asp/Lys pair in MDD may be critical for decarboxylation rather than phosphorylation. PMID:25422158

  15. 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.

  16. 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

  17. Immunological Detection and Quantitation of Tryptophan Decarboxylase in Developing Catharanthus roseus Seedlings 1

    PubMed Central

    Fernandez, Jesus Alvarez; Owen, Terence G.; Kurz, Wolfgang G. W.; De Luca, Vincenzo

    1989-01-01

    l-Tryptophan decarboxylase (TDC) (EC 4.2.1.27) enzyme activity was induced in cell suspension cultures of Catharanthus roseus after treatment with a Pythium aphanidermatum elicitor preparation. The enzyme was extracted from lyophilized cells containing high levels of TDC and the protein was purified to homogeneity. The pure protein was used to produce highly specific polyclonal antibodies, and an enzyme-linked immunosorbent assay (ELISA) was developed to quantitate the level of TDC antigen during seedling development and in leaves of the mature plant. Western immunoblotting of proteins after SDS-PAGE with anti-TDC antibodies detected several immunoreactive proteins (40, 44, 54.8, 55, and 67 kilodaltons) which appeared at different stages during seedling development and in leaves of the mature plant. The major 54.8 and 55 kilodalton antigenic proteins in immunoblots appeared transiently between days 1 to 5 and 5 to 8 of seedling development, respectively. The 54.8 kilodalton protein was devoid of TDC enzyme activity, whereas the appearance of the 55 kilodalton protein coincided with the appearance of this decarboxylase activity. The minor immunoreactive proteins (40, 44, and 67 kilodaltons) appeared after day 5 of seedling development and in older leaves of the mature plant, and their relationship, if any, to TDC is presently unknown. Results suggest that the synthesis and degradation of TDC protein is highly regulated in Catharanthus roseus and that this regulation follows a preset developmental program. Images Figure 3 Figure 5 PMID:16667047

  18. 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.

  19. A preliminary crystallographic analysis of the putative mevalonate diphosphate decarboxylase from Trypanosoma brucei

    SciTech Connect

    Byres, Emma; Martin, David M. A.; Hunter, William N.

    2005-06-01

    The gene encoding the putative mevalonate diphosphate decarboxylase, an enzyme from the mevalonate pathway of isoprenoid precursor biosynthesis, has been cloned from T. brucei. Recombinant protein has been expressed, purified and highly ordered crystals obtained and characterized to aid the structure–function analysis of this enzyme. Mevalonate diphosphate decarboxylase catalyses the last and least well characterized step in the mevalonate pathway for the biosynthesis of isopentenyl pyrophosphate, an isoprenoid precursor. A gene predicted to encode the enzyme from Trypanosoma brucei has been cloned, a highly efficient expression system established and a purification protocol determined. The enzyme gives monoclinic crystals in space group P2{sub 1}, with unit-cell parameters a = 51.5, b = 168.7, c = 54.9 Å, β = 118.8°. A Matthews coefficient V{sub M} of 2.5 Å{sup 3} Da{sup −1} corresponds to two monomers, each approximately 42 kDa (385 residues), in the asymmetric unit with 50% solvent content. These crystals are well ordered and data to high resolution have been recorded using synchrotron radiation.

  20. Catalytic irreversible inhibition of bacterial and plant arginine decarboxylase activities by novel substrate and product analogues.

    PubMed

    Bitonti, A J; Casara, P J; McCann, P P; Bey, P

    1987-02-15

    Arginine decarboxylase (ADC) activity from Escherichia coli and two plant species (oats and barley) was inhibited by five new substrate (arginine) and product (agmatine) analogues. The five compounds, (E)-alpha-monofluoromethyldehydroarginine (delta-MFMA), alpha-monofluoromethylarginine (MFMA), alpha-monofluoromethylagatine (FMA), alpha-ethynylagmatine (EA) and alpha-allenylagmatine (AA), were all more potent inhibitors of ADC activity than was alpha-difluoromethylarginine (DFMA), the only irreversible inhibitor of this enzyme described previously. The inhibition caused by the five compounds was apparently enzyme-activated and irreversible, since the loss of enzyme activity followed pseudo-first-order kinetics, was time-dependent, the natural substrate of ADC (arginine) blocked the effects of the inhibitors, and the inhibition remained after chromatography of inhibited ADC on Sephadex G-25 or on overnight dialysis of the enzyme. DFMA, FMA, delta-MFMA and MFMA were effective at very low concentrations (10 nM-10 microM) at inhibiting ADC activity in growing E. coli. FMA was also shown to deplete putrescine effectively in E. coli, particularly when combined with an inhibitor of ornithine decarboxylase, alpha-monofluoromethyl-putrescine. The potential uses of the compounds for the study of the role of polyamine biosynthesis in bacteria and plants is discussed.

  1. Catalytic irreversible inhibition of bacterial and plant arginine decarboxylase activities by novel substrate and product analogues.

    PubMed Central

    Bitonti, A J; Casara, P J; McCann, P P; Bey, P

    1987-01-01

    Arginine decarboxylase (ADC) activity from Escherichia coli and two plant species (oats and barley) was inhibited by five new substrate (arginine) and product (agmatine) analogues. The five compounds, (E)-alpha-monofluoromethyldehydroarginine (delta-MFMA), alpha-monofluoromethylarginine (MFMA), alpha-monofluoromethylagatine (FMA), alpha-ethynylagmatine (EA) and alpha-allenylagmatine (AA), were all more potent inhibitors of ADC activity than was alpha-difluoromethylarginine (DFMA), the only irreversible inhibitor of this enzyme described previously. The inhibition caused by the five compounds was apparently enzyme-activated and irreversible, since the loss of enzyme activity followed pseudo-first-order kinetics, was time-dependent, the natural substrate of ADC (arginine) blocked the effects of the inhibitors, and the inhibition remained after chromatography of inhibited ADC on Sephadex G-25 or on overnight dialysis of the enzyme. DFMA, FMA, delta-MFMA and MFMA were effective at very low concentrations (10 nM-10 microM) at inhibiting ADC activity in growing E. coli. FMA was also shown to deplete putrescine effectively in E. coli, particularly when combined with an inhibitor of ornithine decarboxylase, alpha-monofluoromethyl-putrescine. The potential uses of the compounds for the study of the role of polyamine biosynthesis in bacteria and plants is discussed. PMID:3297044

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

    PubMed

    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

  3. Stereochemistry of 4-carboxymuconolactone decarboxylase and muconolactone isomerase in the. beta. -ketoadipate pathway

    SciTech Connect

    Whitman, C.P.; Chari, R.V.J.; Ngai, K.L.; Kozarich, J.W.

    1986-05-01

    The protocatechuate and catechol pathways, two separate and parallel branches of the ..beta..-ketoadipate pathway in Pseudomonas putida, converge at a common intermediate - ..beta..-ketoadipate enol-lactone. The enol-lactone is generated by 4-carboxymuconolactone decarboxylase in the protocatechuate pathway while muconolactone isomerase produces it in the catechol pathway. The presence of these enzymes as well as ..beta..-carboxymuconate cycloisomerase and its substrate, ..beta..-carboxy-cis,cis-muconate, in a NMR tube, leads to the following sequence of events. Lactonization of ..beta..-carboxy-cis,cis-muconate produces 4-carboxymuconolactone which decarboxylates enzymatically with deuteration by D/sub 2/O to afford 2-(/sup 2/H)-4-ketoadipate enol-lactone - the substrate for muconolactone isomerase. Further conversion of the monodeuterated enol-lactone by muconolactone isomerase affords muconolactone which is nearly completely deuterated at the 4 position. The proton ricochets between the 2 and 4 positions with concurrent washout while in the 2 position. Based on the known absolute stereochemistry of 4-carboxymuconolactone and muconolactone, these results suggest that both the decarboxylase and isomerase proceed by syn mechanisms, but operate on opposite faces of the common enol-lactone substrate.

  4. 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.

  5. 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...

  6. 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

    ...) and 1 CFR part 51. Copies may be obtained from the National Academy Press, 2101 Constitution Ave. NW... 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...

  7. 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

    ... reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies may be obtained from the National... 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...

  8. 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

    ... reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies may be obtained from the National... 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...

  9. 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

    ... reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies may be obtained from the National... 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...

  10. 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...

  11. 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

    2016-01-01

    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

  12. 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

    ...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and... Bacillus subtilis. The food additive alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation, may be safely used in accordance with the following conditions: (a) The food additive is the enzyme...

  13. Repeated immobilization stress alters rat hippocampal and prefrontal cortical morphology in parallel with endogenous agmatine and arginine decarboxylase levels

    PubMed Central

    Zhu, Meng-Yang; Wang, Wei-Ping; Huang, Jingjing; Feng, Yang-Zheng; Regunathan, Soundar; Bissette, Garth

    2008-01-01

    Agmatine, an endogenous amine derived from decarboxylation of L-arginine catalyzed by arginine decarboxylase, has been proposed as a neurotransmitter or neuromodulator in the brain. In the present study we examined whether agmatine has neuroprotective effects against repeated immobilization-induced morphological changes in brain tissues and possible effects of immobilization stress on endogenous agmatine levels and arginine decarboxylase expression in rat brains. Sprague-Dawley rats were subjected to two hour immobilization stress daily for seven days. This paradigm significantly increased plasma corticosterone levels, and the glutamate efflux in the hippocampus as measured by in vivo microdialysis. Immunohistochemical staining with β-tubulin III showed that repeated immobilization caused marked morphological alterations in the hippocampus and medial prefrontal cortex that were prevented by simultaneous treatment with agmatine (50 mg/kg/day, i.p.). Likewise, endogenous agmatine levels measured by high performance liquid chromatography in the prefrontal cortex, hippocampus, striatum and hypothalamus were significantly increased by immobilization, as compared to controls. The increased endogenous agmatine levels, ranging from 92% to 265% of controls, were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. These results demonstrate that administration of exogenous agmatine protects the hippocampus and medial prefrontal cortex against neuronal insults caused by repeated immobilization. The parallel increase in endogenous brain agmatine and arginine decarboxylase protein levels triggered by repeated immobilization indicates that the endogenous agmatine system may play an important role in adaptation to stress as a potential neuronal self-protection mechanism. PMID:18832001

  14. Insulin and phorbol myristic acetate induce ornithine decarboxylase in Reuber H35 rat hepatoma cells by different mechanisms.

    PubMed

    Goodman, S A; Esau, B; Koontz, J W

    1988-11-01

    Reuber H35 rat hepatoma cells respond to insulin or to tumor promoting phorbol esters with an increase in ornithine decarboxylase enzyme activity. This occurs in a time- and dose-dependent manner with both types of agonist. We report here that the increase in ornithine decarboxylase activity with optimal concentrations of both agonists is additive. Furthermore, the initial increase is dependent on continued RNA and protein synthesis. We also find that both of these agonists cause an increase in mRNA coding for ornithine decarboxylase in a time- and dose-dependent manner which suggests that the increase in enzyme activity can be accounted for by the increase in transcript levels. The difference in the time course of induction by the agonists, the additivity of induction by the two agonists, the differential sensitivity of induction to cycloheximide and RNA synthesis inhibitors, and the observation that phorbol myristic acetate causes a further increase in ornithine decarboxylase activity and transcript levels in cells already maximally induced by insulin suggest that these two agonists act through separate mechanisms.

  15. 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

  16. 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

  17. The Response of Dopa Decarboxylase Activity to Variations in Gene Dosage in Drosophila: A Possible Location of the Structural Gene

    PubMed Central

    Hodgetts, Ross B.

    1975-01-01

    A location of the structural gene(s) for dopa decarboxylase (EC 4.1.1.26) is proposed on the basis of enzyme determinations in a set of duplication-bearing aneuploids, which revealed only one dosage-sensitive region in the Drosophila genome. This region lies between 36EF and 37D on the left arm of chromosome 2. PMID:1126620

  18. Evidence of Two Functionally Distinct Ornithine Decarboxylation Systems in Lactic Acid Bacteria

    PubMed Central

    Romano, Andrea; Trip, Hein; Lonvaud-Funel, Aline; Lolkema, Juke S.

    2012-01-01

    Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterized in vitro, whereas transporters were heterologously expressed in Lactococcus lactis and functionally characterized in vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine and l-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol. PMID:22247134

  19. Bacterial-injection-induced syntheses of N-beta-alanyldopamine and Dopa decarboxylase in the hemolymph of coleopteran insect, Tenebrio molitor larvae.

    PubMed

    Kim, M H; Joo, C H; Cho, M Y; Kwon, T H; Lee, K M; Natori, S; Lee, T H; Lee, B L

    2000-05-01

    Injection of Escherichia coli into larvae of the coleopteran Tenebrio molitor resulted in the appearance of a dopamine-like substance on the electrochemical detector. To characterize this dopamine-like substance, we purified it to homogeneity from the immunized hemolymph and determined its molecular structure to be N-beta-alanyldopamine using the liquid chromatographic/tandem mass spectrometric method. Chemically synthesized N-beta-alanyldopamine showed the same retention time on HPLC as the purified N-beta-alanyldopamine from immunized larvae. To elucidate the molecular mechanism of N-beta-alanyldopamine synthesis in vivo, we examined the enzyme activity of Dopa decarboxylase against E. coli-injected hemolymph of T. molitor larvae. The enzyme activity of Dopa decarboxylase increased dramatically approximately 8 h after injection; Dopa decarboxylase activity of injected larvae being 10-times higher than naive larvae after 24 h. To evaluate the extent of quantitative changes of Dopa decarboxylase in response to bacterial challenge, Tenebrio Dopa decarboxylase was purified to homogeneity from the whole larvae and a cDNA clone for Tenebrio Dopa decarboxylase was isolated. RNA blot hybridization revealed that expression of the Dopa decarboxylase gene was activated transiently 3-8 h after E. coli challenge. Immunoprecipitation experiments showed that Tenebrio Dopa decarboxylase was detected from 8 to 24 h in E. coli-injected larval extract. Thus, bacterial injection into T. molitor larvae might induce transcriptional activation of a Dopa decarboxylase gene, and then synthesis of N-beta-alanyldopamine. The synthesized N-beta-alanyldopamine might be used as a substrate by phenoloxidase during melanin synthesis in the humoral defense response or the melanotic encapsulation reaction of the cellular defense response.

  20. Expression of an oxalate decarboxylase impairs the necrotic effect induced by Nep1-like protein (NLP) of Moniliophthora perniciosa in transgenic tobacco.

    PubMed

    da Silva, Leonardo F; Dias, Cristiano V; Cidade, Luciana C; Mendes, Juliano S; Pirovani, Carlos P; Alvim, Fátima C; Pereira, Gonçalo A G; Aragão, Francisco J L; Cascardo, Júlio C M; Costa, Marcio G C

    2011-07-01

    Oxalic acid (OA) and Nep1-like proteins (NLP) are recognized as elicitors of programmed cell death (PCD) in plants, which is crucial for the pathogenic success of necrotrophic plant pathogens and involves reactive oxygen species (ROS). To determine the importance of oxalate as a source of ROS for OA- and NLP-induced cell death, a full-length cDNA coding for an oxalate decarboxylase (FvOXDC) from the basidiomycete Flammulina velutipes, which converts OA into CO(2) and formate, was overexpressed in tobacco plants. The transgenic plants contained less OA and more formic acid compared with the control plants and showed enhanced resistance to cell death induced by exogenous OA and MpNEP2, an NLP of the hemibiotrophic fungus Moniliophthora perniciosa. This resistance was correlated with the inhibition of ROS formation in the transgenic plants inoculated with OA, MpNEP2, or a combination of both PCD elicitors. Taken together, these results have established a pivotal function for oxalate as a source of ROS required for the PCD-inducing activity of OA and NLP. The results also indicate that FvOXDC represents a potentially novel source of resistance against OA- and NLP-producing pathogens such as M. perniciosa, the causal agent of witches' broom disease of cacao (Theobroma cacao L.).

  1. Short-term UV-B and UV-C radiations preferentially decrease spermidine contents and arginine decarboxylase transcript levels of Synechocystis sp. PCC 6803.

    PubMed

    Jantaro, Saowarath; Pothipongsa, Apiradee; Khanthasuwan, Suparaporn; Incharoensakdi, Aran

    2011-02-01

    To investigate the short term effect of ultraviolet (UV) radiations on changes in pigments and polyamine contents, Synechocystis sp. PCC 6803 cells after exposure to UV-radiation were extracted by dimethylformamide and perchloric acid for pigments and polyamines determination, respectively. Cell growth was slightly decreased after 1 h exposure to UV-A and UV-B radiations. UV-C had little effect on cell growth despite the decrease of photosynthetic rate by about 18%. UV-A and UV-B decreased the contents of chlorophyll a and carotenoids whereas UV-C decreased chlorophyll a but had no effect on carotenoids. Spermidine contents were unaffected by UV-A, in contrast to the reduction of 25 and 50% by UV-B and UV-C, respectively. All three types of UV-radiation particularly reduced perchloric acid-insoluble spermidine. Importantly, putrescine and spermine which accounted for less than 1% of intracellular polyamines were increased by about three- to eight-fold by UV-B and UV-C, respectively. The changes in polyamines contents by UV-B and UV-C were consistent with the changes in transcript levels of arginine decarboxylase mRNA, but not with the protein levels. The decrease in the transcripts of adc2 but not adc1 was observed with UV-B and UV-C treatments.

  2. Relation between coumarate decarboxylase and vinylphenol reductase activity with regard to the production of volatile phenols by native Dekkera bruxellensis strains under 'wine-like' conditions.

    PubMed

    Sturm, M E; Assof, M; Fanzone, M; Martinez, C; Ganga, M A; Jofré, V; Ramirez, M L; Combina, M

    2015-08-01

    Dekkera/Brettanomyces bruxellensis is considered a major cause of wine spoilage, and 4-ethylphenol and 4-ethylguaiacol are the most abundant off-aromas produced by this species. They are produced by decarboxylation of the corresponding hydroxycinnamic acids (HCAs), followed by a reduction of the intermediate 4-vinylphenols. The aim of the present study was to examine coumarate decarboxylase (CD) and vinylphenol reductase (VR) enzyme activities in 5 native D. bruxellensis strains and determine their relation with the production of ethylphenols under 'wine-like' conditions. In addition, biomass, cell culturability, carbon source utilization and organic acids were monitored during 60 days. All strains assayed turned out to have both enzyme activities. No significant differences were found in CD activity, whilst VR activity was variable among the strains. Growth of D. bruxellensis under 'wine-like' conditions showed two growth phases. Sugars were completely consumed during the first growth phase. Transformation of HCAs into ethylphenols also occurred during active growth of the yeast. No statistical differences were observed in volatile phenol levels produced by the strains growing under 'wine-like' conditions, independently of the enzyme activity previously recorded. Furthermore, our results demonstrate a relationship between the physiological state of D. bruxellensis and its ability to produce ethylphenols. Inhibition of growth of D. bruxellensis in wine seems to be the most efficient way to avoid ethylphenol production and the consequent loss of wine quality.

  3. Brewers' yeast pyruvate decarboxylase produces acetoin from acetaldehyde: a novel tool to study the mechanism of steps subsequent to carbon dioxide loss.

    PubMed

    Chen, G C; Jordan, F

    1984-07-31

    A gas-liquid chromatographic technique was developed for the determination of both acetaldehyde and the 3-4% acetoin side product that results from the brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) catalyzed reaction of pyruvic acid. Employing this method enabled the demonstration of the catalysis of acetaldehyde condensation to acetoin by the enzyme. It was found that the acetoin produced enzymatically from pyruvic acid or from acetaldehyde was optically active, thus providing stereochemical information about the reaction. Deuterium kinetic isotope effects (employing CH3CHO and CH3CDO) were determined on the steady-state kinetic parameters to be 4.5 (Vmax) and 3.2 (Vmax/Kappm), respectively. This enabled, for the first time, the estimation of relative kinetic barriers for steps past decarboxylation. It could be concluded that (a) C-H bond scission was part of rate limitation in the enzyme-catalyzed condensation of acetaldehyde to acetoin and that (b) among the steps leading to the release of acetaldehyde, protonation of the key enamine intermediate was part of rate limitation. This latter finding is also directly applicable to the mechanism of pyruvate decarboxylation.

  4. Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both L-ornithine and L-lysine.

    PubMed

    Lee, Y S; Cho, Y D

    2001-12-15

    The cDNA encoding ornithine decarboxylase (ODC; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank AF 323910), and was expressed in Escherichia coli. The amino acid sequence of N. glutinosa ODC showed 90% identity with Datura stramonium ODC, and 44% identity with human ODC. N. glutinosa ODC did not possess the PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein. The purified ODC was a homodimeric protein, having a native M(r) of 92000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both l-ornithine and l-lysine with K(m) values of 562 microM and 1592 microM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by alpha-difluoromethylornithine (K(i) 1.15 microM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine (Lys(95)) and cysteine (Cys(96), Cys(338) and Cys(377)) residues, chosen by examination of the conserved sequence, which were proven by chemical modification to be involved in enzymic activity. Except for Cys(96), each mutation caused a substantial loss in enzyme activity. Most notably, Lys(95) increased the K(m) for l-ornithine by 16-fold and for l-lysine by 3-fold, with 100-fold and 2.8-fold decreases in the k(cat) for ODC and lysine decarboxylase (LDC) activity respectively. The Cys(377)-->Ala mutant possessed a k(cat) that was lowered by 23-fold, and the K(m) value was decreased by 1.4-fold for l-ornithine. The three-dimensional model of ODC protein constructed on the basis of the crystal structure of Trypanosoma brucei, mouse and human ODCs localized the four residues in the active-site cleft. This is the first work carried out on active-site residues of plant ODC, where ODC and LDC

  5. 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

  6. Suppression of ornithine decarboxylase promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

    PubMed

    Tsai, Yo-Hsian; Lin, Kuan-Lian; Huang, Yuan-Pin; Hsu, Yi-Chiang; Chen, Chung-Hwan; Chen, Yuhsin; Sie, Min-Hua; Wang, Gwo-Jaw; Lee, Mon-Juan

    2015-07-22

    Ornithine decarboxylase (ODC) is the rate-limiting enzyme for polyamine biosynthesis. Suppression of ODC by its irreversible inhibitor, α-difluoromethylornithine (DFMO), or by RNA interference through siRNA, enhanced osteogenic gene expression and alkaline phosphatase activity, and accelerated matrix mineralization of human bone marrow-derived mesenchymal stem cells (hBMSCs). Besides, adipogenic gene expression and lipid accumulation was attenuated, indicating that the enhanced osteogenesis was accompanied by down-regulation of adipogenesis when ODC was suppressed. A decrease in the intracellular polyamine content of hBMSCs during osteogenic induction was observed, suggesting that the level of endogenous polyamines is regulated during differentiation of hBMSCs. This study elucidates the role of polyamine metabolism in the lineage commitment of stem cells and provides a potential new indication for DFMO as bone-stimulating drug. PMID:26140984

  7. 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.

  8. 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.

  9. 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

  10. Discovery and characterization of gut microbiota decarboxylases that can produce the neurotransmitter tryptamine

    PubMed Central

    Williams, Brianna B.; Van Benschoten, Andrew H.; Cimermancic, Peter; Donia, Mohamed S.; Zimmermann, Michael; Taketani, Mao; Ishihara, Atsushi; Kashyap, Purna C.; Fraser, James S.; Fischbach, Michael A.

    2014-01-01

    Summary Several recent studies describe the influence of the gut microbiota on host brain and behavior. However, the mechanisms responsible for microbiota-nervous system interactions are unknown. Using a combination of genetics, biochemistry, and crystallography, we identify and characterize two phylogenetically distinct enzymes found in the human microbiome that decarboxylate tryptophan to form the β-arylamine neurotransmitter tryptamine. Although this enzymatic activity is exceedingly rare among bacteria more broadly, analysis of the Human Microbiome Project data demonstrates that at least 10% of the human population harbors at least one bacterium encoding a tryptophan decarboxylase in their gut community. Our results uncover a previously unrecognized enzymatic activity that can give rise to host-modulatory compounds and suggests a potential direct mechanism by which gut microbiota can influence host physiology, including behavior. PMID:25263219

  11. Oral putrescine restores virulence of ornithine decarboxylase-deficient Leishmania donovani in mice

    PubMed Central

    Olenyik, Tamara; Gilroy, Caslin; Ullman, Buddy

    2011-01-01

    Administration of putrescine as a 1% solution in the drinking water ameliorated the profound loss of virulence exhibited by ornithine decarboxylase (ODC) deficient Leishmania donovani in mice. Furthermore, supplying α-difluoromethylornithine, an ODC inhibitor, at 2% in the drinking water reduced but did not eliminate infection with wild type L. donovani in the mouse model. Taken collectively, these findings: 1) demonstrate that oral putrescine can access the phagolysosome of macrophages in which the parasite resides in mice; 2) establish that the loss of virulence due to the Δodc lesion is a consequence of the inability of the mutant parasite to synthesize sufficient polyamines de novo; 3) imply that the L. donovani amastigote cannot access host polyamines in sufficient amounts for survival and growth; 4) and validate ODC as a drug target, although oral administration of DFMO is an unlikely therapeutic paradigm for visceral leishmaniasis. PMID:21182873

  12. Glycine decarboxylase in C3, C4 and C3-C4 intermediate species.

    PubMed

    Schulze, Stefanie; Westhoff, Peter; Gowik, Udo

    2016-06-01

    The glycine decarboxylase complex (GDC) plays a central role in photorespiration. GDC is localized in the mitochondria and together with serine hydroxymethyltransferase it converts two molecules of glycine to one molecule of serine, CO2 and NH3. Overexpression of GDC subunits in the C3 species Arabidopsis thaliana can increase the metabolic flux through the photorespiratory pathway leading to enhanced photosynthetic efficiency and consequently to an enhanced biomass production of the transgenic plants. Changing the spatial expression patterns of GDC subunits was an important step during the evolution of C3-C4 intermediate and likely also C4 plants. Restriction of the GDC activity to the bundle sheath cells led to the establishment of a photorespiratory CO2 pump. PMID:27038285

  13. Heterologous expression of a plant arginine decarboxylase gene in Trypanosoma cruzi.

    PubMed

    Carrillo, Carolina; Serra, María P; Pereira, Claudio A; Huber, Alejandra; González, Nélida S; Algranati, Israel D

    2004-11-01

    Wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity. However, the transformation of these parasites with a recombinant plasmid containing the oat ADC cDNA coding region gave rise to the transient heterologous expression of the enzyme, suggesting the absence of endogenous mechanisms that could inhibit the expression of a hypothetical own ADC gene or the assay used to measure its enzymatic activity. The foreign ADC enzyme expressed in the transgenic T. cruzi was characterized by identification of the products, the stoichiometry of the catalysed reaction, the specific inhibition by alpha-difluoromethylarginine (DFMA) and the study of its metabolic turnover. The half-life of the heterologous ADC activity in T. cruzi was about 150 min. Bioinformatics studies and polymerase chain reaction (PCR) analyses seem to indicate the absence of ADC-like DNA sequences in the wild-type T. cruzi genome.

  14. Preliminary crystallographic data for the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from brewers' yeast.

    PubMed

    Dyda, F; Furey, W; Swaminathan, S; Sax, M; Farrenkopf, B; Jordan, F

    1990-10-15

    Single crystals of the thiamin diphosphate (the vitamin B1 coenzyme)-dependent enzyme pyruvate decarboxylase (EC 4.1.1.1) from brewers' yeast have been grown using polyethylene glycol as a precipitating agent. Crystals of the homotetrameric version alpha 4 of the holoenzyme are triclinic, space group P1, with cell constants a = 81.0, b = 82.4, c = 116.6 A, alpha = 69.5 beta = 72.6, gamma = 62.4 degrees. The crystals are reasonably stable in a rotating anode x-ray beam and diffract to at least 2.5 A resolution. The Vm value of 2.55 A/dalton is consistent with a unit cell containing four subunits with mass of approximately 60 kDa each. Rotation function results with native data indicate strong non-crystallographic 222 symmetry relating the four identical subunits, thus density averaging methods are likely to play a role in the structure determination.

  15. Effect of methionine deprivation on S-adenosylmethionine decarboxylase of tumour cells.

    PubMed

    Tisdale, M J

    1981-07-17

    Transference of Walker carcinoma and TLX5 lymphoma from normal L-methionine-containing medium to medium containing limiting amounts of L-methionine, or L-homocysteine only, caused a 2-fold increase of S-adenosylmethionine decarboxylase activity. Kinetic analysis showed an increase in the V value of the enzyme from 22 to 53 pmol/min per mg protein in media containing only 0.1 mM L-homocysteine, without any alteration in the Km value (0.1 mM). The increase in enzyme activity does not result from (a) a reduction of the intracellular level of S-adenosylmethionine, since cycloleucine, an inhibitor of methionine adenosyltransferase, had no effect on enzyme activity; (b) an increase in intracellular adenosine 3',5' monophosphate (cyclic AMP), since high extracellular concentrations of N6-monobutyryl cyclic AMP had no effect on enzyme activity; (c) an alteration of polyamine levels, since addition of micromolar concentrations of exogenous putrescine, spermidine and spermine did not prevent the induction of S-adenosylmethionine decarboxylase activity in methionine-free media containing 0.1 mM L-homocysteine. The increased enzyme activity appears to be mainly due to enhanced stabilization, since the half-life was increased from 2.45 to 5.0 h in media containing only 0.1 mM L-homocysteine. Induction of enzyme activity is specific to the removal of L-methionine, since no increase occurred in the absence of L-serine or L-glycine, or both, or by reduction of the serum concentrations in the medium.

  16. Decarboxylation of substituted cinnamic acids by lactic acid bacteria isolated during malt whisky fermentation.

    PubMed

    van Beek, S; Priest, F G

    2000-12-01

    Seven strains of Lactobacillus isolated from malt whisky fermentations and representing Lactobacillus brevis, L. crispatus, L. fermentum, L. hilgardii, L. paracasei, L. pentosus, and L. plantarum contained genes for hydroxycinnamic acid (p-coumaric acid) decarboxylase. With the exception of L. hilgardii, these bacteria decarboxylated p-coumaric acid and/or ferulic acid, with the production of 4-vinylphenol and/or 4-vinylguaiacol, respectively, although the relative activities on the two substrates varied between strains. The addition of p-coumaric acid or ferulic acid to cultures of L. pentosus in MRS broth induced hydroxycinnamic acid decarboxylase mRNA within 5 min, and the gene was also induced by the indigenous components of malt wort. In a simulated distillery fermentation, a mixed culture of L. crispatus and L. pentosus in the presence of Saccharomyces cerevisiae decarboxylated added p-coumaric acid more rapidly than the yeast alone but had little activity on added ferulic acid. Moreover, we were able to demonstrate the induction of hydroxycinnamic acid decarboxylase mRNA under these conditions. However, in fermentations with no additional hydroxycinnamic acid, the bacteria lowered the final concentration of 4-vinylphenol in the fermented wort compared to the level seen in a pure-yeast fermentation. It seems likely that the combined activities of bacteria and yeast decarboxylate p-coumaric acid and then reduce 4-vinylphenol to 4-ethylphenol more effectively than either microorganism alone in pure cultures. Although we have shown that lactobacilli participate in the metabolism of phenolic compounds during malt whisky fermentations, the net result is a reduction in the concentrations of 4-vinylphenol and 4-vinylguaiacol prior to distillation.

  17. C-terminal residues of plant glutamate decarboxylase are required for oligomerization of a high-molecular weight complex and for activation by calcium/calmodulin.

    PubMed

    Zik, Moriyah; Fridmann-Sirkis, Yael; Fromm, Hillel

    2006-05-01

    Bacterial glutamate decarboxylase (GAD) is a homohexameric enzyme of about 330 kDa. Plant GAD differs from the bacterial enzyme in having a C-terminal extension of 33 amino acids within which resides a calmodulin (CaM)-binding domain. In order to assess the role of the C-terminal extension in the formation of GAD complexes and in activation by Ca2+/CaM, we examined complexes formed with the purified full-length recombinant petunia GAD expressed in E. coli, and with a 9 amino acid C-terminal deletion mutant (GADDeltaC9). Size exclusion chromatography revealed that the full-length GAD formed complexes of about 580 kDa and 300 kDa in the absence of Ca2+/CaM, whereas in the presence of Ca2+/CaM all complexes shifted to approximately 680 kDa. With deletion of 9 amino acids from the C-terminus (KKKKTNRVC(500)), the ability to bind CaM in the presence of Ca2+, and to purify it by CaM-affinity chromatography was retained, but the formation of GAD complexes larger than 340 kDa and enzyme activation by Ca2+/CaM were completely abolished. Hence, responsiveness to Ca2+/CaM is associated with the formation of protein complexes of 680 kDa, and requires some or all of the nine C-terminal amino acid residues. We suggest that evolution of plant GAD from a bacterial ancestral enzyme involved the formation of higher molecular weight complexes required for activation by Ca2+/CaM.

  18. Critical factors governing the difference in antizyme-binding affinities between human ornithine decarboxylase and antizyme inhibitor.

    PubMed

    Liu, Yen-Chin; Liu, Yi-Liang; Su, Jia-Yang; Liu, Guang-Yaw; Hung, Hui-Chih

    2011-01-01

    Both ornithine decarboxylase (ODC) and its regulatory protein, antizyme inhibitor (AZI), can bind with antizyme (AZ), but the latter has a higher AZ-binding affinity. The results of this study clearly identify the critical amino acid residues governing the difference in AZ-binding affinities between human ODC and AZI. Inhibition experiments using a series of ODC mutants suggested that residues 125 and 140 may be the key residues responsible for the differential AZ-binding affinities. The ODC_N125K/M140K double mutant demonstrated a significant inhibition by AZ, and the IC(50) value of this mutant was 0.08 µM, three-fold smaller than that of ODC_WT. Furthermore, the activity of the AZ-inhibited ODC_N125K/M140K enzyme was hardly rescued by AZI. The dissociation constant (K(d)) of the [ODC_N125K/M140K]-AZ heterodimer was approximately 0.02 µM, which is smaller than that of WT_ODC by approximately 10-fold and is very close to the K(d) value of AZI_WT, suggesting that ODC_N125K/M140K has an AZ-binding affinity higher than that of ODC_WT and similar to that of AZI. The efficiency of the AZI_K125N/K140M double mutant in the rescue of AZ-inhibited ODC enzyme activity was less than that of AZI_WT. The K(d) value of [AZI_K125N/K140M]-AZ was 0.18 µM, nine-fold larger than that of AZI_WT and close to the K(d) value of ODC_WT, suggesting that AZI_K125N/K140M has an AZ-binding affinity lower than that of AZI_WT and similar to that of ODC. These data support the hypothesis that the differences in residues 125 and 140 in ODC and AZI are responsible for the differential AZ-binding affinities. PMID:21552531

  19. Ornithine decarboxylase antizyme finder (OAF): Fast and reliable detection of antizymes with frameshifts in mRNAs

    PubMed Central

    Bekaert, Michaël; Ivanov, Ivaylo P; Atkins, John F; Baranov, Pavel V

    2008-01-01

    Background Ornithine decarboxylase antizymes are proteins which negatively regulate cellular polyamine levels via their affects on polyamine synthesis and cellular uptake. In virtually all organisms from yeast to mammals, antizymes are encoded by two partially overlapping open reading frames (ORFs). A +1 frameshift between frames is required for the synthesis of antizyme. Ribosomes change translation phase at the end of the first ORF in response to stimulatory signals embedded in mRNA. Since standard sequence analysis pipelines are currently unable to recognise sites of programmed ribosomal frameshifting, proper detection of full length antizyme coding sequences (CDS) requires conscientious manual evaluation by a human expert. The rapid growth of sequence information demands less laborious and more cost efficient solutions for this problem. This manuscript describes a rapid and accurate computer tool for antizyme CDS detection that requires minimal human involvement. Results We have developed a computer tool, OAF (ODC antizyme finder) for identifying antizyme encoding sequences in spliced or intronless nucleic acid sequenes. OAF utilizes a combination of profile hidden Markov models (HMM) built separately for the products of each open reading frame constituting the entire antizyme coding sequence. Profile HMMs are based on a set of 218 manually assembled antizyme sequences. To distinguish between antizyme paralogs and orthologs from major phyla, antizyme sequences were clustered into twelve groups and specific combinations of profile HMMs were designed for each group. OAF has been tested on the current version of dbEST, where it identified over six thousand Expressed Sequence Tags (EST) sequences encoding antizyme proteins (over two thousand antizyme CDS in these ESTs are non redundant). Conclusion OAF performs well on raw EST sequences and mRNA sequences derived from genomic annotations. OAF will be used for the future updates of the RECODE database. OAF can also

  20. The Genetics of Dopa Decarboxylase in DROSOPHILA MELANOGASTER I. Isolation and Characterization of Deficiencies That Delete the Dopa-Decarboxylase-Dosage-Sensitive Region and the α-Methyl-Dopa-Hypersensitive Locus

    PubMed Central

    Wright, Theodore R. F.; Hodgetts, Ross B.; Sherald, Allen F.

    1976-01-01

    A detailed cytogenetic investigation of 16 overlapping deficiencies in the 36C-40A region on the left arm of the second chromosome (2L) in Drosophila melanogaster is reported. These deficiencies permit a localization of both the dopa-decarboxylase-dosage-sensitive region and the α-methyl-dopa-hypersensitive locus, l(2)amd, to the same region, 37B10-37C7. PMID:826447

  1. Study of orotidine 5'-monophosphate decarboxylase in complex with the top three OMP, BMP, and PMP ligands by molecular dynamics simulation.

    PubMed

    Jamshidi, Shirin; Jalili, Seifollah; Rafii-Tabar, Hashem

    2015-01-01

    Catalytic mechanism of orotidine 5'-monophosphate decarboxylase (OMPDC), one of the nature most proficient enzymes which provides large rate enhancement, has not been fully understood yet. A series of 30 ns molecular dynamics (MD) simulations were run on X-ray structure of the OMPDC from Saccharomyces cerevisiae in its free form as well as in complex with different ligands, namely 1-(5'-phospho-D-ribofuranosyl) barbituric acid (BMP), orotidine 5'-monophosphate (OMP), and 6-phosphonouridine 5'-monophosphate (PMP). The importance of this biological system is justified both by its high rate enhancement and its potential use as a target in chemotherapy. This work focuses on comparing two physicochemical states of the enzyme (protonated and deprotonated Asp91) and three ligands (substrate OMP, inhibitor, and transition state analog BMP and substrate analog PMP). Detailed analysis of the active site geometry and its interactions is properly put in context by extensive comparison with relevant experimental works. Our overall results show that in terms of hydrogen bond occupancy, electrostatic interactions, dihedral angles, active site configuration, and movement of loops, notable differences among different complexes are observed. Comparison of the results obtained from these simulations provides some detailed structural data for the complexes, the enzyme, and the ligands, as well as useful insights into the inhibition mechanism of the OMPDC enzyme. Furthermore, these simulations are applied to clarify the ambiguous mechanism of the OMPDC enzyme, and imply that the substrate destabilization and transition state stabilization contribute to the mechanism of action of the most proficient enzyme, OMPDC.

  2. Cysteine dioxygenase and cysteine sulfinate decarboxylase genes of the deep-sea mussel Bathymodiolus septemdierum: possible involvement in hypotaurine synthesis and adaptation to hydrogen sulfide.

    PubMed

    Nagasaki, Toshihiro; Hongo, Yuki; Koito, Tomoko; Nakamura-Kusakabe, Ikumi; Shimamura, Shigeru; Takaki, Yoshihiro; Yoshida, Takao; Maruyama, Tadashi; Inoue, Koji

    2015-03-01

    It has been suggested that invertebrates inhabiting deep-sea hydrothermal vent areas use the sulfinic acid hypotaurine, a precursor of taurine, to protect against the toxicity of hydrogen sulfide contained in the seawater from the vent. In this protective system, hypotaurine is accumulated in the gill, the primary site of sulfide exposure. However, the pathway for hypotaurine synthesis in mollusks has not been identified. In this study, we screened for the mRNAs of enzymes involved in hypotaurine synthesis in the deep-sea mussel Bathymodiolus septemdierum and cloned cDNAs encoding cysteine dioxygenase and cysteine sulfinate decarboxylase. As mRNAs encoding cysteamine dioxygenase and cysteine lyase were not detected, the cysteine sulfinate pathway is suggested to be the major pathway of hypotaurine and taurine synthesis. The two genes were found to be expressed in all the tissues examined, but the gill exhibited the highest expression. The mRNA level in the gill was not significantly changed by exposure to sulfides or thiosulfate. These results suggests that the gill of B. septemdierum maintains high levels of expression of the two genes regardless of ambient sulfide level and accumulates hypotaurine continuously to protect against sudden exposure to high level of sulfide. PMID:25501502

  3. Over-expression of mouse ornithine decarboxylase gene under the control of fruit-specific promoter enhances fruit quality in tomato.

    PubMed

    Pandey, Roopali; Gupta, Aarti; Chowdhary, Anuj; Pal, Ram Krishna; Rajam, Manchikatla Venkat

    2015-02-01

    Diamine putrescine (Put) and polyamines; spermidine (Spd) and spermine (Spm) are essential component of every cell because of their involvement in the regulation of cell division, growth and development. The aim of this study is to enhance the levels of Put during fruit development and see its implications in ripening and quality of tomato fruits. Transgenic tomato plants over-expressing mouse ornithine decarboxylase gene under the control of fruit-specific promoter (2A11) were developed. Transgenic fruits exhibited enhanced levels of Put, Spd and Spm, with a concomitant reduction in ethylene levels, rate of respiration and physiological loss of water. Consequently such fruits displayed significant delay of on-vine ripening and prolonged shelf life over untransformed fruits. The activation of Put biosynthetic pathway at the onset of ripening in transgenic fruits is also consistent with the improvement of qualitative traits such as total soluble solids, titratable acids and total sugars. Such changes were associated with alteration in expression pattern of ripening specific genes. Transgenic fruits were also fortified with important nutraceuticals like lycopene, ascorbate and antioxidants. Therefore, these transgenic tomatoes would be useful for the improvement of tomato cultivars through breeding approaches. PMID:25537646

  4. Protein rich diet suppressed net and unidirectional clearances of 6-[F-18]Fluoro-DOPA to striatum but did not alter relative DOPA decarboxylase activity

    SciTech Connect

    Kuwabara, H.; Cumming, P.; Reutens, D.

    1994-05-01

    We examined the effects of competitors in plasma (i.e., large neutral amino acids, LNAA) on the transport across the blood-brain-barrier and decarboxylation of tracer FDOPA in human striatum. We studied 10 healthy, neurologically normal subjects with the Scanditronix PC 2048-15B PET camera for 2 hours following intravenous injection of FDOPA. The subjects were either fasted overnight (n=6, ages:23-42 years) or received protein rich diet ({approximately}50 g protein) 1 hour before the study (n=4, ages:21-40 years). During the study, we obtained time-courses of radioactivity in arterial plasma and separated FDOPA and O-methyl-fluoro-DOPA with HPLC. We estimated, by means of least-squares optimization, the unidirectional blood-brain D clearance (K{sub 1}{sup D}), relative DOPA decarboxylase activity (k{sub 3}{sup D}), and effective vascular volume (V{sub 0}) in striatum while the partition volume (V{sub e}=K{sub 1}/k2) was set to the individual`s estimates of frontal lobe. The net clearance (K{sup D}) was calculated as K{sub 1}{sup D}k{sub 3}{sup D}/(k{sub 2}{sup D}+k{sub 3}{sup D}).

  5. Over-expression of mouse ornithine decarboxylase gene under the control of fruit-specific promoter enhances fruit quality in tomato.

    PubMed

    Pandey, Roopali; Gupta, Aarti; Chowdhary, Anuj; Pal, Ram Krishna; Rajam, Manchikatla Venkat

    2015-02-01

    Diamine putrescine (Put) and polyamines; spermidine (Spd) and spermine (Spm) are essential component of every cell because of their involvement in the regulation of cell division, growth and development. The aim of this study is to enhance the levels of Put during fruit development and see its implications in ripening and quality of tomato fruits. Transgenic tomato plants over-expressing mouse ornithine decarboxylase gene under the control of fruit-specific promoter (2A11) were developed. Transgenic fruits exhibited enhanced levels of Put, Spd and Spm, with a concomitant reduction in ethylene levels, rate of respiration and physiological loss of water. Consequently such fruits displayed significant delay of on-vine ripening and prolonged shelf life over untransformed fruits. The activation of Put biosynthetic pathway at the onset of ripening in transgenic fruits is also consistent with the improvement of qualitative traits such as total soluble solids, titratable acids and total sugars. Such changes were associated with alteration in expression pattern of ripening specific genes. Transgenic fruits were also fortified with important nutraceuticals like lycopene, ascorbate and antioxidants. Therefore, these transgenic tomatoes would be useful for the improvement of tomato cultivars through breeding approaches.

  6. Cysteine dioxygenase and cysteine sulfinate decarboxylase genes of the deep-sea mussel Bathymodiolus septemdierum: possible involvement in hypotaurine synthesis and adaptation to hydrogen sulfide.

    PubMed

    Nagasaki, Toshihiro; Hongo, Yuki; Koito, Tomoko; Nakamura-Kusakabe, Ikumi; Shimamura, Shigeru; Takaki, Yoshihiro; Yoshida, Takao; Maruyama, Tadashi; Inoue, Koji

    2015-03-01

    It has been suggested that invertebrates inhabiting deep-sea hydrothermal vent areas use the sulfinic acid hypotaurine, a precursor of taurine, to protect against the toxicity of hydrogen sulfide contained in the seawater from the vent. In this protective system, hypotaurine is accumulated in the gill, the primary site of sulfide exposure. However, the pathway for hypotaurine synthesis in mollusks has not been identified. In this study, we screened for the mRNAs of enzymes involved in hypotaurine synthesis in the deep-sea mussel Bathymodiolus septemdierum and cloned cDNAs encoding cysteine dioxygenase and cysteine sulfinate decarboxylase. As mRNAs encoding cysteamine dioxygenase and cysteine lyase were not detected, the cysteine sulfinate pathway is suggested to be the major pathway of hypotaurine and taurine synthesis. The two genes were found to be expressed in all the tissues examined, but the gill exhibited the highest expression. The mRNA level in the gill was not significantly changed by exposure to sulfides or thiosulfate. These results suggests that the gill of B. septemdierum maintains high levels of expression of the two genes regardless of ambient sulfide level and accumulates hypotaurine continuously to protect against sudden exposure to high level of sulfide.

  7. Over-expressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation.

    PubMed

    Hamill, J D; Robins, R J; Parr, A J; Evans, D M; Furze, J M; Rhodes, M J

    1990-07-01

    Transformed root cultures of Nicotiana rustica have been generated in which the gene from the yeast Saccharomyces cerevisiae coding for ornithine decarboxylase has been integrated. The gene, driven by the powerful CaMV35S promoter with an upstream duplicated enhancer sequence, shows constitutive expression throughout the growth cycle of some lines, as demonstrated by the analysis of mRNA and enzyme activity. The presence of the yeast gene and enhanced ornithine decarboxylase activity is associated with an enhanced capacity of cultures to accumulate both putrescine and the putrescine-derived alkaloid, nicotine. Even, however, with the very powerful promoter used in this work the magnitude of the changes seen is typically only in the order of 2-fold, suggesting that regulatory factors exist which limit the potential increase in metabolic flux caused by these manipulations. Nevertheless, it is demonstrated that flux through a pathway to a plant secondary product can be elevated by means of genetic manipulation. PMID:2103440

  8. Increase in S-adenosyl-L-methionine decarboxylase activity during the transformation of chick embroy fibroblasts by Rous sarcoma virus.

    PubMed

    Bachrach, U; Weiner, H

    1980-07-15

    The increase in S-adenosyl-L-methionine decarboxylase activity in chick embryo fibroblasts after infection with Rous sarcoma virus has been studied. It has been shown that enzyme levels in transformed cells were two or three times higher than those of the non-infected controls. The activity of this enzyme was not elevated in chick embryo fibroblasts infected with a temperature sensitive mutant of Rous sarcoma virus (RSV-T5) at 42 degrees C, the non-permissive temperature. When the temperature of these infected cultures was shifted from 42 degrees C to 37 degrees C a two- or three-fold increase in decarboxlase activity was detected after 10 to 12 h. The half-live of S-adenosyl-L-methionine decarboxylase was practically identical in normal and RSV-transformed fibroblasts.

  9. Arginine decarboxylase inhibitors reduce the capacity of Trypanosoma cruzi to infect and multiply in mammalian host cells.

    PubMed Central

    Kierszenbaum, F; Wirth, J J; McCann, P P; Sjoerdsma, A

    1987-01-01

    The capacity of blood (trypomastigote) forms of Trypanosoma cruzi to infect mouse peritoneal macrophages or rat heart myoblasts in vitro was inhibited by treatment of the trypomastigotes with DL-alpha-difluoromethylarginine (F2Me Arg), monofluoromethylagmatine, or (E)-alpha-monofluoromethyl-3-4-dehydroarginine--all irreversible inhibitors of arginine decarboxylase. Similar results were obtained when F2MeArg-treated parasites were incubated with rat heart myoblasts. The inhibitory effects were characterized by marked reductions in both the proportion of infected cells and the number of parasites per 100 host cells. The concentrations of the arginine decarboxylase inhibitors that affected infectivity had no detectable effect on either the concentration or motility of the parasite and, therefore, could not have affected the collision frequency. F2MeArg appeared to inhibit the ability of T. cruzi to penetrate the host cells since the drug had no significant effect on the extent of parasite binding to the surface of the host cells. The inhibitory effect of F2MeArg was markedly reduced or abrogated in the presence of either agmatine or putrescine, as would have been expected if F2MeArg acted by inhibiting arginine decarboxylase. Addition of F2MeArg to macrophage or myoblast cultures immediately after infection or at a time when virtually all of the intracellular parasites had transformed into the multiplicative amastigote form, resulted in a markedly reduced parasite growth rate. This effect was also prevented by exogenous agmatine. These results indicate the importance of polyamines and polyamine biosynthesis in the following two important functions of T. cruzi: invasion of host cells and intracellular multiplication. Furthermore, concentrations of the inhibitors tested that affected the parasite did not alter the viability of the host cells, the cellular density of the cultures, or the ability of uninfected myoblasts to grow. Thus, arginine decarboxylase inhibitors may

  10. The gamma-aminobutyric acid shunt contributes to closing the tricarboxylic acid cycle in Synechocystis sp PCC 6803

    SciTech Connect

    Xiong, W; Brune, D; Vermaas, WFJ

    2014-07-16

    A traditional 2-oxoglutarate dehydrogenase complex is missing in the cyanobacterial tricarboxylic acid cycle. To determine pathways that convert 2-oxoglutarate into succinate in the cyanobacterium Synechocystis sp. PCC 6803, a series of mutant strains, Delta sll1981, Delta slr0370, Delta slr1022 and combinations thereof, deficient in 2-oxoglutarate decarboxylase (Sll1981), succinate semialdehyde dehydrogenase (Slr0370), and/or in gamma-aminobutyrate metabolism (Slr1022) were constructed. Like in Pseudomonas aeruginosa, N-acetylornithine aminotransferase, encoded by slr1022, was shown to also function as gamma-aminobutyrate aminotransferase, catalysing gamma-aminobutyrate conversion to succinic semialdehyde. As succinic semialdehyde dehydrogenase converts succinic semialdehyde to succinate, an intact gamma-aminobutyrate shunt is present in Synechocystis. The Delta sll1981 strain, lacking 2-oxoglutarate decarboxylase, exhibited a succinate level that was 60% of that in wild type. However, the succinate level in the Delta slr1022 and Delta slr0370 strains and the Delta sll1981/Delta slr1022 and Delta sll1981/Delta slr0370 double mutants was reduced to 20-40% of that in wild type, suggesting that the gamma-aminobutyrate shunt has a larger impact on metabolite flux to succinate than the pathway via 2-oxoglutarate decarboxylase. C-13-stable isotope analysis indicated that the gamma-aminobutyrate shunt catalysed conversion of glutamate to succinate. Independent of the 2-oxoglutarate decarboxylase bypass, the gamma-aminobutyrate shunt is a major contributor to flux from 2-oxoglutarate and glutamate to succinate in Synechocystis sp. PCC 6803.

  11. Lactic acid bacterial cell factories for gamma-aminobutyric acid.

    PubMed

    Li, Haixing; Cao, Yusheng

    2010-11-01

    Gamma-aminobutyric acid is a non-protein amino acid that is widely present in organisms. Several important physiological functions of gamma-aminobutyric acid have been characterized, such as neurotransmission, induction of hypotension, diuretic effects, and tranquilizer effects. Many microorganisms can produce gamma-aminobutyric acid including bacteria, fungi and yeasts. Among them, gamma-aminobutyric acid-producing lactic acid bacteria have been a focus of research in recent years, because lactic acid bacteria possess special physiological activities and are generally regarded as safe. They have been extensively used in food industry. The production of lactic acid bacterial gamma-aminobutyric acid is safe and eco-friendly, and this provides the possibility of production of new naturally fermented health-oriented products enriched in gamma-aminobutyric acid. The gamma-aminobutyric acid-producing species of lactic acid bacteria and their isolation sources, the methods for screening of the strains and increasing their production, the enzymatic properties of glutamate decarboxylases and the relative fundamental research are reviewed in this article. And the potential applications of gamma-aminobutyric acid-producing lactic acid bacteria were also referred to.

  12. Isotope effect studies of the pyruvate-dependent histidine decarboxylase from Lactobacillus 30a

    SciTech Connect

    Abell, L.M.; O'Leary, M.H.

    1988-08-09

    The decarboxylation of histidine by the pyruvate-dependent histidine decarboxylase of Lactobacillus 30 a shows a carbon isotope effect k/sup 12//k/sup 13/ = 1.0334 +/- 0.0005 and a nitrogen isotope effect k/sup 14//k/sup 15/ = 0.9799 +/- 0.0006 at pH 4.8, 37/sup 0/C. The carbon isotope effect is slightly increased by deuteriation of the substrate and slightly decreased in D/sub 2/O. The observed nitrogen isotope effect indicates that the imine nitrogen in the substrate-Schiff base intermediate complex is ordinarily protonated, and the pH dependence of the carbon isotope effect indicates that both protonated and unprotonated forms of this intermediate are capable of undergoing decarboxylation. As with the pyridoxal 5'-phosphate dependent enzyme, Schiff base formation and decarboxylation are jointly rate-limiting, with the intermediate histidine-pyruvate Schiff base showing a decarboxylation/Schiff base hydrolysis ratio of 0.5-1.0 at pH 4.8. The decarboxylation transition state is more reactant-like for the pyruvate-dependent enzyme than for the pyridoxal 5'-phosphate dependent enzyme. These studies find no particular energetic or catalytic advantage to the use of pyridoxal 5'-phosphate over covalently bound pyruvate in catalysis of the decarboxylation of histidine.

  13. 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.

  14. 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.

  15. 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.

  16. Effects of feeding, fasting, and caerulein treatment on ornithine decarboxylase in rat pancreas.

    PubMed

    Langlois, A; Morisset, J

    1991-09-01

    Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis. We examined circadian variations in pancreatic ODC activity and time-course effects of caerulein in fed and fasted rats. Significant circadian variations in amount of ODC activity were observed. The highest values were obtained during the dark period (1855 +/- 406 pmoles CO2/h), and the lowest during the light period (359 +/- 84 pmoles CO2/h). Caerulein treatment induced hypertrophy and hyperplasia of the pancreas in fed rats; increases in pancreatic ODC activity preceded the rise in protein and DNA contents (447 +/- 44 pmoles CO2/h and 5573 +/- 893 pmoles CO2/h, 6 and 12 h after the first injection of caerulein, respectively). In fasted rats, pancreatic ODC activity was very low (149 +/- 37 pmoles CO2/h) and caerulein treatment induced a transient increase in this activity 12 h after the first injection; hypertrophy but not hyperplasia of the pancreas was observed. In caerulein-treated fasted rats, refeeding during the night following a 48 h fasting period was not enough to increase either ODC activity or DNA content. These findings demonstrate that nutritional status is an important factor in the regulation of ODC activity and, thereby, in caerulein-induced pancreatic growth.

  17. 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. PMID:16663805

  18. Herbacetin Is a Novel Allosteric Inhibitor of Ornithine Decarboxylase with Antitumor Activity.

    PubMed

    Kim, Dong Joon; Roh, Eunmiri; Lee, Mee-Hyun; Oi, Naomi; Lim, Do Young; Kim, Myoung Ok; Cho, Yong-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

    2016-03-01

    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

  19. 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.

  20. Relation of polymorphism of the histidine decarboxylase gene to chronic heart failure in Han Chinese.

    PubMed

    He, Gong-Hao; Cai, Wen-Ke; Meng, Jing-Ru; Ma, Xue; Zhang, Fan; Lu, Jun; Xu, Gui-Li

    2015-06-01

    Histidine decarboxylase (HDC) is a key determinant of the levels of endogenous histamine that has long been recognized to play important pathophysiological roles during development of chronic heart failure (CHF). Meanwhile, certain genetic variants in HDC gene were reported to affect the function of HDC and associated with histamine-related diseases. However, the relation between polymorphisms of HDC gene and CHF risk remains unclear. This study aims to investigate the associations between 2 nonsynonymous HDC polymorphisms (rs17740607 and rs2073440) and CHF. We designed a 2-stage case-control study, in which we genotyped 439 patients with CHF and 467 healthy controls recruited in Xi'an, China, and replicated this study in 413 patients with CHF and 452 healthy subjects in Kunming, China. We also performed in vitro experiments to further validate the functional consequences of variants positively associated with CHF. The rs17740607 polymorphism showed replicated associations with all-cause CHF according to genotype and allele distribution and also under a dominant and additive genetic model after adjusted for traditional cardiovascular-related factors. Functional experiments further demonstrated that rs17740607 polymorphism decreased the HDC activity. In conclusion, HDC rs17740607 polymorphism is at least a partial loss-of-function variant and acts as a protective factor against CHF, which provides novel highlights for investigating the contribution of CHF.

  1. 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.

  2. 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.

  3. 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.

  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. 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

  6. Regulation of human ornithine decarboxylase expression by the c-Myc.Max protein complex.

    PubMed

    Peña, A; Reddy, C D; Wu, S; Hickok, N J; Reddy, E P; Yumet, G; Soprano, D R; Soprano, K J

    1993-12-25

    The presence of a CACGTG element within a region of the human ornithine decarboxylase (ODC) promoter located at -491 to -474 base pairs 5' to the start site of transcription suggested that the c-Myc.Max protein complex may play a role in the regulation of ODC expression during growth. Electrophoretic mobility shift assays and methylation interference analysis showed that the nuclei of WI-38 cells expressing ODC contained proteins that bound to this region of the ODC gene in a manner that correlated with growth-associated ODC expression. Also, use of antibodies against c-Myc and Max and purified recombinant c-Myc and Max protein in the electrophoretic mobility shift assay confirmed that these proteins can specifically bind this portion of the human ODC promoter. Transient transfection studies showed that increase in the level of c-Myc and/or Max led to a significant enhancement of expression of a human ODC promoter-CAT reporter construct. Moreover, treatment of actively growing WI-38 cells with an antisense oligomer to c-Myc reduced the amount of endogenous protein complex formed and the amount of endogenous ODC mRNA expressed. These studies show that the c-Myc.Max protein complex plays a role in the transcriptional regulation of human ODC in vivo.

  7. 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

  8. 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.

  9. Glutamate Decarboxylase 67 Deficiency in a Subset of GABAergic Neurons Induces Schizophrenia-Related Phenotypes

    PubMed Central

    Fujihara, Kazuyuki; Miwa, Hideki; Kakizaki, Toshikazu; Kaneko, Ryosuke; Mikuni, Masahiko; Tanahira, Chiyoko; Tamamaki, Nobuaki; Yanagawa, Yuchio

    2015-01-01

    Decreased expression of the GABA synthetic enzyme glutamate decarboxylase 67 (GAD67) in a subset of GABAergic neurons, including parvalbumin (PV)-expressing neurons, has been observed in postmortem brain studies of schizophrenics and in animal models of schizophrenia. However, it is unclear whether and how the perturbations of GAD67-mediated GABA synthesis and signaling contribute to the pathogenesis of schizophrenia. To address this issue, we generated the mice lacking GAD67 primarily in PV neurons and characterized them with focus on schizophrenia-related parameters. We found that heterozygous mutant mice exhibited schizophrenia-related behavioral abnormalities such as deficits in prepulse inhibition, MK-801 sensitivity, and social memory. Furthermore, we observed reduced inhibitory synaptic transmission, altered properties of NMDA receptor-mediated synaptic responses in pyramidal neurons, and increased spine density in hippocampal CA1 apical dendrites, suggesting a possible link between GAD67 deficiency and disturbed glutamatergic excitatory synaptic functions in schizophrenia. Thus, our results indicate that the mice heterozygous for GAD67 deficiency primarily in PV neurons share several neurochemical and behavioral abnormalities with schizophrenia, offering a novel tool for addressing the underlying pathophysiology of schizophrenia. PMID:25904362

  10. Structural basis of Ornithine Decarboxylase inactivation and accelerated degradation by polyamine sensor Antizyme1

    PubMed Central

    Wu, Donghui; Kaan, Hung Yi Kristal; Zheng, Xiaoxia; Tang, Xuhua; He, Yang; Vanessa Tan, Qianmin; Zhang, Neng; Song, Haiwei

    2015-01-01

    Ornithine decarboxylase (ODC) catalyzes the first and rate-limiting step of polyamine biosynthesis in humans. Polyamines are essential for cell proliferation and are implicated in cellular processes, ranging from DNA replication to apoptosis. Excessive accumulation of polyamines has a cytotoxic effect on cells and elevated level of ODC activity is associated with cancer development. To maintain normal cellular proliferation, regulation of polyamine synthesis is imposed by Antizyme1 (AZ1). The expression of AZ1 is induced by a ribosomal frameshifting mechanism in response to increased intracellular polyamines. AZ1 regulates polyamine homeostasis by inactivating ODC activity and enhancing its degradation. Here, we report the structure of human ODC in complex with N-terminally truncated AZ1 (cAZ1). The structure shows cAZ1 binding to ODC, which occludes the binding of a second molecule of ODC to form the active homodimer. Consequently, the substrate binding site is disrupted and ODC is inactivated. Structural comparison shows that the binding of cAZ1 to ODC causes a global conformational change of ODC and renders its C-terminal region flexible, therefore exposing this region for degradation by the 26S proteasome. Our structure provides the molecular basis for the inactivation of ODC by AZ1 and sheds light on how AZ1 promotes its degradation. PMID:26443277

  11. Glutamate Decarboxylase 67 Deficiency in a Subset of GABAergic Neurons Induces Schizophrenia-Related Phenotypes.

    PubMed

    Fujihara, Kazuyuki; Miwa, Hideki; Kakizaki, Toshikazu; Kaneko, Ryosuke; Mikuni, Masahiko; Tanahira, Chiyoko; Tamamaki, Nobuaki; Yanagawa, Yuchio

    2015-09-01

    Decreased expression of the GABA synthetic enzyme glutamate decarboxylase 67 (GAD67) in a subset of GABAergic neurons, including parvalbumin (PV)-expressing neurons, has been observed in postmortem brain studies of schizophrenics and in animal models of schizophrenia. However, it is unclear whether and how the perturbations of GAD67-mediated GABA synthesis and signaling contribute to the pathogenesis of schizophrenia. To address this issue, we generated the mice lacking GAD67 primarily in PV neurons and characterized them with focus on schizophrenia-related parameters. We found that heterozygous mutant mice exhibited schizophrenia-related behavioral abnormalities such as deficits in prepulse inhibition, MK-801 sensitivity, and social memory. Furthermore, we observed reduced inhibitory synaptic transmission, altered properties of NMDA receptor-mediated synaptic responses in pyramidal neurons, and increased spine density in hippocampal CA1 apical dendrites, suggesting a possible link between GAD67 deficiency and disturbed glutamatergic excitatory synaptic functions in schizophrenia. Thus, our results indicate that the mice heterozygous for GAD67 deficiency primarily in PV neurons share several neurochemical and behavioral abnormalities with schizophrenia, offering a novel tool for addressing the underlying pathophysiology of schizophrenia. PMID:25904362

  12. 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. PMID:24993302

  13. 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

  14. 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).

  15. Role of OsHAL3 protein, a putative 4'-phosphopantothenoylcysteine decarboxylase in rice.

    PubMed

    Zhang, Ning; Wang, Xuechen; Chen, Jia

    2009-01-01

    In this study, we cloned the OsHAL3 gene from rice Oryza sativa. Alignment analysis revealed that OsHAL3 has a high sequence identity to Dfp protein in Escherichia coli and AtHAL3a protein in Arabidopsis thaliana, which have 4'-phosphopantothenoylcysteine decarboxylase (PPC-DC) activity. OsHAL3 can complement mutation in the E. coli dfp gene encoding PPC-DC, so that the mutant strains with OsHAL3 can grow on rich media at 42 degrees C and on VB minimal media at 30 degrees C. Complementation tests with point mutations of OsHAL3 suggested that the conserved Cys176 residue of OsHAL3 is a key active-site residue. The mutant OsHAL3 G180A has a partly reduced activity. Related mRNA-level analysis showed that the OsHAL3 gene is induced by calcium pantothenate in rice. PMID:19232050

  16. 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.

  17. Simultaneous Silencing of Two Arginine Decarboxylase Genes Alters Development in Arabidopsis.

    PubMed

    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

  18. Histidine decarboxylase knockout mice, a genetic model of Tourette syndrome, show repetitive grooming after induced fear

    PubMed Central

    Xu, Meiyu; Li, Lina; Ohtsu, Hiroshi; Pittenger, Christopher

    2015-01-01

    Tics, such as are seen in Tourette syndrome (TS), are common and can cause profound morbidity, but they are poorly understood. Tics are potentiated by psychostimulants, stress, and sleep deprivation. Mutations in the gene histidine decarboxylase (Hdc) have been implicated as a rare genetic cause of TS, and Hdc knockout mice have been validated as a genetic model that recapitulates phenomenological and pathophysiological aspects of the disorder. Tic-like stereotypies in this model have not been observed at baseline but emerge after acute challenge with the psychostimulant D-amphetamine. We tested the ability of an acute stressor to stimulate stereotypies in this model, using tone fear conditioning. Hdc knockout mice acquired conditioned fear normally, as manifest by freezing during the presentation of a tone 48 hours after it had been paired with a shock. During the 30 minutes following tone presentation they showed increased grooming. Heterozygotes exhibited normal freezing and intermediate grooming. These data validate a new paradigm for the examination of tic-like stereotypies in animals without pharmacological challenge and enhance the face validity of the Hdc knockout mouse as a pathophysiologically grounded model of tic disorders. PMID:25841792

  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. Histidine decarboxylase knockout mice, a genetic model of Tourette syndrome, show repetitive grooming after induced fear.

    PubMed

    Xu, Meiyu; Li, Lina; Ohtsu, Hiroshi; Pittenger, Christopher

    2015-05-19

    Tics, such as are seen in Tourette syndrome (TS), are common and can cause profound morbidity, but they are poorly understood. Tics are potentiated by psychostimulants, stress, and sleep deprivation. Mutations in the gene histidine decarboxylase (Hdc) have been implicated as a rare genetic cause of TS, and Hdc knockout mice have been validated as a genetic model that recapitulates phenomenological and pathophysiological aspects of the disorder. Tic-like stereotypies in this model have not been observed at baseline but emerge after acute challenge with the psychostimulant d-amphetamine. We tested the ability of an acute stressor to stimulate stereotypies in this model, using tone fear conditioning. Hdc knockout mice acquired conditioned fear normally, as manifested by freezing during the presentation of a tone 48h after it had been paired with a shock. During the 30min following tone presentation, knockout mice showed increased grooming. Heterozygotes exhibited normal freezing and intermediate grooming. These data validate a new paradigm for the examination of tic-like stereotypies in animals without pharmacological challenge and enhance the face validity of the Hdc knockout mouse as a pathophysiologically grounded model of tic disorders.

  1. Role of the Sulfonium Center in Determining the Ligand Specificity of Human S-Adenosylmethionine Decarboxylase

    SciTech Connect

    Bale, Shridhar; Brooks, Wesley; Hanes, Jeremiah W.; Mahesan, Arnold M.; Guida, Wayne C.; Ealick, Steven E.

    2009-08-13

    S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the polyamine biosynthetic pathway. Inhibition of this pathway and subsequent depletion of polyamine levels is a viable strategy for cancer chemotherapy and for the treatment of parasitic diseases. Substrate analogue inhibitors display an absolute requirement for a positive charge at the position equivalent to the sulfonium of S-adenosylmethionine. We investigated the ligand specificity of AdoMetDC through crystallography, quantum chemical calculations, and stopped-flow experiments. We determined crystal structures of the enzyme cocrystallized with 5{prime}-deoxy-5{prime}-dimethylthioadenosine and 5{prime}-deoxy-5{prime}-(N-dimethyl)amino-8-methyladenosine. The crystal structures revealed a favorable cation-{pi} interaction between the ligand and the aromatic side chains of Phe7 and Phe223. The estimated stabilization from this interaction is 4.5 kcal/mol as determined by quantum chemical calculations. Stopped-flow kinetic experiments showed that the rate of the substrate binding to the enzyme greatly depends on Phe7 and Phe223, thus supporting the importance of the cation-{pi} interaction.

  2. Purification and characterisation of pyruvate decarboxylase from pea seeds (Pisum sativum cv. Miko).

    PubMed

    Mücke, U; König, S; Hübner, G

    1995-02-01

    Pyruvate decarboxylase (PDC) was purified from pea seeds. The catalytically active holoenzyme is an oligomer of two types of subunits with molecular masses of about 65 kDa and 68 kDa, respectively. The active enzyme is a mixture of tetramers, octamers and even higher oligomers. These differences in the quaternary structure compared with PDC from yeast (tetramer) do not result in a different kinetic behaviour. The activity of pea PDC as well as that of yeast PDC is regulated by its substrate pyruvate resulting in a sigmoid shape of the v/S-plot. At the optimum pH of 6.0 a S0.5-value of 1 mM pyruvate is found that increases with rising pH and increasing concentrations of phosphate. The substrate analogue activator pyruvamide activates the enzyme resulting in a hyperbolic v/S-plot. The stability of PDC from pea seeds in solution is about one order of magnitude higher than that of yeast PDC. Despite the described similarities of the two enzymes no significant cross reactivity of the anti-pea PDC antibody with the enzyme from yeast occurs. PMID:7794525

  3. 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.

  4. [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. PMID:27443004

  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. Glucocorticoid hormones downregulate histidine decarboxylase mRNA and enzyme activity in rat lung.

    PubMed

    Zahnow, C A; Panula, P; Yamatodani, A; Millhorn, D E

    1998-08-01

    Histidine decarboxylase (HDC) is the primary enzyme regulating histamine biosynthesis. Histamine contributes to the pathogenesis of chronic inflammatory disorders such as asthma. Because glucocorticoids are effective in the treatment of asthma, we examined the effects of 6 h of exogenously administered dexamethasone (0.5-3,000 microg/kg ip), corticosterone (0.2-200 mg/kg ip), or endogenously elevated corticosterone (via exposure of rats to 10% oxygen) on HDC expression in the rat lung. HDC transcripts were decreased approximately 73% with dexamethasone treatment, 57% with corticosterone treatment, and 50% with exposure to 10% oxygen. Likewise, HDC enzyme activity was decreased 80% by treatment with dexamethasone and corticosterone and 60% by exposure to 10% oxygen. Adrenalectomy prevented the decreases in HDC mRNA and enzyme activity observed in rats exposed to 10% oxygen, suggesting that the adrenal gland is necessary for the mediation of hypoxic effects on HDC gene expression. These results demonstrate that corticosteroids initiate a process that leads to the decrease of HDC mRNA levels and enzyme activity in rat lung. PMID:9700103

  7. 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

  8. Substrate distortion contributes to the catalysis of orotidine 5'-monophosphate decarboxylase

    PubMed Central

    Fujihashi, Masahiro; Ishida, Toyokazu; Kuroda, Shingo; Kotra, Lakshmi P.; Pai, Emil F.; Miki, Kunio

    2014-01-01

    Orotidine 5'-monophosphate decarboxylase (ODCase) accelerates the decarboxylation of orotidine 5'-monophosphate (OMP) to uridine 5'-monophosphate (UMP) by 17 orders of magnitude. Eight new crystal structures with ligand analogues combined with computational analyses of the enzyme’s short-lived intermediates and the intrinsic electronic energies to distort the substrate and other ligands improve our understanding of the still controversially discussed reaction mechanism. In their respective complexes, 6-methyl-UMP displays significant distortion of its methyl substituent bond, 6-amino-UMP shows the competition between the K72 and C6 substituents for a position close to D70, and the methyl- and ethyl-ester of OMP both induce rotation of the carboxylate group substituent out of the plane of the pyrimidine ring. MD and QM/MM computations of the enzyme-substrate (ES) complex also show the bond between the carboxylate group and the pyrimidine ring to be distorted with the distortion contributing a 10–15% decrease of the ΔΔG‡ value. These results are consistent with ODCase using both substrate distortion as well as transition state stabilization, primarily exerted by K72, in its catalysis of the OMP decarboxylation reaction. PMID:24151964

  9. [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.

  10. 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.

  11. The role of arginine decarboxylase in modulating the sensitivity of barley to ozone.

    PubMed

    Rowland-Bamford, A J; Borland, A M; Lea, P J; Mansfield, T A

    1989-01-01

    Polyamines (PA) are known to be involved in the areas of plant physiology and biochemistry which are related to the response of a plant to air pollution. This study examines the role of arginine decarboxylase (ADC), an important rate-limiting enzyme in polyamine synthesis, in barley plants exposed to ozone (O(3)). The activity of ADC increased significantly in O(3)-treated leaves when visible injury was hardly apparent. The increase in ADC activity may be a mechanism to increase the PA levels in O(3)-treated leaves and so minimize the damaging effects of O(3). Supporting this, foliar applications of DL-alpha-difluoromethylarginine (DFMA), a specific inhibitor of ADC, prevented the rise in ADC activity and visible injury was considerable on exposure to O(3). This damage was not due to the foliar sprays, as little visible injury was seen in leaves in the O(3)-free controls. The results are discussed in terms of the roles of PA in conferring O(3) resistance in plants.

  12. Polyamine metabolism and osmotic stress. II. Improvement of oat protoplasts by an inhibitor of arginine decarboxylase.

    PubMed

    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.

  13. Ornithine decarboxylase, kidney size, and the tubular hypothesis of glomerular hyperfiltration in experimental diabetes

    PubMed Central

    Thomson, Scott C.; Deng, Aihua; Bao, Dingjiu; Satriano, Joseph; Blantz, Roland C.; Vallon, Volker

    2001-01-01

    In early diabetes, the kidney grows and the glomerular filtration rate (GFR) increases. This growth is linked to ornithine decarboxylase (ODC). The study of hyperfiltration has focused on microvascular abnormalities, but hyperfiltration may actually result from a prior increase in capacity for proximal reabsorption which reduces the signal for tubuloglomerular feedback (TGF). Experiments were performed in Wistar rats after 1 week of streptozotocin diabetes. Kidney weight, ODC activity, and GFR were correlated in diabetic and control rats given difluoromethylornithine (DFMO; Marion Merrell Dow, Cincinnati, Ohio, USA) to inhibit ODC. We assessed proximal reabsorption by micropuncture, using TGF as a tool for manipulating single-nephron GFR (SNGFR), then plotting proximal reabsorption versus SNGFR. ODC activity was elevated 15-fold in diabetic kidneys and normalized by DFMO, which also attenuated hyperfiltration and hypertrophy. Micropuncture data revealed an overall increase in proximal reabsorption in diabetic rats too great to be accounted for by glomerulotubular balance. DFMO prevented the overall increase in proximal reabsorption. These data confirm that ODC is required for the full effect of diabetes on kidney size and proximal reabsorption in early streptozotocin diabetes and are consistent with the hypothesis that diabetic hyperfiltration results from normal physiologic actions of TGF operating in a larger kidney, independent of any primary malfunction of the glomerular microvasculature. PMID:11160138

  14. Histidine Decarboxylases and Their Role in Accumulation of Histamine in Tuna and Dried Saury▿

    PubMed Central

    Kanki, Masashi; Yoda, Tomoko; Tsukamoto, Teizo; Baba, Eiichiroh

    2007-01-01

    Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae. PMID:17220267

  15. Biochemical and genetic characterization of the Enterococcus faecalis oxaloacetate decarboxylase complex.

    PubMed

    Repizo, Guillermo D; Blancato, Víctor S; Mortera, Pablo; Lolkema, Juke S; Magni, Christian

    2013-05-01

    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.

  16. Simultaneous Silencing of Two Arginine Decarboxylase Genes Alters Development in Arabidopsis.

    PubMed

    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.

  17. Induction of the Arginine Decarboxylase ADC2 Gene Provides Evidence for the Involvement of Polyamines in the Wound Response in Arabidopsis1

    PubMed Central

    Perez-Amador, Miguel A.; Leon, Jose; Green, Pamela J.; Carbonell, Juan

    2002-01-01

    Polyamines are small ubiquitous molecules that have been involved in nearly all developmental processes, including the stress response. Nevertheless, no direct evidence of a role of polyamines in the wound response has been described. We have studied the expression of genes involved in polyamine biosynthesis in response to mechanical injury. An increase in the expression of the arginine decarboxylase 2 (ADC2) gene in response to mechanical wounding and methyl jasmonate (JA) treatment in Arabidopsis was detected by using DNA microarray and RNA gel-blot analysis. No induction was observed for the ADC1 gene or other genes coding for spermidine and spermine synthases, suggesting that ADC2 is the only gene of polyamine biosynthesis involved in the wounding response mediated by JA. A transient increase in the level of free putrescine followed the increase in the mRNA level for ADC2. A decrease in the level of free spermine, coincident with the increase in putrescine after wounding, was also observed. Abscisic acid effected a strong induction on ADC2 expression and had no effect on ADC1 expression. Wound-induction of ADC2 mRNA was not prevented in the JA-insensitive coi1 mutant. The different pattern of expression of ADC2 gene in wild-type and coi1 mutant might be due to the dual regulation of ADC2 by abscisic acid and JA signaling pathways. This is the first direct evidence of a function of polyamines in the wound-response, and it opens a new aspect of polyamines in plant biology. PMID:12428010

  18. Role of cysteines in the activation and inactivation of brewers' yeast pyruvate decarboxylase investigated with a PDC1-PDC6 fusion protein.

    PubMed

    Zeng, X; Farrenkopf, B; Hohmann, S; Dyda, F; Furey, W; Jordan, F

    1993-03-16

    Possible roles of the Cys side chains in the activation and inactivation mechanisms of brewers' yeast pyruvate decarboxylase were investigated by comparing the behavior of the tetrameric enzyme pdc1 containing four cysteines/subunit (positions 69, 152, 221, and 222) with that of a fusion enzyme (pdc1-6, a result of spontaneous gene fusion between PDC1 and PDC6 genes) that is 84% identical in sequence with pdc1 and has only Cys221 (the other three Cys being replaced by aliphatic side chains). The two forms of the enzyme are rather similar so far as steady-state kinetic parameters and substrate activation are considered, as tested for activation by the substrate surrogate pyruvamide. Therefore, if a cysteine is responsible for substrate activation, it must be Cys221. The inactivation of the two enzymes was tested with several inhibitors. Methylmethanethiol sulfonate, a broad spectrum sulfhydryl reagent, could substantially inactivate both enzymes, but was slightly less effective toward the fusion enzyme. (p-Nitrobenzoyl)formic acid is an excellent alternate substrate, whose decarboxylation product p-nitrobenzaldehyde inhibited both enzymes possibly at a Cys221, the only one still present in the fusion enzyme. Exposure of the fusion enzyme, just as of pdc1, to (E)-2-oxo-4-phenyl-3-butenoic acid type inhibitors/alternate substrates enabled detection of the enzyme-bound enamine intermediate at 440 nm. However, unlike pdc1, the fusion enzyme was not irreversibly inactivated by these substrates. These substrates are now known to cause inactivation of pdc1 with concomitant modification of one Cys of the four [Zeng, X.; Chung, A.; Haran, M.; Jordan, F. (1991) J. Am. Chem. Soc. 113, 5842-49].(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Identification in Haloferax volcanii of phosphomevalonate decarboxylase and isopentenyl phosphate kinase as catalysts of the terminal enzyme reactions in an archaeal alternate mevalonate pathway.

    PubMed

    Vannice, John C; Skaff, D Andrew; Keightley, Andrew; Addo, James K; Wyckoff, Gerald J; Miziorko, Henry M

    2014-03-01

    Mevalonate (MVA) metabolism provides the isoprenoids used in archaeal lipid biosynthesis. In synthesis of isopentenyl diphosphate, the classical MVA pathway involves decarboxylation of mevalonate diphosphate, while an alternate pathway has been proposed to involve decarboxylation of mevalonate monophosphate. To identify the enzymes responsible for metabolism of mevalonate 5-phosphate to isopentenyl diphosphate in Haloferax volcanii, two open reading frames (HVO_2762 and HVO_1412) were selected for expression and characterization. Characterization of these proteins indicated that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate (in a reaction analogous to that of Methanococcus jannaschii MJ0044). The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate monophosphate, a reaction that has been proposed but never demonstrated by direct experimental proof, which is provided in this account. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to our knowledge, the first identification and characterization of any phosphomevalonate decarboxylase. PMID:24375100

  20. 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

  1. Overexpression of ornithine decarboxylase decreases ventricular systolic function during induction of cardiac hypertrophy.

    PubMed

    Giordano, Emanuele; Hillary, Rebecca A; Vary, Thomas C; Pegg, Anthony E; Sumner, Andrew D; Caldarera, Claudio M; Zhang, Xue-Qian; Song, Jianliang; Wang, JuFang; Cheung, Joseph Y; Shantz, Lisa M

    2012-02-01

    Ornithine decarboxylase (ODC), the first enzyme of polyamine metabolism, is rapidly upregulated in response to agents that induce a pathological cardiac hypertrophy. Transgenic mice overexpressing ODC in the heart (MHC-ODC mice) experience a much more dramatic left ventricular hypertrophy in response to β-adrenergic stimulation with isoproterenol (ISO) compared to wild-type (WT) controls. ISO also induced arginase activity in transgenic hearts but not in controls. The current work studies the cooperation between the cardiac polyamines and L-arginine (L-Arg) availability in MHC-ODC mice. Although ISO-induced hypertrophy is well-compensated, MHC-ODC mice administered L-Arg along with ISO showed a rapid onset of systolic dysfunction and died within 48 h. Myocytes isolated from MHC-ODC mice administered L-Arg/ISO exhibited reduced contractility and altered calcium transients, suggesting an alteration in [Ca(2+)] homeostasis, and abbreviated action potential duration, which may contribute to arrhythmogenesis. The already elevated levels of spermidine and spermine were not further altered in MHC-ODC hearts by L-Arg/ISO treatment, suggesting alternative L-Arg utilization pathways lead to dysregulation of intracellular calcium. MHC-ODC mice administered an arginase inhibitor (Nor-NOHA) along with ISO died almost as rapidly as L-Arg/ISO-treated mice, while the iNOS inhibitor S-methyl-isothiourea (SMT) was strongly protective against L-Arg/ISO. These results point to the induction of arginase as a protective response to β-adrenergic stimulation in the setting of high polyamines. Further, NO generated by exogenously supplied L-Arg may contribute to the lethal consequences of L-Arg/ISO treatment. Since considerable variations in human cardiac polyamine and L-Arg content are likely, it is possible that alterations in these factors may influence myocyte contractility.

  2. Inhibition of histidine decarboxylase ablates the autocrine tumorigenic effects of histamine in human cholangiocarcinoma

    PubMed Central

    Francis, Heather; DeMorrow, Sharon; Venter, Julie; Onori, Paolo; White, Mellanie; Gaudio, Eugenio; Francis, Taylor; Greene, John F; Tran, Steve; Meininger, Cynthia J; Alpini, Gianfranco

    2011-01-01

    Background In several tumours the endogenous activity of histidine decarboxylase (HDC), the enzyme stimulating histamine synthesis, sustains the autocrine trophic effect of histamine on cancer progression. Cholangiocarcinoma is a biliary cancer with limited treatment options. Histamine interacts with four G-protein coupled receptors, H1–H4 histamine receptors (HRs). Objective To determine the effects of histamine stimulation and inhibition of histamine synthesis (by modulation of HDC) on cholangiocarcinoma growth. Methods In vitro studies were performed using multiple human cholangiocarcinoma lines. The expression levels of the histamine synthetic machinery and HRs were evaluated along with the effects of histamine stimulation and inhibition on cholangiocarcinoma proliferation. A xenograft tumour model was used to measure tumour volume after treatment with histamine or inhibition of histamine synthesis by manipulation of HDC. Vascular endothelial growth factor (VEGF) expression was measured in cholangiocarcinoma cells concomitant with the evaluation of the expression of CD31 in endothelial cells in the tumour microenvironment. Results Cholangiocarcinoma cells display (1) enhanced HDC and decreased monoamine oxidase B expression resulting in increased histamine secretion; and (2) increased expression of H1–H4 HRs. Inhibition of HDC and antagonising H1HR decreased histamine secretion in Mz-ChA-1 cells. Long-term treatment with histamine increased proliferation and VEGF expression in cholangiocarcinoma that was blocked by HDC inhibitor and the H1HR antagonist. In nude mice, histamine increased tumour growth (up to 25%) and VEGF expression whereas inhibition of histamine synthesis (by reduction of HDC) ablated the autocrine stimulation of histamine on tumour growth (~80%) and VEGF expression. No changes in angiogenesis (evaluated by changes in CD31 immunoreactivity) were detected in the in vivo treatment groups. Conclusion The novel concept that an autocrine loop

  3. A tyrosine decarboxylase catalyzes the initial reaction of the salidroside biosynthesis pathway in Rhodiola sachalinensis.

    PubMed

    Zhang, Ji-Xing; Ma, Lan-Qing; Yu, Han-Song; Zhang, Hong; Wang, Hao-Tian; Qin, Yun-Fei; Shi, Guang-Lu; Wang, You-Nian

    2011-08-01

    Salidroside, the 8-O-β-D-glucoside of tyrosol, is the main bioactive component of Rhodiola species and is found mainly in the plant roots. It is well known that glucosylation of tyrosol is the final step in the biosynthesis of salidroside; however, the biosynthetic pathway of tyrosol and its regulation are less well understood. A summary of the results of related studies revealed that the precursor of tyrosol might be tyramine, which is synthesized from tyrosine. In this study, a cDNA clone encoding tyrosine decarboxylase (TyrDC) was isolated from Rhodiola sachalinensis A. Bor using rapid amplification of cDNA ends. The resulting cDNA was designated RsTyrDC. RNA gel-blot analysis revealed that the predominant sites of expression in plants are the roots and high levels of transcripts are also found in callus tissue culture. Functional analysis revealed that tyrosine was best substrate of recombinant RsTyrDC. The over-expression of the sense-RsTyrDC resulted in a marked increase of tyrosol and salidroside content, but the levels of tyrosol and salidroside were 274 and 412%, respectively, lower in the antisense-RsTyrDC transformed lines than those in the controls. The data presented here provide in vitro and in vivo evidence that the RsTyrDC can regulate the tyrosol and salidroside biosynthesis, and the RsTyrDC is most likely to have an important function in the initial reaction of the salidroside biosynthesis pathway in R. sachalinensis.

  4. Down-regulation of hypusine biosynthesis in Plasmodium by inhibition of S-adenosyl-methionine-decarboxylase.

    PubMed

    Blavid, Robert; Kusch, Peter; Hauber, Joachim; Eschweiler, Ute; Sarite, Salem Ramadan; Specht, Sabine; Deininger, Susanne; Hoerauf, Achim; Kaiser, Annette

    2010-02-01

    An important issue facing global health today is the need for new, effective and affordable drugs against malaria, particularly in resource-poor countries. Moreover, the currently available antimalarials are limited by factors ranging from parasite resistance to safety, compliance, cost and the current lack of innovations in medicinal chemistry. Depletion of polyamines in the intraerythrocytic phase of P. falciparum is a promising strategy for the development of new antimalarials since intracellular levels of putrescine, spermidine and spermine are increased during cell proliferation. S-adenosyl-methionine-decarboxylase (AdoMETDC) is a key enzyme in the biosynthesis of spermidine. The AdoMETDC inhibitor CGP 48664A, known as SAM486A, inhibited the separately expressed plasmodial AdoMETDC domain with a Km( i ) of 3 microM resulting in depletion of spermidine. Spermidine is an important precursor in the biosynthesis of hypusine. This prompted us to investigate a downstream effect on hypusine biosynthesis after inhibition of AdoMETDC. Extracts from P. falciparum in vitro cultures that were treated with 10 microM SAM 486A showed suppression of eukaryotic initiation factor 5A (eIF-5A) in comparison to the untreated control in two-dimensional gel electrophoresis. Depletion of eIF-5A was also observed in Western blot analysis with crude protein extracts from the parasite after treatment with 10 microM SAM486A. A determination of the intracellular polyamine levels revealed an approximately 27% reduction of spemidine and a 75% decrease of spermine while putrescine levels increased to 36%. These data suggest that inhibition of AdoMetDc provides a novel strategy for eIF-5A suppression and the design of new antimalarials. PMID:19949824

  5. 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.

  6. The Arginine Decarboxylase Pathways of Host and Pathogen Interact to Impact Inflammatory Pathways in the Lung

    PubMed Central

    Dalluge, Joseph J.; Welchlin, Cole W.; Hughes, John; Han, Wei; Blackwell, Timothy S.; Laguna, Theresa A.; Williams, Bryan J.

    2014-01-01

    The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic. PMID:25350753

  7. 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.

  8. Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells.

    PubMed Central

    McConlogue, L; Dana, S L; Coffino, P

    1986-01-01

    We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. All showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four- to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA. Images PMID:3023951

  9. Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius.

    PubMed

    Van Zyl, L J; Taylor, M P; Eley, K; Tuffin, M; Cowan, D A

    2014-02-01

    This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75 × 10(5) M(-1) s(-1) at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45-55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35 ± 0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.

  10. 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

  11. Diversity of plasmids encoding histidine decarboxylase gene in Tetragenococcus spp. isolated from Japanese fish sauce.

    PubMed

    Satomi, Masataka; Furushita, Manabu; Oikawa, Hiroshi; Yano, Yutaka

    2011-07-15

    Nineteen isolates of histamine producing halophilic bacteria were isolated from four fish sauce mashes, each mash accumulating over 1000 ppm of histamine. The complete sequences of the plasmids encoding the pyruvoyl dependent histidine decarboxylase gene (hdcA), which is harbored in histamine producing bacteria, were determined. In conjunction, the sequence regions adjacent to hdcA were analyzed to provide information regarding its genetic origin. As reference strains, Tetragenococcus halophilus H and T. muriaticus JCM10006(T) were also studied. Phenotypic and 16S rRNA gene sequence analyses identified all isolates as T. halophilus, a predominant histamine producing bacteria present during fish sauce fermentation. Genetic analyses (PCR, Southern blot, and complete plasmid sequencing) of the histamine producing isolates confirmed that all the isolates harbored approximately 21-37 kbp plasmids encoding a single copy of the hdc cluster consisting of four genes related to histamine production. Analysis of hdc clusters, including spacer regions, indicated >99% sequence similarity among the isolates. All of the plasmids sequenced encoded traA, however genes related to plasmid conjugation, namely mob genes and oriT, were not identified. Two putative mobile genetic elements, ISLP1-like and IS200-like, respectively, were identified in the up- and downstream region of the hdc cluster of all plasmids. Most of the sequences, except hdc cluster and two adjacent IS elements, were diverse among plasmids, suggesting that each histamine producers harbored a different histamine-related plasmid. These results suggested that the hdc cluster was not spread by clonal dissemination depending on the specific plasmid and that the hdc cluster in tetragenococcal plasmid was likely encoded on transformable elements. PMID:21616548

  12. Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium.

    PubMed

    Vaccaro, J A; Crane, E J; Harris, T K; Washabaugh, M W

    1995-10-01

    Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) exhibits biphasic kinetics with 52 +/- 11% of the PDC reacting with k1 = (1.0 +/- 0.3) x 10(-2) s-1 and 48 +/- 12% of the PDC reacting with k2 = (1.1 +/- 0.6) x 10(-1) s-1 when TDP (KTDP = 0.5 +/- 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 +/- 0.5) x 10(-2) s-1 upon addition of Mg2+ (KMg2+ = 0.2 +/- 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 < or = 3-fold and has no effect on the value of k1.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Substrate activation of brewers' yeast pyruvate decarboxylase is abolished by mutation of cysteine 221 to serine.

    PubMed

    Baburina, I; Gao, Y; Hu, Z; Jordan, F; Hohmann, S; Furey, W

    1994-05-10

    Brewers' yeast pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate and Mg(II)-dependent enzyme, isolated from Saccharomyces cerevisiae possesses four cysteines/subunit at positions 69, 152, 221, and 222. Earlier studies conducted on a variant of the enzyme with a single Cys at position 221 (derived from a gene that was the product of spontaneous fusion) showed that this enzyme is still subject to substrate activation [Zeng, X., Farrenkopf, B., Hohmann, S., Jordan, F., Dyda, F., & Furey, W. (1993) Biochemistry 32, 2704-2709], indicating that if Cys was responsible for this activation, it had to be C221. To further test the hypothesis, the C221S and C222S single and the C221S-C222S double mutants were constructed. It is clearly shown that the mutation at C221, but not at C222, leads to abolished substrate activation according to a number of kinetic criteria, both steady state and pre steady state. On the basis of the three-dimensional structure of the enzyme [Dyda, F., Furey, W., Swaminathan, S., Sax, M., Farrenkopf, B., Jordan, F. (1993) Biochemistry 32, 6165-6170], it is obvious that while C221 is located on the beta domain, whereas thiamin diphosphate is wedged at the interface of the alpha and gamma domains, addition of pyruvate or pyruvamide as a hemiketal adduct to the sulfur of C221 can easily bridge the gap between the beta and alpha domains. In fact, residues in one or both domains must be dislocated by this adduct formation. It is very likely that regulation as expressed in substrate activation is transmitted via this direct contact made between the two domains in the presence of the activator.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. [Ornithine decarboxylase in mammalian organs and tissues at hibernation and artificial hypobiosis].

    PubMed

    Logvinovich, O S; Aksenova, G E

    2013-01-01

    Ornithine decarboxylase (ODC, EC 4.1.1.17.) is a short-lived and dynamically regulated enzyme of polyamines biosynthesis. Regulation of functional, metabolic and proliferative state of organs and tissues involves the modifications of the ODC enzymatic activity. The organ-specific changes in ODC activity were revealed in organs and tissues (liver, spleen, bone marrow, kidney, and intestinal mucosa) of hibernating mammals - squirrels Spermophilus undulates - during the hibernating season. At that, a positive correlation was detected between the decline and recovery of the specialized functions of organs and tissues and the respective modifications of ODC activity during hibernation bouts. Investigation of changes in ODC activity in organs and tissues of non-hibernating mammals under artificial hypobiosis showed that in Wistar rats immediately after exposure to hypothermia-hypoxia-hypercapnia (hypobiosis) the level of ODC activity was low in thymus, spleen, small intestine mucosa, neocortex, and liver. The most marked reduction in enzyme activity was observed in actively proliferating tissues: thymus, spleen, small intestine mucosa. In bone marrow of squirrels, while in a state of torpor, as well as in thymus of rats after exposure to hypothermia-hypoxia-hypercapnia, changes in the ODC activity correlated with changes in the rate of cell proliferation (by the criterion of cells distribution over cell cycle). The results obtained, along with the critical analysis of published data, indicate that the ODC enzyme is involved in biochemical adaptation of mammals to natural and artificial hypobiosis. A decline in the ODC enzymatic activity indicates a decline in proliferative, functional, and metabolic activity of organs and tissues of mammals (bone marrow, mucosa of small intestine, thymus, spleen, neocortex, liver, kidneys) when entering the state of hypobiosis.

  15. Chemically-induced formation of an inhibitor of hepatic uroporphyrinogen decarboxylase in inbred mice with iron overload.

    PubMed Central

    Smith, A G; Francis, J E

    1987-01-01

    An inhibitor of hepatic uroporphyrinogen decarboxylase (EC 4.1.1.37) was demonstrated in heat-treated extracts of livers from C57BL/10ScSn mice with iron overload after a single dose (100 mg/kg; 350 mumol/kg) of hexachlorobenzene (HCB). Inhibition was not due to accumulated uroporphyrin since this could be removed by a SEP-PAK C18 cartridge without affecting inhibitor activity. The presence of the inhibitor could be first demonstrated 2 weeks after mice received HCB and before major elevation of hepatic porphyrin levels. Maximum inhibitory potential was reached at about 8 weeks and was still detected 25 weeks after the chemical, thus paralleling the depression of enzyme activity reported previously [Smith, Francis, Kay, Greig & Stewart (1986) Biochem. J. 238, 871-878]. The inhibitor was not detected following treatment of mice with either iron or HCB alone or after the decarboxylase activity was destroyed in vitro by the combination of uroporphyrin and light. The formation of the inhibitor by inbred mouse strains nominally Ah-responsive (C57BL/6J, C57BL/10ScSn, BALB/c, C3H/HeJ, CBA/J and A/J) and Ah-nonresponsive (SWR, AKR, 129, SJL, LP and DBA/2) did not correlate fully with their reported Ah-phenotype. There was a correlation amongst the Ah-responsive strains only, with hepatic ethoxyphenoxazone de-ethylase activity induced in parallel experiments by treatment with beta-naphthoflavone. De-ethylase activity induced by HCB, however, was considerably less than that with beta-naphthoflavone, which has not been reported as porphyrogenic. Other polyhalogenated chemicals, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,2',3',4'-hexachlorobiphenyl and hexabromobenzene, also caused the formation of the inhibitor of uroporphyrinogen decarboxylase. PMID:3675556

  16. 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.

  17. A known and a novel mutation in the glycine decarboxylase gene in a newborn with classic nonketotic hyperglycinemia.

    PubMed

    Beijer, P; Lichtenbelt, K D; Hofstede, F C; Nikkels, P G J; Lemmers, P; de Vries, L S

    2012-06-01

    A term neonate displayed typical features of nonketotic hyperglycinemia (NKH). Conventional magnetic resonance imaging showed corpus callosum hypoplasia and increased signal intensity of the white matter. Magnetic resonance proton spectroscopy revealed high cerebral glycine levels. The liquor/plasma glycine ratio was increased. Genetic testing detected a known and a novel mutation in the glycine decarboxylase gene, leading to the classic form of glycine encephalopathy. Prenatal genetic testing in the subsequent pregnancy showed that this fetus was not affected. As features of neonatal NKH may not be very specific, recognition of the disease may be difficult. An overview of clinical, electroencephalography, and neuroimaging findings is given in this article. PMID:22610665

  18. Study of orotidine 5'-monophosphate decarboxylase in complex with the top three OMP, BMP, and PMP ligands by molecular dynamics simulation.

    PubMed

    Jamshidi, Shirin; Jalili, Seifollah; Rafii-Tabar, Hashem

    2015-01-01

    Catalytic mechanism of orotidine 5'-monophosphate decarboxylase (OMPDC), one of the nature most proficient enzymes which provides large rate enhancement, has not been fully understood yet. A series of 30 ns molecular dynamics (MD) simulations were run on X-ray structure of the OMPDC from Saccharomyces cerevisiae in its free form as well as in complex with different ligands, namely 1-(5'-phospho-D-ribofuranosyl) barbituric acid (BMP), orotidine 5'-monophosphate (OMP), and 6-phosphonouridine 5'-monophosphate (PMP). The importance of this biological system is justified both by its high rate enhancement and its potential use as a target in chemotherapy. This work focuses on comparing two physicochemical states of the enzyme (protonated and deprotonated Asp91) and three ligands (substrate OMP, inhibitor, and transition state analog BMP and substrate analog PMP). Detailed analysis of the active site geometry and its interactions is properly put in context by extensive comparison with relevant experimental works. Our overall results show that in terms of hydrogen bond occupancy, electrostatic interactions, dihedral angles, active site configuration, and movement of loops, notable differences among different complexes are observed. Comparison of the results obtained from these simulations provides some detailed structural data for the complexes, the enzyme, and the ligands, as well as useful insights into the inhibition mechanism of the OMPDC enzyme. Furthermore, these simulations are applied to clarify the ambiguous mechanism of the OMPDC enzyme, and imply that the substrate destabilization and transition state stabilization contribute to the mechanism of action of the most proficient enzyme, OMPDC. PMID:24559040

  19. Role of a Guanidinium Cation-Phosphodianion Pair in Stabilizing the Vinyl Carbanion Intermediate of Orotidine 5'-Phosphate Decarboxylase-Catalyzed Reactions.†

    PubMed Central

    Goryanova, Bogdana; Goldman, Lawrence M.; Amyes, Tina L.; Gerlt, John A; Richard, John P.

    2013-01-01

    The side chain cation of Arg235 provides a 5.6 and 2.6 kcal/mol stabilization of the transition states for orotidine 5'-monophosphate decarboxylase from Saccharomyces cerevisiae (OMPDC) catalyzed reactions of OMP and 5-fluoroorotidine 5'-monophosphate (FOMP), respectively, a 7.2 kcal/mol stabilization of the vinyl carbanion-like transition state for enzyme-catalyzed exchange of the C-6 proton of 5-fluorouridine 5'-monophosphate (FUMP), but no stabilization of the transition states for enzyme-catalyzed decarboxylation of truncated substrates 1-(β-d-erythrofuranosyl)orotic acid and 1-(β-d-erythrofuranosyl) 5-fluorouracil. These observations show that the transition state stabilization results from formation of a protein cation-phosphodianion pair, and that there is no detectable stabilization from an interaction between the side chain and the pyrimidine ring of substrate. The 5.6 kcal/mol side chain interaction with the transition state for the decarboxylation reaction is 50% of the total 11.2 kcal/mol transition state stabilization by interactions with the phosphodianion of OMP, while the 7.2 kcal/mol side-chain interaction with the transition state for the deuterium exchange reaction is a larger 78% of the total 9.2 kcal/mol transition state stabilization by interactions with the phosphodianion of FUMP. The effect of the R235A mutation on the enzyme-catalyzed deuterium exchange is expressed predominantly as a change in the turnover number kex while the effect on the enzyme-catalyzed decarboxylation of OMP is expressed predominantly as a change in the Michaelis constant Km. These results are rationalized by a mechanism in which the binding of OMP, compared with FUMP, provides a larger driving force for conversion of OMPDC from an inactive open conformation to a productive, active, closed conformation. PMID:24053466

  20. Overexpression of 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases causes growth defects possibly due to abnormal auxin transport in Arabidopsis.

    PubMed

    Kim, Bokyung; Kim, Gyusik; Fujioka, Shozo; Takatsuto, Suguru; Choe, Sunghwa

    2012-07-01

    Sterols play crucial roles as membrane components and precursors of steroid hormones (e.g., brassinosteroids, BR). Within membranes, sterols regulate membrane permeability and fluidity by interacting with other lipids and proteins. Sterols are frequently enriched in detergent-insoluble membranes (DIMs), which organize molecules involved in specialized signaling processes, including auxin transporters. To be fully functional, the two methyl groups at the C-4 position of cycloartenol, a precursor of plant sterols, must be removed by bifunctional 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases (3βHSD/D). To understand the role of 3βHSD/D in Arabidopsis development, we analyzed the phenotypes of knock-out mutants and overexpression lines of two 3βHSD/D genes (At1g47290 and At2g26260). Neither single nor double knock-out mutants displayed a noticeable phenotype; however, overexpression consistently resulted in plants with wrinkled leaves and short inflorescence internodes. Interestingly, the internode growth defects were opportunistic; even within a plant, some stems were more severely affected than others. Endogenous levels of BRs were not altered in the overexpression lines, suggesting that the growth defect is not primarily due to a flaw in BR biosynthesis. To determine if overexpression of the sterol biosynthetic genes affects the functions of membrane-localized auxin transporters, we subjected plants to the auxin efflux carrier inhibitor, 1-N-naphthylphthalamic acid (NPA). Where-as the gravity vectors of wild-type roots became randomly scattered in response to NPA treatment, those of the overexpression lines continued to grow in the direction of gravity. Overexpression of the two Arabidopsis 3βHSD/D genes thus appears to affect auxin transporter activity, possibly by altering sterol composition in the membranes.

  1. L-methionine decarboxylase from Dryopteris filix-mas: Purification, characterization, substrate specificity, abortive transamination of the coenzyme, and stereochemical courses of substrate decarboxylation and coenzyme transamination

    SciTech Connect

    Stevenson, D.E.; Akhtar, M.; Gani, D. )

    1990-08-21

    L-Methionine decarboxylase from the male fern Dryopteris filix-mas has been purified 256-fold from acetone powder extracts to very near homogeneity. The enzyme is membrane-associated and requires detergent for solubilization during the initial extraction. The enzyme is a homodimer of subunit M{sub r} 57,000 and shows a pH optimum at {approximately} 5.0 with 20 mM (2S)-methionine as substrate. A wide range of straight- and branched-chain (2S)-alkylamino acids are substrates for the enzyme. The values for the rate of decarboxylation, V{sub max}, and for the apparent Michaelis constant, K{sub m}, however, vary with structure and with the chirality at C-3. The pH dependence of V and V/K has been examined for three substrates: (2S)-methionine, valine, and leucine. The occurrence of the abortive reaction was confirmed by showing that ({sup 35}S)methionine is converted to labeled 3-(methylthio)propionaldehyde while (4{prime}-{sup 3}H)PLP is converted to labeled pyridoxamine 5{prime}-phosphate (PMP). The decarboxylation of (2S)-methionine gave 3(methylthio)-1-aminopropane. Preparation of the N-camphanamide derivative of the amine allowed the C-1 methylene protons to be distinguished by {sup 1}H NMR spectroscopy. Synthetic samples of the camphanamide were prepared in which each of the C-1 methylene protons was replaced by deuterium. When tritiated pyridoxal phosphate was incubated with the enzyme, tritiated pyridoxamine phosphate was formed. These results are used to construct possible mechanistic schemes for both reactions, decarboxylation and transamination. The position and possible identities of active-site proton donors are discussed.

  2. Detection and Time Course of Formation of Major Thiamin Diphosphate-Bound Covalent Intermediates Derived from a Chromophoric Substrate Analogue on Benzoylformate Decarboxylase

    SciTech Connect

    Chakraborty, Sumit; Nemeria, Natalia S.; Balakrishnan, Anand; Brandt, Gabriel S.; Kneen, Malea M.; Yep, Alejandra; McLeish, Michael J.; Kenyon, George L.; Petsko, Gregory A.; Ringe, Dagmar; Jordan, Frank

    2009-04-02

    The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with {lambda}{sub max} at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with {lambda}{sub max} at 437 nm. The time course of formation/depletion of the PKB-ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB-ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with {lambda}{sub max} at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 {angstrom}) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2{alpha} atom, rather than of the enamine, and supporting the assignment of the {lambda}{sub max} at 473 nm to the PAA-ThDP adduct. The structure of the PAA-ThDP covalent complex is the first example of a product-ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.

  3. Inhibition of cytochrome P450 isozymes and ornithine decarboxylase activities by polysaccharides from soybeans fermented with Phellinus igniarius or Agrocybe cylindracea.

    PubMed

    Shon, Yun-Hee; Nam, Kyung-Soo

    2004-01-01

    Polysaccharides (5, 10, 25, 50 and 100 microg ml(-1)) from soybeans and soybeans fermented with Phellinus igniarius or Agrocybe cylindracea inhibited cytochrome P450 1A1, cytochrome P450 1A2 and cytochrome P450 2B1 activities in rat liver microsomes. The polysaccharides (5, 10 and 25 microg ml(-1)) also suppressed 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity. The most potent inhibitors of cytochrome P450 isozymes and ornithine decarboxylase activities were the polysaccharides from soybeans fermented with Agrocybe cylindracea. PMID:15000485

  4. Induction of dopa (3,4-dihydroxyphenylalanine) decarboxylase in blowfly integument by ecdysone. A demonstration of synthesis of the enzyme de novo.

    PubMed Central

    Fragoulis, E G; Sekeris, C E

    1975-01-01

    The activity of the enzyme dopa (3,4-dihydroxyphenylalanine) decarboxylase, present in the epidermis cells of blowfly larvae, increases during the late third instar under the influence of the steroid hormone, ecdysone. By using the double-labelling technique and immune precipitation with univalent antibody to dopa decarboxylase, we demonstrated that the increase in enzyme activity was due to a stimulation of synthesis of enzyme molecules de novo. In this respect, the action of ecdysone is similar to the action of other steroid hormones. Images PLATE 1 PLATE 2 PMID:807198

  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. Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxylase results in a dark brown phenotype and stunted growth.

    PubMed

    Kanjanaphachoat, Parawee; Wei, Bi-Yin; Lo, Shuen-Fang; Wang, I-Wen; Wang, Chang-Sheng; Yu, Su-May; Yen, Ming-Liang; Chiu, Sheng-Hsien; Lai, Chien-Chen; Chen, Liang-Jwu

    2012-04-01

    A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxylase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxylase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5' hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, darkbrown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the darkbrown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.

  7. Arginine Decarboxylase of Oats Is Clipped from a Precursor into Two Polypeptides Found in the Soluble Enzyme 1

    PubMed Central

    Malmberg, Russell L.; Smith, Katherine E.; Bell, Erin; Cellino, Michael L.

    1992-01-01

    We have examined soluble oat (Avena sativa) arginine decarboxylase by probing its structure with polyclonal antibodies that separately recognize amino-terminal and carboxyl-terminal antigens and with a monoclonal antibody that immunoprecipitates enzyme activity. These experiments indicated that oat arginine decarboxylase is clipped from a 66,000-D precursor polypeptide into 42,000- and 24,000-D produce polypeptides. Both of these are found in the enzyme and may be held together by disulfide bonds. A full-length precursor protein could not be detected in plants but could be produced by expression of the cDNA in Escherichia coli. Analysis of the expression of the cDNA in E. coli, with antibodies and using pulse labeling with [35S]methionine, indicated that the bulk of the expressed protein was the full-length 66,000-D form. Small amounts of 42,000- and 24,000-D polypeptides could also be detected. A reconstruction experiment, adding a radioactively labeled full-length protein isolated from E. coli to powdered oat leaves, supported the idea that the protein extraction method used for western blots was not likely to result in artifactual proteolytic degradation. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:16652937

  8. 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.

  9. Filarial parasites possess an antizyme but lack a functional ornithine decarboxylase.

    PubMed

    Kurosinski, Marc-André; Lüersen, Kai; Ndjonka, Dieudonne; Younis, Abuelhassan Elshazly; Brattig, Norbert W; Liebau, Eva

    2013-06-01

    In eukaryotes, the key player in polyamine metabolism is the ornithine decarboxylase (ODC) that catalyses the first and rate limiting step in cellular polyamine synthesis. The half life of ODC is strictly regulated by the antizyme (AZ), which promotes its degradation. Older reports on the polyamine situation in filarial parasites indicate a lack of ornithine decarboxylation activity and an increased uptake of polyamines. Our in silico analysis of the Brugia malayi genome revealed only an ODC-like protein that lacks essential residues. Consequently, the recombinant protein had no enzymatic ODC activity. Furthermore, only ODC-like genes were found in the available draft genomes of other filarial parasites. In this ODC-free scenario, we set out to investigate the AZ of O. volvulus (OvAZ). The expression of the recombinant protein allowed us to analyse the localization of OvAZ in different O. volvulus stages as well as to identify it as target for the human humoral immune response. Strong immunostaining was observed in the outer zone of the uterine epithelium as well as in the uterus lumen around the periphery of the developing parasite, indicating a potential role of the OvAZ in the control of polyamine levels during embryonic development. By employing a novel in vivo method using Caenorhabditis elegans, we postulate that the OvAZ enters the secretory pathway. Even though the ODCs are absent in filarial parasites, OvAZ has the ability to bind to various ODCs, thereby demonstrating the functionality of the conserved AZ-binding domains. Finally, pull-down assays show an interaction between B. malayi AZ and the B. malayi ODC-like protein, indicating that the B. malayi ODC-like protein might function as an AZI. Taken together, our results suggest that filarial species do not possess the ODC while retaining the ODC-regulatory proteins AZ and AZI. It is tempting to speculate that both proteins are retained for the regulation of polyamine transport systems. PMID:23474393

  10. 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.

  11. 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

  12. Heavy atom isotope effects on the reaction catalyzed by the oxalate decarboxylase from Bacillus subtilis.

    PubMed

    Reinhardt, Laurie A; Svedruzic, Drazenka; Chang, Christopher H; Cleland, W Wallace; Richards, Nigel G J

    2003-02-01

    Oxalate decarboxylase (OxDC) catalyzes a remarkable transformation in which the C-C bond in oxalate is cleaved to give carbon dioxide and formate. Like the native OxDC isolated from Aspergillus niger, the recombinant, bacterial OxDC from Bacillus subtilis contains Mn(II) in its resting state and requires catalytic dioxygen for activity. The most likely mechanism for OxDC-catalyzed C-C bond cleavage involves the participation of free radical intermediates, although this hypothesis remains to be unequivocally demonstrated. Efforts to delineate the catalytic mechanism have been placed on a firm foundation by the high-resolution crystal structure of recombinant, wild type B. subtilis OxDC (Anand et al., Biochemistry 2002, 41, 7659-7669). We now report the results of heavy-atom kinetic isotope effect measurements for the OxDC-catalyzed decarboxylation of oxalate, in what appear to be the first detailed studies of the mechanism employed by OxDC. At pH 4.2, the OxDC-catalyzed formation of formate and CO(2) have normal (13)C isotope effects of 1.5% +/- 0.1% and 0.5% +/- 0.1%, respectively, while the (18)O isotope effect on the formation of formate is 1.1% +/- 0.2% normal. Similarly at pH 5.7, the production of formate and CO(2) exhibits normal (13)C isotope effects of 1.9% +/- 0.1% and 0.8% +/- 0.1%, respectively, and the (18)O isotope effect on the formation of formate is 1.0% +/- 0.2% normal. The (18)O isotope effect on the formation of CO(2), however, 0.7% +/- 0.2%, is inverse at pH 5.7. These results are consistent with a multistep model in which a reversible, proton-coupled, electron transfer from bound oxalate to the Mn-enzyme gives an oxalate radical, which decarboxylates to yield a formate radical anion. Subsequent reduction and protonation of this intermediate then gives formate.

  13. Mechanism of the orotidine 5'-monophosphate decarboxylase-catalyzed reaction: evidence for substrate destabilization.

    PubMed

    Chan, Kui K; Wood, B McKay; Fedorov, Alexander A; Fedorov, Elena V; Imker, Heidi J; Amyes, Tina L; Richard, John P; Almo, Steven C; Gerlt, John A

    2009-06-23

    The reaction catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) involves a stabilized anionic intermediate, although the structural basis for the rate acceleration (k(cat)/k(non), 7.1 x 10(16)) and proficiency [(k(cat)/K(M))/k(non), 4.8 x 10(22) 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 (>or=10(10)). The origin of the "missing" contribution, or=10(10)), 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 [Wu, N., Mo, Y., Gao, J., and Pai, E. F. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 2017-2022]. 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 k(cat) for decarboxylation of FOMP (a more reactive substrate analogue) but have little effect on the value of k(ex) 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 a vis the active site residues. We propose that the differential effects of substitutions for Asp 70 of MtOMPDC on decarboxylation and

  14. Solvent-derived protons in catalysis by brewers' yeast pyruvate decarboxylase.

    PubMed

    Harris, T K; Washabaugh, M W

    1995-10-31

    Catalysis of proton transfer to thiamin diphosphate (TDP) and 2-(1-hydroxyethyl)thiamin diphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determining the solvent discrimination tritium isotope effect, (kH/kT)disc, on the reaction of pyruvate to form acetaldehyde in the presence of the nonsubstrate allosteric effector pyruvamide. The fractionation factors for TDP C(2)-L (phi C(2) = 0.98 +/- 0.06) and HETDP C(alpha)-L (phi C(alpha) = 1.01 +/- 0.07) (L = H or D) do not contribute significantly to observed enzymic isotopic discrimination. The value of (kH/kT)disc = 1.0 for reprotonation of TDP C(2)-L under single-turnover conditions ([E] > [S]) is consistent with C(2)-hydron transfer via a catalytic group (phi = 1) equilibrated with solvent. [1-L]Acetaldehyde formation under transient steady-state ([E] < [S]) conditions shows solvent discrimination tritium isotope effects that increase over the range (kH/kT)disc = 0.39 (single turnover) to 0.86 (ten turnovers). The 2-fold increase in the value of (kH/kT)disc for the [1-L]acetaldehyde product under steady-state compared to single-turnover conditions is attributed to a fractionation factor of phi 1 = 0.88 +/- 0.06 for the residue(s) involved in C(alpha)-hydron transfer to form HETDP. This provides evidence that catalysis of acetaldehyde formation by PDC involves specific protonation of both HETDP C(alpha)-L and TDP C(2)-L (phi 2 = 1.0 +/- 0.1) and requires at least two catalytic groups. Values of phi < or = 1 for protonation of TDP C(2)-L and HETDP C(alpha)-L provide no evidence that the exocyclic 4'-amino or -imino group (phi > or = 1.2) provides significant intramolecular catalysis in the enzyme-bound coenzyme.

  15. 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

  16. Differential Regulation of Glutamic Acid Decarboxylase Gene Expression after Extinction of a Recent Memory vs. Intermediate Memory

    ERIC Educational Resources Information Center

    Sangha, Susan; Ilenseer, Jasmin; Sosulina, Ludmila; Lesting, Jorg; Pape, Hans-Christian

    2012-01-01

    Extinction reduces fear to stimuli that were once associated with an aversive event by no longer coupling the stimulus with the aversive event. Extinction learning is supported by a network comprising the amygdala, hippocampus, and prefrontal cortex. Previous studies implicate a critical role of GABA in extinction learning, specifically the GAD65…

  17. Identification of a Tyrosine Decarboxylase Gene (tdcA) in Streptococcus thermophilus 1TT45 and Analysis of Its Expression and Tyramine Production in Milk▿

    PubMed Central

    La Gioia, Federica; Rizzotti, Lucia; Rossi, Franca; Gardini, Fausto; Tabanelli, Giulia; Torriani, Sandra

    2011-01-01

    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. PMID:21131517

  18. Experiment K-7-21: Effect of Microgravity on 1: Metabolic Enzymes of Type 1 and Type 2 Muscle Fibers, and on 2: Metabolic Enzymes, Neurotransmitter Amino Acids, and Neurotransmitter Associated Enzymes in Selected Regions of the Central Nervous System. Part 2; The Distribution of Selected Enzymes and Amino Acids in the Hippocampal Formation

    NASA Technical Reports Server (NTRS)

    Lowry, O. H.; Krasnov, I.; Ilyina-Kakueva, E. I.; Nemeth, P. M.; McDougal, D. B., Jr.; Choksi, R.; Carter, J. G.; Chi, M. M. Y.; Manchester, J. K.; Pusateri, M. E.

    1994-01-01

    Six key metabolic enzymes plus glutaminase and glutamate decarboxylase, as well as glutamate, aspartate and GABA, were measured in 11 regions of the hippocampal formation of synchronous, flight and tail suspension rats. Major differences were observed in the normal distribution patterns of each enzyme and amino acid, but no substantive effects of either microgravity or tail suspension on these patterns were clearly demonstrated.

  19. The Roles of Organic Acids in C4 Photosynthesis

    PubMed Central

    Ludwig, Martha

    2016-01-01

    Organic acids are involved in numerous metabolic pathways in all plants. The finding that some plants, known as C4 plants, have four-carbon dicarboxylic acids as the first product of carbon fixation showed these organic acids play essential roles as photosynthetic intermediates. Oxaloacetate (OAA), malate, and aspartate (Asp) are substrates for the C4 acid cycle that underpins the CO2 concentrating mechanism of C4 photosynthesis. In this cycle, OAA is the immediate, short-lived, product of the initial CO2 fixation step in C4 leaf mesophyll cells. The malate and Asp, resulting from the rapid conversion of OAA, are the organic acids delivered to the sites of carbon reduction in the bundle-sheath cells of the leaf, where they are decarboxylated, with the released CO2 used to make carbohydrates. The three-carbon organic acids resulting from the decarboxylation reactions are returned to the mesophyll cells where they are used to regenerate the CO2 acceptor pool. NADP-malic enzyme-type, NAD-malic enzyme-type, and phosphoenolpyruvate carboxykinase-type C4 plants were identified, based on the most abundant decarboxylating enzyme in the leaf tissue. The genes encoding these C4 pathway-associated decarboxylases were co-opted from ancestral C3 plant genes during the evolution of C4 photosynthesis. Malate was recognized as the major organic acid transferred in NADP-malic enzyme-type C4 species, while Asp fills this role in NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type plants. However, accumulating evidence indicates that many C4 plants use a combination of organic acids and decarboxylases during CO2 fixation, and the C4-type categories are not rigid. The ability to transfer multiple organic acid species and utilize different decarboxylases has been suggested to give C4 plants advantages in changing and stressful environments, as well as during development, by facilitating the balance of energy between the two cell types involved in the C4 pathway of CO2

  20. The Roles of Organic Acids in C4 Photosynthesis.

    PubMed

    Ludwig, Martha

    2016-01-01

    Organic acids are involved in numerous metabolic pathways in all plants. The finding that some plants, known as C4 plants, have four-carbon dicarboxylic acids as the first product of carbon fixation showed these organic acids play essential roles as photosynthetic intermediates. Oxaloacetate (OAA), malate, and aspartate (Asp) are substrates for the C4 acid cycle that underpins the CO2 concentrating mechanism of C4 photosynthesis. In this cycle, OAA is the immediate, short-lived, product of the initial CO2 fixation step in C4 leaf mesophyll cells. The malate and Asp, resulting from the rapid conversion of OAA, are the organic acids delivered to the sites of carbon reduction in the bundle-sheath cells of the leaf, where they are decarboxylated, with the released CO2 used to make carbohydrates. The three-carbon organic acids resulting from the decarboxylation reactions are returned to the mesophyll cells where they are used to regenerate the CO2 acceptor pool. NADP-malic enzyme-type, NAD-malic enzyme-type, and phosphoenolpyruvate carboxykinase-type C4 plants were identified, based on the most abundant decarboxylating enzyme in the leaf tissue. The genes encoding these C4 pathway-associated decarboxylases were co-opted from ancestral C3 plant genes during the evolution of C4 photosynthesis. Malate was recognized as the major organic acid transferred in NADP-malic enzyme-type C4 species, while Asp fills this role in NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type plants. However, accumulating evidence indicates that many C4 plants use a combination of organic acids and decarboxylases during CO2 fixation, and the C4-type categories are not rigid. The ability to transfer multiple organic acid species and utilize different decarboxylases has been suggested to give C4 plants advantages in changing and stressful environments, as well as during development, by facilitating the balance of energy between the two cell types involved in the C4 pathway of CO2

  1. Catalysis by Orotidine 5′-Monophosphate Decarboxylase: Effect of 5-Fluoro and 4′-Substituents on the Decarboxylation of Two-Part Substrates†

    PubMed Central

    Goryanova, Bogdana; Spong, Krisztina; Amyes, Tina L.; Richard, John P.

    2013-01-01

    The syntheses of two novel truncated analogs of the natural substrate orotidine 5′-monophosphate (OMP) for orotidine 5′-monophosphate decarboxylase (OMPDC) with enhanced reactivity towards decarboxylation are reported: 1-(β-D-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5′-deoxy-5-fluoroorotidine (5′-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M−1 s−1) and 1-(β-D-erythrofuranosyl)orotic acid (EO, 0.026 M−1 s−1) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO32−), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-bound substrate. Evidence is presented that there is a change in rate-limiting step from the chemical step of decarboxylation for the phosphite-activated reaction of EO, to closure of the phosphate gripper loop and an enzyme conformational change at the ternary E·FEO·HPO32− complex for the reaction of FEO. The 4′-CH3 and 4′-CH2OH groups of 5′-dFO and orotidine, respectively, result in identical destabilizations of the transition state for the unactivated decarboxylation of 2.9 kcal/mol. By contrast, the 4′-CH3 group of 5′-dFO and the 4′-CH2OH group of orotidine result in very different 4.7 and 8.3 kcal/mol destabilizations of the transition state for the phosphite-activated decarboxylation. Here, the destabilizing effect of the 4′-CH3 substituent at 5′-dFO is masked by the rate-limiting conformational change that depresses the third-order rate constant for the phosphite-activated reaction of the parent substrate FEO. PMID

  2. Catalysis by orotidine 5'-monophosphate decarboxylase: effect of 5-fluoro and 4'-substituents on the decarboxylation of two-part substrates.

    PubMed

    Goryanova, Bogdana; Spong, Krisztina; Amyes, Tina L; Richard, John P

    2013-01-22

    The syntheses of two novel truncated analogs of the natural substrate orotidine 5'-monophosphate (OMP) for orotidine 5'-monophosphate decarboxylase (OMPDC) with enhanced reactivity toward decarboxylation are reported: 1-(β-d-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5'-deoxy-5-fluoroorotidine (5'-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M⁻¹ s⁻¹) and 1-(β-d-erythrofuranosyl)orotic acid (EO, 0.026 M⁻¹ s⁻¹) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO₃²⁻), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-bound substrate. Evidence is presented that there is a change in the rate-limiting step from the chemical step of decarboxylation for the phosphite-activated reaction of EO, to closure of the phosphate gripper loop and an enzyme conformational change at the ternary E•FEO•HPO₃²⁻ complex for the reaction of FEO. The 4'-CH₃ and 4'-CH₂OH groups of 5'-dFO and orotidine, respectively, result in identical destabilizations of the transition state for the unactivated decarboxylation of 2.9 kcal/mol. By contrast, the 4'-CH₃ group of 5'-dFO and the 4'-CH₂OH group of orotidine result in very different 4.7 and 8.3 kcal/mol destabilizations of the transition state for the phosphite-activated decarboxylation. Here, the destabilizing effect of the 4'-CH₃ substituent at 5'-dFO is masked by the rate-limiting conformational change that depresses the third-order rate constant for the phosphite-activated reaction of the parent substrate FEO.

  3. Occurrence of a novel L-2,4-diaminobutyrate decarboxylase activity in some species of Enterobacteriaceae, and purification and characterization of the enzymes of Enterobacter aerogenes and Serratia marcescens.

    PubMed

    Yamamoto, S; Mutoh, N; Ikai, H; Nagasaka, M

    1996-10-01

    L-2,4-Diaminobutyrate decarboxylase (DABA DC) is a novel enzyme yielding 1,3-diaminopropane (DAP) from DABA, which has previously been purified from strains of the genera Vibrio and Acinetobacter. In this study, we also detected DABA DC activity in the species of Enterobacteriaceae: E. aerogenes, E. cloacae, E. agglomerans, Serratia marcescens, S. liquefaciens, Klebsiella pneumoniace, K. oxytoca and Citrobacter freundii, all of which produced DAP in sufficient amounts. Subsequently, the DABA DCs of E. aerogenes and S. marcescens were purified to homogeneity and characterized. Two separate enzymes had similar properties with respect to chromatographic behaviors, and were a dimer with subunits of identical molecular mass of about 51 kDa. The maximal activity of each enzyme was obtained at pH 8.0-8.25. Both enzymes required pyridoxal 5'-phosphate and Mg2+ for full activity, and were highly specific for L-DABA. There was immunological similarity, but not identity between these proteins, as determined by Ouchterlony double diffusion analysis with antiserum against the E. aerogenes DABA DC. They showed the same N-terminal amino acid sequence up to the 8th residue (S-K-L-N-P-I-L-A-). These enzymes were different in molecular mass, N-terminal amino acid sequence and antigenicity from DABA DCs of Acientobacter and Vibrio species.

  4. Construction of a brewer's yeast having alpha-acetolactate decarboxylase gene from Acetobacter aceti ssp. xylinum integrated in the genome.

    PubMed

    Yamano, S; Kondo, K; Tanaka, J; Inoue, T

    1994-02-14

    alpha-Acetolactate decarboxylase (ALDC) gene from Acetobacter aceti ssp. xylinum has several possible initiation codons in the N-terminus. To determine the initiation codon of the ALDC giving the highest expression levels, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was linked just upstream of each possible initiation codon. The ALDC whose translation starts 130 bp downstream from the first ATG codon had the highest activity in yeast cells. When expression levels of the ALDC gene were compared using three strong yeast promoters of glycolytic genes, alcohol dehydrogenase I (ADC1), phosphoglycerate kinase (PGK) and GPD, the GPD promoter was the strongest. The ALDC gene was integrated in a ribosomal RNA gene of a brewer's yeast by co-transformation with an expression plasmid of G418-resistance gene. The laboratory-scale growth test confirmed that the total diacetyl concentration was reduced in wort.

  5. 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.

  6. 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.

  7. Autoregulation of the Kluyveromyces lactis pyruvate decarboxylase gene KlPDC1 involves the regulatory gene RAG3.

    PubMed

    Ottaviano, Daniela; Micolonghi, Chiara; Tizzani, Lorenza; Lemaire, Marc; Wésolowski-Louvel, Micheline; De Stefano, Maria Egle; Ranieri, Danilo; Bianchi, Michele M

    2014-07-01

    In the yeast Kluyveromyces lactis, the pyruvate decarboxylase gene KlPDC1 is strongly regulated at the transcription level by different environmental factors. Sugars and hypoxia act as inducers of transcription, while ethanol acts as a repressor. Their effects are mediated by gene products, some of which have been characterized. KlPDC1 transcription is also strongly repressed by its product--KlPdc1--through a mechanism called autoregulation. We performed a genetic screen that allowed us to select and identify the regulatory gene RAG3 as a major factor in the transcriptional activity of the KlPDC1 promoter in the absence of the KlPdc1 protein, i.e. in the autoregulatory mechanism. We also showed that the two proteins Rag3 and KlPdc1 interact, co-localize in the cell and that KlPdc1 may control Rag3 nuclear localization.

  8. Epidermal growth factor induces biphasic activation of ornithine decarboxylase in human stomach-derived KATO-III cells.

    PubMed

    Ishikawa, T; Mitsuhashi, M; Ichikawa, Y; Tarnawski, A

    1994-01-01

    Effect of epidermal growth factor (EGF) on ornithine decarboxylase (ODC) was examined in human gastric cancer-derived KATO-III cells, because 125I-EGF binding studies indicated a presence of specific binding sites for EGF on these cells. Upon stimulation with EGF, both ODC mRNA expression and ODC enzyme activity were significantly increased in KATO-III cells. However, unlike in other cellular systems, both EGF-induced ODC mRNA expression and ODC enzyme activation were biphasic with the peaks at 15 +/- 10 min and 2.1 +/- 1.5 hrs (mean +/- SE) for mRNA, and 3.1 +/- 1.5 and 7.7 +/- 1.8 hrs (mean +/- SE) for enzyme activity, respectively. Therefore, KATO-III cell line may provide a unique model for the biochemical analysis of EGF action on ODC activation. PMID:8190004

  9. Light-dependent and tissue-specific expression of the H-protein of the glycine decarboxylase complex.

    PubMed Central

    Srinivasan, R; Oliver, D J

    1995-01-01

    Glycine decarboxylase is a mitochondrial enzyme complex, which is the site of photorespiratory CO2 and NH3 release. Although the proteins that constitute the complex are located within the mitochondria, because of their intimate association with photosynthesis their expression is controlled by light. Comparisons of the kinetics of mRNA accumulation between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and the H-protein of glycine decarboxylase during the greening of etiolated Arabidopsis thaliana suggest that their expression is controlled in parallel. A genomic clone for the H-protein (gdcH) was isolated from Arabidopsis and sequenced. The upstream region from -856 to +62 was fused to the beta-glucuronidase (GUS) reporter gene, and this construct was transformed into tobacco. This 5' upstream regulatory region appears to control GUS expression in a manner very similar to that of the endogenous H-protein gene. Constructs with deletions in the 5' upstream region were transformed into tobacco. These deletions revealed that light-dependent and tissue-specific expression was largely controlled by a 259-bp region between -376 and -117 bp. This region contains several putative GT boxes with the GGTTAA consensus core sequence. Once these strong light-dependent elements were removed, a second level of control was revealed. In constructs in which the gdcH 5' regulatory region was shortened to -117 bp or less, there was more GUS activity in the roots than in the leaves, and in dark-grown plants than in light-grown plants. This suggests that more proximal control elements may be responsible for the constitutive low levels of gene expression noted in all nonphotosynthetic tissues. PMID:7480320

  10. Engineering Salidroside Biosynthetic Pathway in Hairy Root Cultures of Rhodiola crenulata Based on Metabolic Characterization of Tyrosine Decarboxylase

    PubMed Central

    Zeng, Lingjiang; Liu, Xiaoqiang; Qiu, Fei; Zheng, Weilie; Quan, Hong; Liao, Zhihua; Chen, Min; Huang, Wenlin; Liu, Wanhong; Wang, Qiang

    2013-01-01

    Tyrosine decarboxylase initializes salidroside biosynthesis. Metabolic characterization of tyrosine decarboxylase gene from Rhodiola crenulata (RcTYDC) revealed that it played an important role in salidroside biosynthesis. Recombinant 53 kDa RcTYDC converted tyrosine into tyramine. RcTYDC gene expression was induced coordinately with the expression of RcUDPGT (the last gene involved in salidroside biosynthesis) in SA/MeJA treatment; the expression of RcTYDC and RcUDPGT was dramatically upregulated by SA, respectively 49 folds and 36 folds compared with control. MeJA also significantly increased the expression of RcTYDC and RcUDPGT in hairy root cultures. The tissue profile of RcTYDC and RcUDPGT was highly similar: highest expression levels found in stems, higher expression levels in leaves than in flowers and roots. The gene expressing levels were consistent with the salidroside accumulation levels. This strongly suggested that RcTYDC played an important role in salidroside biosynthesis in R. crenulata. Finally, RcTYDC was used to engineering salidroside biosynthetic pathway in R. crenulata hairy roots via metabolic engineering strategy of overexpression. All the transgenic lines showed much higher expression levels of RcTYDC than non-transgenic one. The transgenic lines produced tyramine, tyrosol and salidroside at higher levels, which were respectively 3.21–6.84, 1.50–2.19 and 1.27–3.47 folds compared with the corresponding compound in non-transgenic lines. In conclusion, RcTYDC overexpression promoted tyramine biosynthesis that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside. PMID:24124492

  11. Identification and transcript analysis of two glutamate decarboxylase genes, CsGAD1 and CsGAD2, reveal the strong relationship between CsGAD1 and citrate utilization in citrus fruit.

    PubMed

    Liu, Xiao; Hu, Xiao-Mei; Jin, Long-Fei; Shi, Cai-Yun; Liu, Yong-Zhong; Peng, Shu-Ang

    2014-09-01

    Glutamate decarboxylase (GAD, EC 4.1.1.15) has been suggested to be a key, regulatory point in the biosynthesis of γ-aminobutyrate (GABA) and in the utilization of citric acid through GABA shunt pathway. In this study we discovered two GAD genes, named as CsGAD1 and CsGAD2, in citrus genome database and then successfully cloned. Both CsGAD1 and CsGAD2 have a putative pyridoxal 5-phosphate binding domain in the middle region and a putative calmodulin-binding domain at the carboxyl terminus. Gene structure analysis showed that much difference exists in the size of exons and introns or in cis-regulatory elements in promoter region between the two GAD genes. Gene expression indicated that CsGAD1 transcript was predominantly expressed in flower and CsGAD2 transcript was predominantly expressed in fruit juice sacs; in the ripening fruit, CsGAD1 transcript level was at least 2-time higher than CsGAD2 transcript level. Moreover, CsGAD1 transcript level was increased significantly along with the increase of GAD activity and accompanied by a significant decrease of titratable acid (TA), suggesting that it is CsGAD1 rather than CsGAD2 plays a role in the citric acid utilization during fruit ripening. In addition, injection of abscisic acid and foliar spray of K2SO4 significantly increased the TA content of Satsuma mandarin, and significantly decreased GAD activity as well as CsGAD1 transcript, further suggesting the important role of CsGAD1 in the citrate utilization of citrus fruit.

  12. Metabolic engineering of acid resistance elements to improve acid resistance and propionic acid production of Propionibacterium jensenii.

    PubMed

    Guan, Ningzi; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Chen, Jian; Liu, Long

    2016-06-01

    Propionic acid (PA) and its salts are widely used in the food, pharmaceutical, and chemical industries. Microbial production of PA by propionibacteria is a typical product-inhibited process, and acid resistance is crucial in the improvement of PA titers and productivity. We previously identified two key acid resistance elements-the arginine deaminase and glutamate decarboxylase systems-that protect propionibacteria against PA stress by maintaining intracellular pH homeostasis. In this study, we attempted to improve the acid resistance and PA production of Propionibacterium jensenii ATCC 4868 by engineering these elements. Specifically, five genes (arcA, arcC, gadB, gdh, and ybaS) encoding components of the arginine deaminase and glutamate decarboxylase systems were overexpressed in P. jensenii. The activities of the five enzymes in the engineered strains were 26.7-489.0% higher than those in wild-type P. jensenii. The growth rates of the engineered strains decreased, whereas specific PA production increased significantly compared with those of the wild-type strain. Among the overexpressed genes, gadB (encoding glutamate decarboxylase) increased PA resistance and yield most effectively; the PA resistance of P. jensenii-gadB was more than 10-fold higher than that of the wild-type strain, and the production titer, yield, and conversion ratio of PA reached 10.81 g/L, 5.92 g/g cells, and 0.56 g/g glycerol, representing increases of 22.0%, 23.8%, and 21.7%, respectively. We also investigated the effects of introducing these acid resistance elements on the transcript levels of related enzymes. The results showed that the expression of genes in the engineered pathways affected the expression of the other genes. Additionally, the intracellular pools of amino acids were altered as different genes were overexpressed, which may further contribute to the enhanced PA production. This study provides an effective strategy for improving PA production in propionibacteria; this

  13. Suppressive effects of dietary curcumin on the increased activity of renal ornithine decarboxylase in mice treated with a renal carcinogen, ferric nitrilotriacetate.

    PubMed

    Okazaki, Yasumasa; Iqbal, Mohammad; Okada, Shigeru

    2005-06-10

    Curcumin, a natural, biologically active compound extracted from rhizomes of Curcuma species, has been shown to act as a biological response modifier in various disorders. We have reported previously that the dietary supplementation of curcumin enhances the activities of antioxidant and phase II metabolizing enzymes in mice (M. Iqbal, S.D. Sharma, Y. Okazaki, M. Fujisawa, S. Okada, Dietary supplementation of curcumin enhances antioxidant and phase II metabolizing enzymes in ddY mice: possible role in protection against chemical carcinogenesis and toxicity, Pharmacol and Toxicol. 92 (2003) 33_38.) and inhibits ferric nitrilotriacetate (Fe-NTA) induced oxidative injury of lipids and DNA in vitro (M. Iqbal, Y. Okazaki, S. Okada, In vitro curcumin modulates Ferric Nitrilotriacetate (Fe-NTA) and hydrogen peroxide (H(2)O(2))-induced peroxidation of microsomal membrane lipids and DNA damage, Teratogenesis Carcinogenesis and Mutagenesis Supplement 23 (2003) 151-160.). In our present study, Fe-NTA, a known complete renal carcinogen, which generate ROS in vivo, was given intraperitoneally to mice and curcumin was tested for its ability to inhibits oxidative stress and the activity of ornithine decarboxylase (ODC) as well as histopathological changes in the kidney. Substantial changes in glutathione, antioxidant enzymes as well as changes in phase II metabolizing enzymes were observed in the kidney at 12 h after treatment with Fe-NTA (9.0 mg Fe/kg body weight). Effect of oxidative stress induced by Fe-NTA were also demonstrated by the increase in lipid peroxidation as monitored by formation of thiobarbituric acid-reactive substances and 4-hydroxy-2-nonenal (HNE)-modified proteins in kidney. Likewise, the level of protein carbonyl contents, an indicator of protein oxidation was also increased after Fe-NTA administration. However, the changes in these parameters were restored to normal in curcumin-pretreated mice. The ODC activity in the kidney was significantly increased by Fe

  14. Enhanced flux of substrates into polyamine biosynthesis but not ethylene in tomato fruit engineered with yeast S-adenosylmethionine decarboxylase gene.

    PubMed

    Lasanajak, Yi; Minocha, Rakesh; Minocha, Subhash C; Goyal, Ravinder; Fatima, Tahira; Handa, Avtar K; Mattoo, Autar K

    2014-03-01

    S-adenosylmethionine (SAM), a major substrate in 1-C metabolism is a common precursor in the biosynthetic pathways of polyamines and ethylene, two important plant growth regulators, which exhibit opposing developmental effects, especially during fruit ripening. However, the flux of various substrates including SAM into the two competing pathways in plants has not yet been characterized. We used radiolabeled (14)C-Arg, (14)C-Orn, L-[U-(14)C]Met, (14)C-SAM and (14)C-Put to quantify flux through these pathways in tomato fruit and evaluate the effects of perturbing these pathways via transgenic expression of a yeast SAM decarboxylase (ySAMDC) gene using the fruit ripening-specific promoter E8. We show that polyamines in tomato fruit are synthesized both from Arg and Orn; however, the relative contribution of Orn pathway declines in the later stages of ripening. Expression of ySAMDC reversed the ripening associated decline in spermidine (Spd) and spermine (Spm) levels observed in the azygous control fruit. About 2- to 3-fold higher levels of labeled-Spd in transgenic fruit (556HO and 579HO lines) expressing ySAMDC confirmed the enzymatic function of the introduced gene. The incorporation of L-[U-(14)C]Met into Spd, Spm, ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) was used to determine Met-flux into these metabolites. The incorporation of (14)C-Met into Spd/Spm declined during ripening of the control azygous fruit but this was reversed in fruits expressing ySAMDC. However, incorporation of (14)C-Met into ethylene or ACC during ripening was not altered by the expression of ySAMDC in the fruit. Taken together these results show that: (1) There is an inverse relationship between the production of higher polyamines and ethylene during fruit ripening, (2) the inverse relationship between higher polyamines and ethylene is modulated by ySAMDC expression in that the decline in Spd/Spm during fruit ripening can be reversed without significantly altering ethylene

  15. Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae

    PubMed Central

    Gong, Zheng; Tang, M. Matt; Wu, Xueliang; Phillips, Nancy; Galkowski, Dariusz; Jarvis, Gary A.; Fan, Huizhou

    2016-01-01

    Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis. PMID:26808268

  16. Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae.

    PubMed

    Gong, Zheng; Tang, M Matt; Wu, Xueliang; Phillips, Nancy; Galkowski, Dariusz; Jarvis, Gary A; Fan, Huizhou

    2016-01-01

    Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis.

  17. Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae.

    PubMed

    Gong, Zheng; Tang, M Matt; Wu, Xueliang; Phillips, Nancy; Galkowski, Dariusz; Jarvis, Gary A; Fan, Huizhou

    2016-01-01

    Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis. PMID:26808268

  18. 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.

  19. A metabolic strategy to enhance long-term survival by Phx1 through stationary phase-specific pyruvate decarboxylases in fission yeast

    PubMed Central

    Kim, Ji-Yoon; K