• Title, Summary, Keyword: GTP cyclohydrolase I

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Biochemical Characterization of Oligomerization of Escherichia coli GTP Cyclohydrolase I

  • Lee, Soo-Jin;Ahn, Chi-Young;Park, Eung-Sik;Hwang, Deog-Su;Yim, Jeong-Bin
    • BMB Reports
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    • v.35 no.3
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    • pp.255-261
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    • 2002
  • GTP cyclohydrolase I (E.C. 3.5.4.16) is a homodecameric protein that catalyzes the conversion of GTP to 7,8-dihydroneopterin triphosphate (H2NTP), the initial step in the biosynthesis of pteridines. It was proposed that the enzyme complex could be composed of a dimer of two pentamers, or a pentamer of tightly associated dimers; then the active site of the enzyme was located at the interface of three monomers (Nar et al. 1995a, b). Using mutant enzymes that were made by site-directed mutagenesis, we showed that a decamer of GTP cyclohydrolase I should be composed of a pentamer of five dimers, and that the active site is located between dimers, as analyzed by a series of size exclusion chromatography and the reconstitution experiment. We also show that the residues Lys 136, Arg139, and Glu152 are of particular importance for the oligomerization of the enzyme complex from five dimers to a decamer.

Affinity Labeling of E. coli GTP Cyclohydrolase I by a Dialdehyde Derivative of Guanosine Triphosphate

  • Ahn, Chi-Young;Park, Sang-Ick;Kim, Ju-Myeong;Yim, Jeong-Bin
    • BMB Reports
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    • v.28 no.1
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    • pp.72-78
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    • 1995
  • Time-dependent inactivation of E. coli GTP cyclohydrolase I with a 2',3'-dialdehyde derivative of GTP (oGTP) was directed to the active site of the enzyme, and was dependent on the concentration of oGTP. The kinetics of inactivation were biphasic with a rapid reaction occurring immediately upon exposure of the enzyme to oGTP followed by a slow rate of inactivation. The $K_i$ value of oGTP for the enzyme was 0.25 mM. Inactivation was prevented by preincubation of the enzyme with GTP, the substrate of the enzyme. At 100% inactivation, 2.3 mol of [8.5'-$^3H$]oGTP were bound per each enzyme subunit, which consists of two identical polypeptides. The active site residue which reacted with the affinity label was lysine. oGTP interacted selectively with the ${\varepsilon}$-amino group of lysine in the GTP-binding site to form a morpholine-like structure which was stable without sodium borohydride treatment. However, triphosphate group was eliminated during the hydrolysis step. To identify the active site of the enzyme, [8.5'-$^3H$]oGTP-labeled enzyme was cleaved by endoproteinase Lys-C, and the $^3H$-labeled peptide was purified by HPLC. The amino acid sequence of the active site peptide was Pro-Ser-Leu-Ser-Lys, which corresponds to the aminoterminal sequence of the enzyme.

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The Origin of Ribityl Side Chain of Riboflavin in Ashbya gossypii (Ashbua gossypii에서의 리보플라빈 측쇄의 기원)

  • 최원자;임정빈
    • Korean Journal of Microbiology
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    • v.23 no.3
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    • pp.167-171
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    • 1985
  • In order to investigate the origin of the ribityl froup of riboflavin and the involvement of GTP cyclohydrolase II in the riboflavin pathway, we studied the incorporation of $^{14}C-labeled$ guanosine using a well known riboflavin over producer, Ashbya gossypii.Cells were grown in a media containing $(U- ^{14}C)$ guanosine and the riboflavin and GMP were isolated and purifired by column chromatography. The isolated compounds, riboflavin and GMP were labeled in the ribityl and ribosyl side chain and the isoalloxazine and guannine moiety. By comparing the specific radioactivity of each compound we reached a conclusion that the ribose of guanosine is converted directly to the rivityl moiety of riboflavin. The results indicate that biosynthesis of the vitamin begins at the level of a guanosine compound and also suppory the involvement of GTP cyclohydrolase II in one of the early steps in the biosynthetic pathway.

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Purification and Characterization of GTP Cyclohydrolase I from Streptomyces tubercidicus, a Producer of Tubercidin

  • Yoo, Jin-Cheol;Han, Ji-Man;Ko, Ok-Hyun;Bang, Hee-Jae
    • Archives of Pharmacal Research
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    • v.21 no.6
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    • pp.692-697
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    • 1998
  • GTP cyclohydrolase I catalyzing the first reaction in the biosynthesis of pterin moiety of folic acid in bacteria, was purified from Streptomyces tubercidicus by at least 203-fold with a yield of 32% to apparent homogeneity, using ammonium sulfate fractionation, DEAE-cellulose, Sepharose CL-6B, and hydroxylapatite column chromatography. The molecular weight of the native enzyme was estimated to be 230,000 daltons by gel permeation chromatography. The purified enzyme gave a single band on sodium dodesyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was apparently 58,000 daltons. These results indicate that the enzyme consists of four subunits with the same molecular weight. The $K_m$ and $V_{max}$ values for GTP of the purified enzyme were determined to be 80${\mu}$M and 90nmol/min (mg protein), respectively. The optimum pH and temperature for the enzyme reaction were pH 7.5-8.5 and $40-42^{\circ}C$, respectively. Coenzyme or metal ion was not required for the enzyme activity. The enzyme activity was inhibited by most divalent cations, while it was slightly activated by potassium ion. In case of nucleotides, CTP, GMP, GDP, and UTP inhibited enzyme activity, among which GDP exhibited the strongest inhibitory effect.

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The Functions of the Riboflavin Genes in the lux Operon from Photobacterium Species (Photobacterium Species의 lux 오페론에서 발견된 Riboflavin 생합성 유전자들의 기능)

  • 이찬용;임종호
    • Korean Journal of Microbiology
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    • v.38 no.3
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    • pp.173-179
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    • 2002
  • The functions of riboflavin synthesis genes ( ribI,II,III and IV) found immediately downstream of luxG in the lux operon from Photobacterium species were identified using the biochemical and genetical analysis. The ribI-III gene codes for protein corresponding to that coded by the second (riboflavin synthase), third (3,4-dihydroxy 2-butanone 4-phosphate synthase/GTP cyclohydrolase II) and fourth (lumazine synthase) gene, respectively, of Bacillus subtilis rib operon with the respective gene procuct sharing 41-50% amino acid sequence identity. Unexpectedly, the sequence of the ribIV product of Photobacterium phosphoreum does not correspond in sequence to the protein encoded by the fifth rib gene of Bacillus subtilis. Instead the gene (ribIV) codes for a polypeptide similar in sequence to GTP cyclohydrolase II of Escherichia coli and the carboxy terminal domain of the third rib gene from Bacillus subtilis. Complementation of Escherichia coli riboflavin auxotrophs showed that the function of the gene products of ribII and ribIV are DHBP synthase and GTP cyclohydrolase II, respectively. In addition the experiment, showing that increase in thermal stability of riboflavin synthase coded by ribIon coexpression with ribIII, provided indirect evidence that the latter gene codes for lumazine synthase.

Analysis of Two Promoters that Control the Expression of the GTP cyclohydrolase I Gene in Drosophila melanogaster

  • Byun, Jaegoo;Yoon, Jaeseung;Baek, Kwanghee
    • Molecules and Cells
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    • v.27 no.5
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    • pp.583-589
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    • 2009
  • GTP cyclohydrolase I (GTPCH) is a key enzyme in the de novo synthesis of tetrahydrobiopterin. Previously, the Drosophila melanogaster GTPCH gene has been shown to be expressed from two different promoters (P1 and P2). In our study, the 5'-flanking DNA regions required for P1 and P2 promoter activities were characterized using transient expression assay. The DNA regions between -98 and +31, and between -73 and +35 are required for efficient P1 and P2 promoter activities, respectively. The regions between -98 and -56 and between -73 and -41 may contain critical elements required for the expression of GTPCH in Drosophila. By aligning the nucleotide sequences in the P1 and P2 promoter regions of the Drosophila melanogaster and Drosophila virilrs GTPCH genes, several conserved elements including palindromic sequences in the regions critical for P1 and P2 promoter activities were identified. Western blot analysis of transgenic flies transformed using P1 or P2 promoter-lacZ fusion plasmids further revealed that P1 promoter expression is restricted to the late pupae and adult developmental stages but that the P2 promoter driven expression of GTPCH is constitutive throughout fly development. In addition, X-gal staining of the embryos and imaginal discs of transgenic flies suggests that the P2 promoter is active from stage 13 of embryo and is generally active in most regions of the imaginal discs at the larval stages.

Biosynthesis of Tetrahydrobiopterin in an Aquatic Fungus, Allomyces macrogynus (물곰팡이 Allomyces macrogynus에서 Tetrahydrobiopterin의 생합성)

  • Lee, Soo Woong;Park, Young Shik
    • Korean Journal of Microbiology
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    • v.34 no.4
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    • pp.243-247
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    • 1998
  • Comparative analyses of intracellular biopterin contents and its biosynthetic enzymes were performed in Allomyces macrogynus. Biopterin content in fresh weight was 14-fold higher in mycelium than in zoospore. Enzyme activities of GTP cyclohydrolase I and 6-pyruvoyltetrahydropterin synthase in ammonium sulfate fractions were approximately 2-fold higher in mycelium. On the other hand, sepiapterin reductase (SR) activity was 10 fold higher in zoospore. Northern blot assay also demonstrated that SR transcript was abundant in zoospore. These results suggest a possible involvement of tetrahydrobiopterin in cellular differentiation of Allomyces macrogyllus as well as provide an experimental basis to elucidate the physiological function of SR in this organism.

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Human Embryonic Stem Cells Co-Transfected with Tyrosine Hydroxylase and GTP Cyclohydrolase I Relieve Symptomatic Motor Behavior in a Rat Model of Parkinson′s Disease

  • Kil, Kwang-Soo;Lee, Chang-Hyun;Shin, Hyun-Ah;Cho, Hwang-Yoon;Yoon, Ji-Yeon;Lee, Gun-Soup;Lee, Young-Jae;Kim, Eun-Young;Park, Se-Pill
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • pp.101-101
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    • 2003
  • Main strategy for a treatment of Parkinson's disease (PD), due to a progressive degeneration of dopaminergic neurons, is a pharmaceutical supplement of dopamine derivatives or ceil replacement therapy. Both of these protocols have pros and cons; former exhibiting a dramatic relief but causing a severe side effects on long-term prescription and latter also having a proven effectiveness but having availability and ethical problems Embryonic stem (ES) cells have several characteristics suitable for this purpose. To investigate a possibility of using ES cells as a carrier of therapeutic gene(s), human ES (hES, MB03) cells were transfected with cDNAs coding for tyrosine hydroxylase (TH) in pcDNA3.1 (+) and the transfectants were selected using neomycin (250 $\mu /ml$). Expression of TH being confirmed, two of the positive clone (MBTH2 & 8) were second transfected with GTP cyclohydrolase 1 (GTPCH 1) in pcDNA3.1 (+)-hyg followed by selection with hygromycin-B (150 $\mu /ml$) and RT-PCR confirmation. By immune-cytochemistry, these genetically modified but undifferentiated dual drug-resistant cells were found to express few of the neuronal markers, such as NF200, $\beta$-tubulin, and MAP2 as well as astroglial marker GFAP. This results suggest that over-production of BH4 by ectopically expressed GTPCH I may be involved in the induction of those markers. Transplantation of the cells into striatum of 6-OHDA- denervated PD animal model relieved symptomatic rotational behaviors of the animals. Immunohistochemical analyses showed the presence of human cells within the striatum of the recipients. These results suggest a possibility of using hES cells as a carrier of therapeutic gene(s).

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Alteration of Striatal Tetrahydrobiopterin in Iron-Induced Unilateral Model of Parkinson's Disease

  • Aryal, Bijay;Lee, Jin-Koo;Kim, Hak Rim;Kim, Hyung-Gun
    • The Korean Journal of Physiology and Pharmacology
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    • v.18 no.2
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    • pp.129-134
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    • 2014
  • It has been suggested that transition metal ions such as iron can produce an oxidative injuries to nigrostriatal dopaminergic neurons, like Parkinson's disease (PD) and subsequent compensative increase of tetrahydrobiopterin ($BH_4$) during the disease progression induces the aggravation of dopaminergic neurodegeneration in striatum. It had been established that the direct administration of $BH_4$ into neuron would induce the neuronal toxicity in vitro. To elucidate a role of $BH_4$ in pathogenesis in the PD in vivo, we assessed the changes of dopamine (DA) and $BH_4$ at striatum in unilateral intranigral iron infused PD rat model. The ipsistriatal DA and $BH_4$ levels were significantly increased at 0.5 to 1 d and were continually depleting during 2 to 7 d after intranigral iron infusion. The turnover rate of $BH_4$ was higher than that of DA in early phase. However, the expression level of GTP-cyclohydrolase I mRNA in striatum was steadily increased after iron administration. These results suggest that the accumulation of intranigral iron leads to generation of oxidative stress which damage to dopaminergic neurons and causes increased release of $BH_4$ in the dopaminergic neuron. The degenerating dopaminergic neurons decrease the synthesis and release of both $BH_4$ and DA in vivo that are relevance to the progression of PD. Based on these data, we propose that the increase of $BH_4$ can deteriorate the disease progression in early phase of PD, and the inhibition of $BH_4$ increase could be a strategy for PD treatment.

Isolation and properties of protease Pi in escherichia coli (대장균 세포내 단백질 분해효소, protease Pi의 정제와 특성)

  • 이영섭;곽태환;임정빈;정진하
    • Korean Journal of Microbiology
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    • v.24 no.2
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    • pp.119-126
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    • 1986
  • A periplasmic endoprotease, named protease Pi, was purified to homogeneity from Escherkchia coli by conventional procedure with insulin as substrate. This enzyme degrades insulin and glucagon to trichloroacetic acid-soluble meterials, but shows little or no hydrolysis of bovine serum albumin, casein or globin. Its molecular weight was 110, 000 when determined by gel filtration on Sephacryl S-300 and was 105, 000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, it appears to be single polypeptide. This snzyme is metalloprotease, since it is completely inhibited by o-phenanthroline and can be activated by addition of divalent metal cations, such as $Mg^{2+}\;and\;Co^{2+}$. It is destinct from protease Ci, a cytoplasmic insulin degrading enzyme, since protease Pi is localized to the periplasm. Since protease Pi selectively degrades GTP cyclohydrolase I, it appears to play a role in the regulation of pteridine biosynthesis.

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