DOCSLIB.ORG

Substrate Specificity and Reaction Mechanism of Putrescine Oxidase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

J. Biochem. 86, 97-104 (1979) Substrate Specificity and Reaction Mechanism of Putrescine Oxidase Masato OKADA, Seiichi KAWASHIMA, and Kazutomo IMAHORI Department of Biochemistry, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, January 9, 1979 Putrescine oxidase [EC 1.4.3.4] of Micrococcus rubens oxidizes many kinds of synthetic poly amines: triamines (spermidine types), tetramines (spermine types), and N-substituted putres cines. Polyamines possessing terminal 4-aminobutylimino groups in their structures were more active as substrates. Putreanine was oxidized at a rate comparable to that of putrescine, and was converted to 1-pyrroline and 8-alanine. Activities and Km values for polyamines were affected by the substituent attached to the 4-aminobutylimino group of the polyamine, and especially by its methylene chain length. It was also found that two types of oxidation occurred in the oxidation of polyamines by putrescine oxidase. When the moieties attached to the 4-aminobutylimino groups in polyamines were less hydrophobic, these polyamines were oxidized at the secondary amino groups to form 1-pyrroline. Polyamines which contained a hydrophobic substituent attached to the 4-aminobutylimino group were oxidized at the ter minal primary amino group of the 4-aminobutylimino moiety to form ammonia. N,N•Œ- Bis(4-aminobutyl)-1,3-diaminopropane ([‡U,4-3-4]) and N-(4-aminobutyl)-N•Œ-(3-aminopropyl) 1,3-diaminopropane ([‡U,4-3-3]) were oxidized to form 1-pyrrolinium salt derivatives as a result of oxidation of the terminal primary amino groups. It was concluded that the essential struc ture for substrates of putrescine oxidase is a 4-aminobutylimino group (NH2(CH2),NH-). Putrescine oxidase [EC 1.4.3.4], putrescine: oxygen which catalyze the degradation of spermidine oxidoreductase (deaminating) (flavin containing), also oxidize spermine (4-10). from Micrococcus rubens has a high substrate Swain and DeSa proposed a model for the specificity for amines (1). This enzyme oxidized active site of this enzyme based on studies of the putrescine, cadaverine, spermidine, and 1,6 interactions between the enzyme and competitive diaminohexane (1-3), but is exceptional in that it inhibitors: the enzyme has an anionic point and a does not oxidize spermine (1), since many enzymes hydrophobic region in the active site (3). How ever, this model failed to explain why putrescine oxidase oxidizes spermidine, but not spermine. Abbreviations: Me, methyl; Et, ethyl; n-Pr, n-propyl; The substrate specificity for polyamines other n-Bu, n-butyl; n-Am, n-amyl; [‡T, x-y], NH2(CH2) x- NH(CH2)yNH2; [‡V, x-y-z], NH2(CH2)xNH(CH2) than diamines, spermidine and spermine has not y- NH(CH2)zNH2; [‡V, R], NH2(CH2)4NH-R; [‡W, X], examined. In order to characterize the properties NH2(CH2)4NH(CH2)2-X. of the active site in more detail, it is necessary to Vol. 86, No. 1, 1979 97 98 M. OKADA, S. KAWASHIMA, and K. IMAHORI examine the substrate specificity for many poly 12 N HCl : H2O (60: 20: 20, v/v) (15). The amines. amines were detected by spraying 0.1 % ninhydrin In this paper, we report the activities of in acetone. IR spectra were taken with a Hitachi putrescine oxidase towards many kinds of synthetic EPI-G2 spectrophotometer, and NMR spectra polyamines (triamines, tetramines, and N-sub with a Hitachi Perkin-Elmer R-20A spectrometer stituted putrescine) and the structures of their at 90 MHz using tetramethylsilane as an external oxidation products. Based on these results, the standard. Liquid chromatographic analyses of essential structures of polyamines for activity as polyamines and their oxidation products were substrates, as well as the binding properties of the carried out using a Hitachi 835 amino acid ana enzyme, are discussed. lyzer in the buffer system with suitable modification (16). Activity of Putrescine Oxidase towards Poly MATERIALS AND METHODS amines-The maximal velocity (Vmax) and pH Materials-Putrescine oxidase of Micrococcus optimum of putrescine oxidase for polyamines rubens was purified to homogeneity by an affinity were determined from the amount of hydrogen chromatographic procedure (11). The specific peroxide evolved using the coupling assay method activity of the enzyme used in this study was with peroxidase and o-dianisidine (17) in 0.1 M 34.7 ƒÊmol/min/mg protein. Peroxidase and cata borate buffer at pH 7.5-10.5. lase were purchased from Boehringer, Mannheim, Km Values for Polyamines-Km values for Germany. Putrescine (free base and hydro polyamines were estimated from the competitive chloride salt), spermidine trihydrochloride, and inhibition by 1,8-diaminooctane of the oxidation spermine tetrahydrochloride were the products of of the polyamines. First, Km for putrescine was Nakarai Chemicals Ltd., Japan. Other diamines, determined from Lineweaver-Burk plots based on dimethylenetriamine, trimethylenetetramine, and the above assay method. Next, the Ki value for N-(3-aminopropyl)-1,3-diaminopropane were pur 1,8-diaminooctane, which is a competitive inhibitor chased from Tokyo Chemical Industry Co., Japan. of putrescine oxidase (3), was determined (Ki= Other polyamines were synthesized in our labo 9.2•~10-6M). The oxidation velocities for poly ratory (12). Other chemicals were of the highest amines in the presence (vi) and absence (v) of grade available. 1,8-diaminooctane at various concentrations (i) 1-(3-Hydroxy-l-oxoinden-2-yl)-1-pyrrolinium were measured, then Km values for the polyamines Hydroxide, Inner Salt ([V])-This compound were calculated from v/vi vs. i plots based on the (13) was prepared by the condensation reaction of equation: v/vi=1+i/Ki[Km/(Ki+s)], where Ki is proline with ninhydrin according to the method the inhibitor constant of 1,8-diaminooctane and of Grassmann and Arnim (14). NMR (in CDCl3) s is the concentration of polyamine. S 9.76 (s, 1H, imine proton), 8.00 (2d, 4H J=2Hz, Determination of 1-Pyrroline-l-Pyrroline indene ring protons), 5.43 (t, 2H, J=8Hz, 5-H), formed by the oxidation of polyamines was deter 3.60 (m, 2H, 3-H), 2.80 (quin, 2H, J=8Hz, 4-H). mined according to the method described by Holmstedt et al. (18, 19), except that a value of 2.1 •~ 103 M-1 • cm-1 was used for the molar extinc tion coefficient of the adduct between 1-pyrroline and o-aminobenzaldehyde at 435 nm (18). The reaction mixture contained 0.5 ml of 0.1 M borate (pH 9.0), 10 pl of 50 mM polyamine (0.50 ƒÊmol), and 20 ƒÊl (ca. 1.0 unit) of putrescine oxidase. After incubation for 16 h, 0.1 ml of 0.6 M phosphate (pH 5.5) was added to the incubation mixture. After 1 h, the absorbance at 435 nm was mea Measurement-TLC was carried out on sured. Avicel SF cellulose (Funakoshi Pharmaceutical Determination of Ammonia-Ammonia was Co.) in the following solvent system : 1-propanol: determined with an amino acid analyzer using J. Biochem. SUBSTRATE SPECIFICITY OF PUTRESCINE OXIDASE 99 ammonium sulfate as a standard. Nessler's reagent TABLE I. Vmax, Km, and pH optimum values of (20) was not applicable in this case because a putrescine oxidase for polyamines, including diamines. The reaction mixtures (0.555-0.570 ml) contained 2.5 precipitate was formed in some cases. The ƒÊ mol of polyamines, and 0.06-1.10 units of purified enzymatic reactions were carried out by methods putrescine oxidase. Vmax and Km were determined by similar to that described for the determination of the methods described in " MATERIALS AND 1-pyrroline. When the reaction stopped, 5011 METHODS." Vmax values are relative ratios based aliquots were withdrawn from the incubation on the velocity of putrescine oxidation at pH 9.0. mixtures and diluted to 550 pl with water. These diluted mixtures were used as samples for the amino acid analyzer. RESULTS Activities of Putrescine Oxidase towards Poly amines-Vmax, pH optimum, and Km for various polyamines were determined, as summarized in Table I; most of the polyamines examined were active as substrates, and polyamines possessing 4. aminobutylimino groups were more active. This agrees with the model which Swain and DeSa proposed for the active site of putrescine oxidase (3): the length of the 4-aminobutylimino group corresponds to the distance from the active (cata lytic) point to the anionic point which has a binding role. It is interesting that the activity for [IV, COOH] (putreanine) (21) is comparable to that for putrescine, in spite of its higher Km value. So far, no enzymes other than putrescine oxidase have been reported that can degrade putreanine. On the other hand, Km values for polyamines tend to decrease with increasing methylene chain length in their structures. This tendency is clear when Km values for the members of the [‡T,4-y] or [‡V,R] group are compared. This is comparable to the known tendency that Ki values for com petitive inhibitors of putrescine oxidase decrease as the methylene chain length increases (3), sug gesting the existence of a hydrophobic interaction between the enzyme and the substituent on the 4-aminobutylimino group in polyamines. Formation of 1-Pyrroline and Ammonia on Polyamine Oxidation by Putrescine Oxidase Putrescine oxidase oxidizes putrescine to form 1-pyrroline, ammonia, and hydrogen peroxide, and converts spermidine to 1-pyrroline and 1,3 diaminopropane, generating hydrogen peroxide ND: Not determined because the formation of H2O2 (1, 2). However, it has not been clarified whether all the [I,4-y], [‡U,4-y-4], [‡V,R], and [IV,X] types was observed only for a short initial period. Other of polyamines are also oxidized to form 1-pyrroline polyamines, [I, 2-2], [‡U, 2-2-2], [‡U, 4-8-4], and [‡U, 6-6-6] were not active as substrates.
Recommended publications
  • Polyamines and Transglutaminases: Biological, Clinical, and Biotechnological Perspectives

    Polyamines and Transglutaminases: Biological, Clinical, and Biotechnological Perspectives

    Amino Acids (2014) 46:475–485 DOI 10.1007/s00726-014-1688-0 EDITORIAL Polyamines and transglutaminases: biological, clinical, and biotechnological perspectives Enzo Agostinelli Received: 3 January 2014 / Accepted: 27 January 2014 / Published online: 20 February 2014 Ó Springer-Verlag Wien 2014 Preface Europe. The ancient name of Istanbul was Bisantium, a city founded by Greeks in 659 B.C. on the banks of the The history of polyamines dates back to the fifteenth cen- Bosporus. Bisantium was renamed Constantinopolis in tury when spermine was discovered by Antoni van Leeu- honor of the Roman emperor Constantine I, becoming a wenhoek [born in Delft, Holland (1632–1723)], and yet it center of Greek culture and Christianity. Throughout its took many years before serious attention was given to long history, Istanbul (the old Constantinopolis) was the understanding the role of spermine or other polyamines in capital of three important empires: Roman, Byzantine, and the biology of living cells. It is now clear that regulation of Ottoman. Today, Istanbul as one of the largest cities in the polyamine homeostasis is complex and has excited poly- world is also one of the European capitals of culture while amine researchers who have continued to focus on this its historic areas are part of the UNESCO list of World productive area of research. Therefore, enough new Cultural Heritage. research findings have prompted to organize conferences This Special Issue of amino acids brings together 28 and congresses worldwide to disseminate the new knowl- peer-reviewed
  • The Role of Polyamine Uptake Transporters on Growth and Development of Arabidopsis Thaliana

    The Role of Polyamine Uptake Transporters on Growth and Development of Arabidopsis Thaliana

    THE ROLE OF POLYAMINE UPTAKE TRANSPORTERS ON GROWTH AND DEVELOPMENT OF ARABIDOPSIS THALIANA Jigarkumar Patel A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2015 Committee: Paul Morris, Advisor Wendy D Manning Graduate Faculty Representative Vipaporn Phuntumart Scott Rogers Ray Larsen © 2015 Jigarkumar Patel All Rights Reserved iii ABSTRACT Paul Morris, Advisor Transgenic manipulation of polyamine levels has provided compelling evidence that polyamines enable plants to respond to environmental cues by activation of stress and developmental pathways. Here we show that the chloroplasts of A. thaliana and soybeans contain both an arginine decarboxylase, and an arginase/agmatinase. These two enzymes combine to synthesize putrescine from arginine. Since the sequences of plant arginases show conservation of key residues and the predicted 3D structures of plant agmatinases overlap the crystal structure of the enzyme from Deinococcus radiodurans, we suggest that these enzymes can synthesize putrescine, whenever they have access to the substrate agmatine. Finally, we show that synthesis of putrescine by ornithine decarboxylase takes place in the ER. Thus A. thaliana has two, and soybeans have three separate pathways for the synthesis of putrescine. This study also describes key changes in plant phenotypes in response to altered transport of polyamines. iv Dedicated to my father, Jayantilal Haribhai Patel v ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Paul F. Morris, for helping me learn and grow during my Ph.D. Dr. Morris has an open door policy, and he was always available to answer my questions and provide helpful suggestions.
  • Defining Novel Plant Polyamine Oxidase Subfamilies Through

    Defining Novel Plant Polyamine Oxidase Subfamilies Through

    Bordenave et al. BMC Evolutionary Biology (2019) 19:28 https://doi.org/10.1186/s12862-019-1361-z RESEARCHARTICLE Open Access Defining novel plant polyamine oxidase subfamilies through molecular modeling and sequence analysis Cesar Daniel Bordenave1, Carolina Granados Mendoza2, Juan Francisco Jiménez Bremont3, Andrés Gárriz1 and Andrés Alberto Rodríguez1* Abstract Background: The polyamine oxidases (PAOs) catabolize the oxidative deamination of the polyamines (PAs) spermine (Spm) and spermidine (Spd). Most of the phylogenetic studies performed to analyze the plant PAO family took into account only a limited number and/or taxonomic representation of plant PAOs sequences. Results: Here, we constructed a plant PAO protein sequence database and identified four subfamilies. Subfamily PAO back conversion 1 (PAObc1) was present on every lineage included in these analyses, suggesting that BC-type PAOs might play an important role in plants, despite its precise function is unknown. Subfamily PAObc2 was exclusively present in vascular plants, suggesting that t-Spm oxidase activity might play an important role in the development of the vascular system. The only terminal catabolism (TC) PAO subfamily (subfamily PAOtc) was lost in Superasterids but it was present in all other land plants. This indicated that the TC-type reactions are fundamental for land plants and that their function could being taken over by other enzymes in Superasterids. Subfamily PAObc3 was the result of a gene duplication event preceding Angiosperm diversification, followed by a gene extinction in Monocots. Differential conserved protein motifs were found for each subfamily of plant PAOs. The automatic assignment using these motifs was found to be comparable to the assignment by rough clustering performed on this work.
  • Structure and Mechanism of the Propionibacterium Acnes Polyunsaturated Fatty Acid Isomerase

    Structure and Mechanism of the Propionibacterium Acnes Polyunsaturated Fatty Acid Isomerase

    Structure and mechanism of the Propionibacterium acnes polyunsaturated fatty acid isomerase Alena Liavonchanka*, Ellen Hornung*, Ivo Feussner*, and Markus Georg Rudolph†‡ Departments of *Plant Biochemistry and †Molecular Structural Biology, University of Go¨ttingen, D-37077 Go¨ttingen, Germany Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved December 20, 2005 (received for review November 23, 2005) Conjugated linoleic acids (CLAs) affect body fat gain, carcinogen- biochemical data on a structural basis and to determine how FAD esis, insulin resistance, and lipid peroxidation in mammals. Several can catalyze the nonredox PUFA isomerization. isomers of CLA exist, of which the (9Z,11E) and (10E,12Z) isomers We determined the crystal structure of PAI to define the active have beneficial effects on human metabolism but are scarce in site and extract a structure-based mechanism for polyenoic fatty foods. Bacterial polyunsaturated fatty acid isomerases are prom- acid isomerization. PAI is an FAD-containing monomer consisting ising biotechnological catalysts for CLA production. We describe six of three intricately connected domains. The N-terminal domain crystal structures of the Propionibacterium acnes polyunsatu- shares similarity with modules found in other FAD-binding pro- rated fatty acid isomerase PAI in apo- and product-bound forms. teins, and the overall fold is in part similar to yeast polyamine The three-domain flavoprotein has previously undescribed folds oxidase (11). The geometry of the substrate-binding pocket deter- outside the FAD-binding site. Conformational changes in a hydro- mined for the PAI–LA complex reveals that fatty acid is bound with phobic channel toward the active site reveal a unique gating mech- the methyl end inside and delineates the residues that are involved anism for substrate specificity.
  • Crystallographic Snapshots of the Complete Reaction Cycle of Nicotine Degradation by an Amine Oxidase of the Monoamine Oxidase (MAO) Family

    Crystallographic Snapshots of the Complete Reaction Cycle of Nicotine Degradation by an Amine Oxidase of the Monoamine Oxidase (MAO) Family

    Crystallographic snapshots of the complete reaction cycle of nicotine degradation by an amine oxidase of the monoamine oxidase (MAO) family Galina Kachalovaa, Karl Deckerb, Andrew Holtc, and Hans D. Bartunika,1 aMax Planck Unit for Structural Molecular Biology, Notkestrasse 85, 22607 Hamburg, Germany; bInstitut für Biochemie und Molekularbiologie, Albert-Ludwig University, Stefan-Meier-Strasse 17, 79104 Freiburg im Breisgau, Germany; and cDepartment of Pharmacology, School of Molecular and Systems Medicine, Faculty of Medicine and Dentistry, 9-58 Medical Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2H7 Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved February 4, 2011 (received for review November 9, 2010) FAD-linked oxidases constitute a class of enzymes which catalyze alditol oxidase (17). However, substrate and product were found dehydrogenation as a fundamental biochemical reaction, followed to coexist in the active site and could not be distinguished, despite by reoxidation of reduced flavin. Here, we present high-resolution the high resolution reached in these studies. crystal structures showing the flavoenzyme 6-hydroxy-L-nicotine Here, we describe discrete structural states populated during oxidase in action. This enzyme was trapped during catalytic degra- productive enzymatic turnover in crystalline 6HLNO. The flavo- dation of the native substrate in a sequence of discrete reaction enzyme is a close structural neighbor (15) of human MAO-A states corresponding to the substrate-reduced enzyme, a complex and MAO-B. It is involved in the degradation of nicotine in of the enzyme with the intermediate enamine product and forma- the soil bacterium Arthrobacter nicotinovorans, which starts with tion of the final aminoketone product.
  • Distribution of Membrane-Bound Monoamine Oxidase in Bacteria

    Distribution of Membrane-Bound Monoamine Oxidase in Bacteria

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1979, p. 565-569 Vol. 38, No. 4 0099-2240/79/10-0565/05$02.00/0 Distribution of Membrane-Bound Monoamine Oxidase in Bacteria YOSHIKATSU MUROOKA,* NOBUYUKI DOI, AND TOKUYA HARADA The Institute ofScientific and Industrial Research, Osaka University, Yamadakami, Suita, Osaka (565), Japan Received for publication 22 March 1979 The distribution of membrane-bound monoamine oxidase in 30 strains of various bacteria was studied. Monoamine oxidase was determined by using an ammonia-selective electrode; analyses were sensitive and easy to perform. The enzyme was found in some strains of the family Enterobacteriaceae, such as Klebsiella, Enterobacter, Escherichia, Salmonella, Serratia, and Proteus. Among strains of other families of bacteria tested, only Pseudomonas aeruginosa IFO 3901, Micrococcus luteus IFO 12708, and Brevibacterium ammoniagenes IAM 1641 had monoamine oxidase activity. In all of these bacteria except B. ammoniagenes, monoamine oxidase was induced by tyramine and was highly specific for tyramine, octopamine, dopamine, and norepinephrine. The enzyme in two strains oxidized histamine or benzylamine. Correlations between the distri- butions of membrane-bound monoamine oxidase and arylsulfatase synthesized in the presence of tyramine were discussed. Monoamine oxidase catalyzes the oxidative monoamine oxidase had broad substrate speci- deamination of monoamines by the following ficity, the enzyme in bacteria can be assayed reaction: R-CH2NH2 + 02+ H20 -+ R-CHO + conveniently by potentiometric measurement of NH3 + H202. ammonia formation with an ammonia-selective The enzyme usually has a broad substrate electrode by the method used for the brain en- specificity in animals and plays a major role in zyme by Meyerson et al.
  • A 30 Å Long U-Shaped Catalytic Tunnel in the Crystal Structure of Polyamine

    A 30 Å Long U-Shaped Catalytic Tunnel in the Crystal Structure of Polyamine

    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Research Article 265 A 30 Å long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase Claudia Binda1, Alessandro Coda1, Riccardo Angelini2, Rodolfo Federico2, Paolo Ascenzi2 and Andrea Mattevi1* Background: Polyamines are essential for cell growth and differentiation; Addresses: 1Dipartimento di Genetica e compounds interfering with their metabolism are potential anticancer agents. Microbiologia, Università di Pavia, Via Abbiategrasso 207, I-27100 Pavia, Italy and Polyamine oxidase (PAO) plays a central role in polyamine homeostasis. The 2Dipartimento di Biologia, Università Roma Tre, enzyme utilises an FAD cofactor to catalyse the oxidation of the secondary Viale Guglielmo Marconi 446, I-00146 Roma, Italy. amino groups of spermine and spermidine. *Corresponding author. E-mail: [email protected] Results: The first crystal structure of a polyamine oxidase has been determined to a resolution of 1.9 Å. PAO from Zea mays contains two domains, which define Key words: enzyme catalysis, flavoenzymes, a remarkable 30 Å long U-shaped catalytic tunnel at their interface. The structure monoamine oxidase, polyamine oxidase, X-ray of PAO in complex with the inhibitor MDL72527 reveals the residues forming the crystallography catalytic machinery and unusual enzyme-inhibitor CH...O H bonds. A ring of Received: 19 October 1998 glutamate and aspartate residues surrounding one of the two tunnel openings Revisions requested: 3 December 1998 contributes to the steering of the substrate towards the inside of the tunnel. Revisions received: 16 December 1998 Accepted: 18 December 1998 Conclusions: PAO specifically oxidises substrates that have both primary and Published: 24 February 1999 secondary amino groups.
  • Activation of Polyamine Catabolism by N1,N11-Diethylnorspermine Leads to Cell Death in Glioblastoma

    Activation of Polyamine Catabolism by N1,N11-Diethylnorspermine Leads to Cell Death in Glioblastoma

    431-440 4/7/07 11:31 Page 431 INTERNATIONAL JOURNAL OF ONCOLOGY 31: 431-440, 2007 431 Activation of polyamine catabolism by N1,N11-diethylnorspermine leads to cell death in glioblastoma RONGCAI JIANG1,7*, WOONYOUNG CHOI1*, ASAD KHAN2,8, KENNETH HESS3, EUGENE W. GERNER5, ROBERT A. CASERO Jr6, W.K. ALFRED YUNG4, STANLEY R. HAMILTON1 and WEI ZHANG1 Departments of 1Pathology, 2Pediatric Oncology, 3Biostatistics, and 4Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030; 5Department of Cell Biology and Anatomy, Arizona Cancer Center, University of Arizona, Tucson, AZ 85724; 6Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA Received February 5, 2007; Accepted March 26, 2007 Abstract. Glioblastoma multiforme (GBM) is one of the with N1-acetylpolymine oxidase (APAO)/spermine oxidase most therapeutically refractory human cancers. Elevated (SMO) inhibitor. Though mitochondrial damage was induced, cellular polyamine levels are a common feature of cancer neither activation of the caspase cascade nor cytochrome c cells, including GBM cells, and the polyamine pathway redistribution between the mitochondria and cytoplasm has been explored as a potential therapeutic target to inhibit was observed. Systemic DENSPM treatment of mice with polyamine biosynthesis or activate polyamine catabolism. intracerebral GBM led to longer survival. Taken together, In this study, we investigated the effect of N1,N11-diethyl- our studies indicate that DENSPM kills GBM cells through norspermine (DENSPM), a spermine analog that activates induction of SSAT coupled with H2O2 production, which is a polyamine catabolism, in GBM cells. The in vitro cell culture potential target for GBM therapy. experiments showed that DENSPM increased the sub-G1 apoptotic cell population in GBM cell lines but caused minimal Introduction cytotoxicity in normal astrocytes.
  • Control of the Polyamine Biosynthesis Pathway by G2-Quadruplexes

    Control of the Polyamine Biosynthesis Pathway by G2-Quadruplexes

    RESEARCH ARTICLE Control of the polyamine biosynthesis pathway by G2-quadruplexes Helen Louise Lightfoot1, Timo Hagen1, Antoine Cle´ ry2,3, Fre´ de´ ric Hai-Trieu Allain2, Jonathan Hall1* 1Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland; 2Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland; 3Biomolecular NMR spectroscopy platform, ETH Zurich, Zurich, Switzerland Abstract G-quadruplexes are naturally-occurring structures found in RNAs and DNAs. Regular RNA G-quadruplexes are highly stable due to stacked planar arrangements connected by short loops. However, reports of irregular quadruplex structures are increasing and recent genome-wide studies suggest that they influence gene expression. We have investigated a grouping of G2-motifs in the UTRs of eight genes involved in polyamine biosynthesis, and concluded that several likely form novel metastable RNA G-quadruplexes. We performed a comprehensive biophysical characterization of their properties, comparing them to a reference G-quadruplex. Using cellular assays, together with polyamine-depleting and quadruplex-stabilizing ligands, we discovered how some of these motifs regulate and sense polyamine levels, creating feedback loops during polyamine biosynthesis. Using high-resolution 1H-NMR spectroscopy, we demonstrated that a long- looped quadruplex in the AZIN1 mRNA co-exists in salt-dependent equilibria with a hairpin structure. This study expands the repertoire of regulatory G-quadruplexes and demonstrates how they act in unison to control metabolite homeostasis. DOI: https://doi.org/10.7554/eLife.36362.001 *For correspondence: [email protected] Introduction Polyamines (PAs) are small poly-cationic molecules present at millimolar concentrations in cells Competing interests: The (Lightfoot and Hall, 2014).
  • University of Groningen Exploring the Cofactor-Binding and Biocatalytic

    University of Groningen Exploring the Cofactor-Binding and Biocatalytic

    University of Groningen Exploring the cofactor-binding and biocatalytic properties of flavin-containing enzymes Kopacz, Malgorzata IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2014 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Kopacz, M. (2014). Exploring the cofactor-binding and biocatalytic properties of flavin-containing enzymes. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 29-09-2021 Exploring the cofactor-binding and biocatalytic properties of flavin-containing enzymes Małgorzata M. Kopacz The research described in this thesis was carried out in the research group Molecular Enzymology of the Groningen Biomolecular Sciences and Biotechnology Institute (GBB), according to the requirements of the Graduate School of Science, Faculty of Mathematics and Natural Sciences.
  • Whthesis.Pdf (1.486Mb)

    Whthesis.Pdf (1.486Mb)

    INVESTIGATION OF A POSSIBLE MULTI-ENZYME COMPLEX INVOLVED IN NICOTINE BIOSYNTHESIS IN ROOTS OF TOBACCO (Nicotiana tabacum) by WILLIAM HEIM Thesis submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment for the degree of MASTER OF LIFE SCIENCES in THE DEPARTMENT OF PLANT PATHOLOGY, PHYSIOLOGY, AND WEED SCIENCE with a Major Emphasis in PLANT PHYSIOLOGY APPROVED: ___________________________________ __________________ John Jelesko, Chairperson Date ___________________________________ __________________ Carole Cramer, Member Date ___________________________________ __________________ Fabricio Medina-Bolivar, Member Date August, 2003 Blacksburg, Virginia Keywords: Nicotiana tabacum, nicotine biosynthesis, diamine oxidase, N-methylputrescine oxidase, S-adenosylhomocysteine hydrolase, multi-enzyme complex, metabolon 2003 by William Heim ALL RIGHTS RESERVED INVESTIGATION OF A POSSIBLE MULTI-ENZYME COMPLEX INVOLVED IN NICOTINE BIOSYNTHESIS IN ROOTS OF TOBACCO (Nicotiana tabacum) by William Heim (Dr. John Jelesko, Chairperson) Department of Plant Pathology, Physiology, and Weed Science ABSTRACT N-methylputrescine oxidase (MPO) is a member of the diamine oxidase (DAO) class of enzymes believed to be responsible for synthesis of the alkaloid nicotine in the roots of Nicotiana tabacum (Mizusaki et al., 1972). A purportedly pure MPO protein from tobacco root culture extracts was used to generate immune antiserum in rabbits (McLauchlan et al., 1993). In an attempt to clone a cDNA encoding MPO, we used this antiserum to screen a tobacco cDNA expression library. Unexpectedly, two previously unreported genes with strong homology to members of a gene family encoding S-adenosylhomocysteine hydrolase (SAHH) in N. sylvestris and a gene encoding SAHH in N. tabacum were cloned instead. SAHH is an enzyme of the S-adenosylmethionine (SAM) recycling pathway, which also includes SAM synthetase (SAMS) and methionine synthase (MS).
  • Interactions of Natural Polyamines with Mammalian Proteins

    Interactions of Natural Polyamines with Mammalian Proteins

    Article in press - uncorrected proof BioMol Concepts, Vol. 2 (2011), pp. 79–94 • Copyright ᮊ by Walter de Gruyter • Berlin • New York. DOI 10.1515/BMC.2011.007 Review Interactions of natural polyamines with mammalian proteins Inge Schuster1 and Rita Bernhardt2,* ecules, such as phospholipids or nucleotides, thereby mas- 1 Institute for Theoretical Chemistry, University Vienna, sively affecting their structures and functions (9, 10). A-1090 Vienna, Austria Accordingly, polyamines were found to modulate numerous 2 Institute of Biochemistry, Saarland University, Campus regulatory processes in model systems that range from cel- B2.2, D-66123 Saarbru¨cken, Germany lular signaling to the control of gene expression and trans- lation and offer explanations for the obvious involvement of * Corresponding author polyamines in the regulation of cell growth, differentiation, e-mail: [email protected] and death wreviewed in (1, 11–14)x. The broad actions of polyamines on essential life functions are reflected by their effects on global gene expression. A recent study in a yeast Abstract mutant, an eukaryotic system comprising a three times small- er genome than mammalians, showed a significant regulation The ubiquitously expressed natural polyamines putrescine, of some 10% of the genome in (direct or indirect) response spermidine, and spermine are small, flexible cationic com- to spermidine or spermine (15). The essential requirement pounds that exert pleiotropic actions on various regulatory for polyamines and balanced levels of polyamines is under- systems and, accordingly, are essentially involved in diverse lined from studying knockouts that lack distinct genes life functions. These roles of polyamines result from their involved in polyamine synthesis and are not viable (10).