DOCSLIB.ORG

Site-Directed Mutagenesis and Ruthenium Labeling of Oxalate Decarboxylase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Site-Directed Mutagenesis and Ruthenium Labeling of Oxalate Decarboxylase Site-Directed Mutagenesis and Ruthenium Labeling of Oxalate Decarboxylase Timothy DeMason Undergraduate Honors Thesis – Spring 2018 Department of Chemistry, University of Florida 2 Contents List of Abbreviations .................................................................................................................... 3 Abstract ......................................................................................................................................... 4 Introduction ................................................................................................................................... 5 Methods......................................................................................................................................... 11 Results and Discussion ................................................................................................................. 16 Calculation of Ru—Mn Electron Transfer ....................................................................... 16 Mutagenesis of K375C/C383A ......................................................................................... 18 Synthesis of Ru-OxDC ..................................................................................................... 22 Conclusion .................................................................................................................................... 25 Acknowledgements ....................................................................................................................... 26 References ..................................................................................................................................... 26 3 List of Abbreviations bis-tris 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol bpy 2,2’-bipyridine DMSO dimethyl sulfoxide DTT dithiothreitol E. coli Escherichia coli FDH Formate Dehydrogenase IA-phen 5-iodoacetamido-1,10-phenanthroline IPTG isopropyl β-D-1-thiogalactopyranoside LRET Long Range Electron Transfer mM mili Molar μM micro Molar MS Mass Spectrometry NAD+ nicotinamide adenine dinucleotide OxDC Oxalate Decarboxylase PCET Proton Coupled Electron Transfer PCR Polymerase Chain Reaction PDB Protein Data Bank Ru-OxDC Ruthenium Modified Oxalate Decarboxylase Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol WT Wild Type Oxalate Decarboxylase 4 Abstract Oxalate is a toxic dicarboxylic acid that is decomposed into carbon dioxide and formate by Bacillus subtilis Oxalate Decarboxylase (OxDC). Each monomer of OxDC contains two manganese ions, one at the N-terminal end and one at the C-terminal end. Evidence suggests that the C-terminal manganese plays a functional role in the mechanism of catalysis, which can be further investigated by studying a ruthenium labeled OxDC. A K375C/C383A OxDC mutant was generated, which places a cysteine close to the C-terminal manganese for labeling with the 2+ thiol reactive ruthenium compound [Ru(bpy)2(IA-phen)] . Marcus theory calculations predict a tunneling time of 660 ns between the ruthenium and C-terminal manganese ions. The K375C/C383A OxDC mutant displayed typical wild type Michaelis-Menten kinetics, -1 with KM = 10 ± 2 mM and kcat = 90 ± 13 s . Attempts at labeling K375C/C383A OxDC with 2+ [Ru(bpy)2(IA-phen)] were unsuccessful. The failure of the labeling reaction appears to be due 2+ to an inability of [Ru(bpy)2(IA-phen)] to react with K375C/C383A OxDC rather than nonspecificity of the reaction. Future labeling with other thiol reactive ligands should be attempted. 5 Introduction Oxalic acid is a dicarboxylic acid with pKa’s of 1.2 and 4.2.1 Oxalic acid is produced in plants through several biochemical pathways including the activity of glyoxalate oxidase and isocitrate lysase.2,3 Oxalate is known to precipitate in the presence of divalent cations.1 Calcium oxalate is one of the more important salts since it is relatively insoluble and is present in about 60% of kidney stones.4 Developing ways of reducing the degree of oxalate accumulation in the kidneys is of medical importance. The buildup of calcium oxalate deposits can also cause problems in the manufacturing of paper.5,6 Oxalate Decarboxylase (OxDC) is an oxalate degrading enzyme found in Bacillus subtilis and other organisms. The enzyme catalyzes the degradation of oxalate into formate and carbon dioxide in 99.8% of turnovers (decarboxylase pathway), and two equivalents of carbon dioxide and hydrogen peroxide in the rest (oxidase pathway), as shown in Schemes 1A and 1B.1 – O O O O2 A CO2 + – O OH H O – O O B + O H 2 CO H O + 2 + 2 + 2 2 O OH Scheme 1: A) Degradation of oxalate into carbon dioxide and formate and B) of oxalate into carbon dioxide and hydrogen peroxide. Native OxDC exists as a homo-hexamer formed from a dimer of trimers (see Figure 1C). The structure of the monomer is shown in Figures 1A and 1B and consists of two β-barrel domains, one at the N-terminal end (shown in green in Figures 1A and 1B) and another at the C- 6 terminal end (shown in blue in Figures 1A and 1B). Inside each β-barrel rests a manganese ion bound to three histidines and one glutamate, leaving two sites free for other small ligands. A) C) B) Figure 1: The crystal structure of Bacillus subtilis OxDC showing the manganese ions in purple. A) Monomer structure as viewed from the side. B) Monomer viewed from the top. C) Hexamer viewed from the top with one trimer in blue and the other in orange. These figures were generated in PyMOL using the 1UW8 PDB file. The mechanism of OxDC catalysis of oxalate is still not completely understood. Figure 2 illustrates the current proposed mechanism for catalysis. First, mono-protonated oxalate and dioxygen bind to the N-terminal manganese, with the dioxygen generating Mn3+.1 Then, glutamate-162 removes the remaining acidic proton from oxalate, while simultaneously an electron is transferred from oxalate to the manganese, i.e. proton coupled electron transfer (PCET).1 Heterolytic cleavage of the oxalate carbon-carbon bond follows, liberating carbon dioxide and producing a carbon dioxide radical anion still bound to the manganese.1 Finally, PCET occurs again, reducing the carbon dioxide radical anion while oxidizing the Mn2+ to 7 regenerate Mn3+. Concurrently, glutamate-162 protonates the carbon of the carbon dioxide radical anion to form formate.1 A problem with this mechanism is that it places a dioxygen radical and carbon dioxide radical anion in close proximity. These two radicals are expected to react to form - peroxycarbonate (HCO4 ) which may then decompose with proton uptake to produce hydrogen peroxide and carbon dioxide as is the case in the oxidase pathway.7 However, the products of the oxidase pathway are only observed in 0.2% of turnovers.1 Figure 2: Current proposed mechanism for OxDC. The N-terminal manganese binds oxalate (and oxygen as a co-catalyst) while it cycles between Mn2+ and Mn3+. From (9) with permission from Elsevier. While the C-terminal manganese was originally thought to only play a structural role, further investigation into the OxDC mechanism suggests that the C-terminal manganese may play a role in catalysis. The presence of a stacked tryptophan dimer between the N- and C- terminal manganese of adjacent monomers suggests that electron transfer between the two manganese ions is possible. Substitution of tryptophan-96 and tryptophan-274 with 8 phenylalanine or tyrosine leads to significantly reduced catalytic capability indicating their importance in catalysis.1,8 Additionally, EPR spin trapping studies of a flexible lid mutant suggest that the carbon dioxide radical anions and superoxide radicals are produced in separate locations.9 Since oxalate is known to bind at the N-terminal manganese, perhaps oxygen binds to the C-terminal manganese.1 A new mechanism for OxDC catalysis is shown in Figure 3, proposing the C-terminal manganese site as the temporary electron sink through the use of long range electron transfer (LRET).1010 C-terminal N-terminal O O Glu Glu Glu Glu 280 101 280 101 – His + HC O - O O His 319 O 140 OH2 2 4 His 319 O His 140 2+ 2+ 2+ 2+ Mn Mn Mn Mn His 275 OH2 His 97 OH2 His 275 OH2 His 97 O OH O His O His 273 His 95 His 273 95 – – Glu O Glu162 O 162 LRET + H+ OH OH Glu Glu Glu Glu 280 101 – 280 101 – O PCET O O His 319 O His 140 O His 319 O His 140 2+ 2+ oxidation 2+ 3+ Mn Mn + Mn Mn C – His 275 OH2 His 97 O O His 275 OH2 His 97 O OH His O His His 273 95 His 273 95 O HO Glu162 – O Glu162 - - CO2 - CHOO - + HC2O4 OH OH Glu Glu Glu 280 101 PCET 280 Glu101 His His 319 O 140 reduction His 319 O His 140 2+ 2+ 2+ 3+ H Mn Mn – Mn Mn C His His – 275 OH2 97 O O His 275 OH2 His 97 O O His His His 273 95 O His 273 95 O – HO Glu162 O Glu162 Figure 3: New proposed mechanism for OxDC, including the possibility of long range electron transfer, assuming oxalate binds in a bi-dentate fashion. From (10). 9 Selective oxidation of the C-terminal manganese could be used to further study the possibility of LRET. Ruthenium modified proteins have been used previously to study the electron transfer properties of metalloproteins such as azurin, cytochrome c, and myoglobin.11 These modified proteins employ the use of the so-called “flash-quench” technique shown in Scheme 2A. A ruthenium(II) diimine complex, such as tris(bipyridine)ruthenium(II), is excited with a “flash” of light at 450 nm to produce an excited state which is then “quenched” by either an oxidant or reductant to produce ruthenium(III) or ruthenium(I), respectively.12 The oxidized or reduced ruthenium can then either remove or inject
Load more

Recommended publications
  • Proteomic Analysis of Mycelial Proteins from Magnaporthe Oryzae Under Nitrogen Starvation
    Proteomic analysis of mycelial proteins from Magnaporthe oryzae under nitrogen starvation X.-G. Zhou1,2, P. Yu 2, C. Dong2, C.-X. Yao2, Y.-M. Ding2, N. Tao2 and Z.-W. Zhao1 1State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming, China 2Key Laboratory of Southwestern Crop Gene Resources and Germplasm Innovation, Ministry of Agriculture and Yunnan Provincial Key Laboratory of Agricultural Biotechnology, Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming, China Corresponding author: Z.W. Zhao E-mail: [email protected] Genet. Mol. Res. 15 (2): gmr.15028637 Received March 23, 2016 Accepted April 11, 2016 Published May 13, 2016 DOI http://dx.doi.org/10.4238/gmr.15028637 ABSTRACT. Magnaporthe oryzae is an important model system in studies of plant pathogenic fungi, and nitrogen is a key nutrient source affecting microbial growth and development. In order to understand how nitrogen stress causes changes in mycelial proteins, we analyzed differentially expressed mycelial proteins from the M. oryzae virulent strain CH-63 using two-dimensional electrophoresis and mass spectrometry in complete medium or under nitrogen starvation conditions. A total of 975 ± 70 and 1169 ± 90 protein spots were detected in complete medium and under nitrogen starvation conditions, respectively. Forty-nine protein spots exhibited at least 2-fold up- regulation or down-regulation at the protein level according to PDQuest7.4. Moreover, 43 protein spots were successfully identified by matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry. Among these spots, 6 proteins were functionally unknown and 37 proteins were categorized into 5 groups according to Genetics and Molecular Research 15 (2): gmr.15028637 ©FUNPEC-RP www.funpecrp.com.br X.-G.
    [Show full text]
  • Oxdc Antibody Rabbit Polyclonal Antibody Catalog # ABV11223
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 OxdC Antibody Rabbit Polyclonal Antibody Catalog # ABV11223 Specification OxdC Antibody - Product Information Application WB Primary Accession O34714 Reactivity Human Host Rabbit Clonality Polyclonal Isotype Rabbit IgG Calculated MW 43566 OxdC Antibody - Additional Information Gene ID 938620 Positive Control Western Blot: Recombinant protein Application & Usage Western blot: 1-4 Western blot of Oxalate decarboxylase µg/ml. antibody. Lane 1: rb- Oxalate decarboxylase - Other Names 10 ng. Lane 2: rb- Oxalate decarboxylase - 50 YvrK ng Target/Specificity OxdC OxdC Antibody - Background Antibody Form Oxalate decarboxylase (OxdC, EC4.1.1.2) is a Liquid manganese-containing enzyme, which decomposes oxalic acid and oxalate. With Appearance OxdC catalysis, oxalate is split into formate Colorless liquid and CO2. This enzyme belongs to the family of lyases, specifically the carboxy-lyases, which Formulation 100 µg (0.5 mg/ml) of antibody in PBS pH cleave carbon-carbon bonds. The systematic 7.2 containing 0.01 % BSA, 0.01 % name of this enzyme class is oxalate thimerosal, and 50 % glycerol. carboxy-lyase (formate-forming). This enzyme is also called oxalate carboxy-lyase. The Handling enzyme is composed of two cupin domains, The antibody solution should be gently each of which contains a Mn (II) ion. This mixed before use. enzyme participates in glyoxylate and dicarboxylate metabolism. This enzyme has Reconstitution & Storage been recognized for diagnostics in diverse -20 °C biotechnological applications such as the clinical assay of oxalate in blood and urine, Background Descriptions therapeutics, process industry, and agriculture to lower oxalate levels in foods and the environment.
    [Show full text]
  • Proteo-Metabolomic Investigation of Transgenic Rice Unravels Metabolic
    www.nature.com/scientificreports OPEN Proteo-metabolomic investigation of transgenic rice unravels metabolic alterations and Received: 27 November 2018 Accepted: 24 June 2019 accumulation of novel proteins Published: xx xx xxxx potentially involved in defence against Rhizoctonia solani Subhasis Karmakar1, Karabi Datta1, Kutubuddin Ali Molla2,3, Dipak Gayen4, Kaushik Das1, Sailendra Nath Sarkar1 & Swapan K. Datta1 The generation of sheath blight (ShB)-resistant transgenic rice plants through the expression of Arabidopsis NPR1 gene is a signifcant development for research in the feld of biotic stress. However, to our knowledge, regulation of the proteomic and metabolic networks in the ShB-resistant transgenic rice plants has not been studied. In the present investigation, the relative proteome and metabolome profles of the non–transformed wild-type and the AtNPR1-transgenic rice lines prior to and subsequent to the R. solani infection were investigated. Total proteins from wild type and transgenic plants were investigated using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS). The metabolomics study indicated an increased abundance of various metabolites, which draws parallels with the proteomic analysis. Furthermore, the proteome data was cross-examined using network analysis which identifed modules that were rich in known as well as novel immunity-related prognostic proteins, particularly the mitogen-activated protein kinase 6, probable protein phosphatase 2C1, probable trehalose-phosphate phosphatase 2 and heat shock protein. A novel protein, 14–3– 3GF14f was observed to be upregulated in the leaves of the transgenic rice plants after ShB infection, and the possible mechanistic role of this protein in ShB resistance may be investigated further.
    [Show full text]
  • Oxalic Acid Degradation by a Novel Fungal Oxalate Oxidase from Abortiporus Biennis Marcin Grąz1*, Kamila Rachwał2, Radosław Zan2 and Anna Jarosz-Wilkołazka1
    Vol. 63, No 3/2016 595–600 http://dx.doi.org/10.18388/abp.2016_1282 Regular paper Oxalic acid degradation by a novel fungal oxalate oxidase from Abortiporus biennis Marcin Grąz1*, Kamila Rachwał2, Radosław Zan2 and Anna Jarosz-Wilkołazka1 1Department of Biochemistry, Maria Curie-Skłodowska University, Lublin, Poland; 2Department of Genetics and Microbiology, Maria Curie-Skłodowska University, Lublin, Poland Oxalate oxidase was identified in mycelial extracts of a to formic acid and carbon dioxide (Mäkelä et al., 2002). basidiomycete Abortiporus biennis strain. Intracellular The degradation of oxalate via action of oxalate oxidase enzyme activity was detected only after prior lowering (EC 1.2.3.4), described in our study, is atypical for fun- of the pH value of the fungal cultures by using oxalic or gi and was found predominantly in higher plants. The hydrochloric acids. This enzyme was purified using size best characterised oxalate oxidase originates from cereal exclusion chromatography (Sephadex G-25) and ion-ex- plants (Dunwell, 2000). Currently, only three oxalate oxi- change chromatography (DEAE-Sepharose). This enzyme dases of basidiomycete fungi have been described - an exhibited optimum activity at pH 2 when incubated at enzyme from Tilletia contraversa (Vaisey et al., 1961), the 40°C, and the optimum temperature was established at best characterised so far enzyme from Ceriporiopsis subver- 60°C. Among the tested organic acids, this enzyme ex- mispora (Aguilar et al., 1999), and an enzyme produced by hibited specificity only towards oxalic acid. Molecular Abortiporus biennis (Grąz et al., 2009). The enzyme from mass was calculated as 58 kDa. The values of Km for oxa- C.
    [Show full text]
  • Information to Users
    INFORMATION TO USERS While the most advanced technology has been used to photograph and reproduce this manuscript, the quality of the reproduction is heavily dependent upon the quality of the material submitted. For example: • Manuscript pages may have indistinct print. In such cases, the best available copy has been filmed. • Manuscripts may not always be complete. In such cases, a note will indicate that it is not possible to obtain missing pages. • Copyrighted material may have been removed from the manuscript. In such cases, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, and charts) are photographed by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each oversize page is also filmed as one exposure and is available, for an additional charge, as a standard 35mm slide or as a 17”x 23” black and white photographic print. Most photographs reproduce acceptably on positive microfilm or microfiche but lack the clarity on xerographic copies made from the microfilm. For an additional charge, 35mm slides of 6”x 9” black and white photographic prints are available for any photographs or illustrations that cannot be reproduced satisfactorily by xerography. 8710037 Poikey, Leonard Andrew THE USE OF IMMOBILIZED OXALATE OXIDASE IN AN ANALYTICAL ASSAY FOR URINARY OXALATE AND IN AN EXTRACORPOREAL SHUNT TREATMENT FOR HYPEROXALURIA The Ohio State University Ph.D. 1987 University Microfilms I nternsition300 el N. Zeeb Road, Ann Arbor, Ml 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy.
    [Show full text]
  • Oxygen-Reducing Enzymes in Coatings and Films for Active Packaging |
    Kristin Johansson | Oxygen-reducing enzymes in coatings and films for active packaging | | Oxygen-reducing enzymes in coatings and films for active packaging Kristin Johansson Oxygen-reducing enzymes in coatings and films for active packaging Oxygen-reducing enzymes This work focused on investigating the possibility to produce oxygen-scavenging packaging materials based on oxygen-reducing enzymes. The enzymes were incorporated into a dispersion coating formulation applied onto a food- in coatings and films for packaging board using conventional laboratory coating techniques. The oxygen- reducing enzymes investigated included a glucose oxidase, an oxalate oxidase active packaging and three laccases originating from different organisms. All of the enzymes were successfully incorporated into a coating layer and could be reactivated after drying. For at least two of the enzymes, re-activation after drying was possible not only Kristin Johansson by using liquid water but also by using water vapour. Re-activation of the glucose oxidase and a laccase required relative humidities of greater than 75% and greater than 92%, respectively. Catalytic reduction of oxygen gas by glucose oxidase was promoted by creating 2013:38 an open structure through addition of clay to the coating formulation at a level above the critical pigment volume concentration. For laccase-catalysed reduction of oxygen gas, it was possible to use lignin derivatives as substrates for the enzymatic reaction. At 7°C all three laccases retained more than 20% of the activity they
    [Show full text]
  • Direct Spectrophotometric Determination of Serum and Urinary Oxalate with Oxalate Oxidase1) by G. Kohlbecker
    Kohibecker and Bute: Direct spcctrophotometric determination of oxalate 1103 J. Gin. Chem. Clin. Biochem. Vol. 19,1981, pp. 1103-1106 Direct Spectrophotometric Determination of Serum and Urinary Oxalate with Oxalate Oxidase1) By G. Kohlbecker Institut f r Molekularbiologie und Biochemie, Freie Universit t Berlin and M. Butz Urologische Klinik und Poliklinik, Klinikum St eglitz, Freie Universit t Berlin (Received May 20, 1981) Summary: A new enzymatic method for direct photometric determination of oxalate in serum and urine is described, using oxalate oxidase. The resulting H202 is measured with a coupled enzyme system of catalase and aldehyde de- hydrogenase. Percentage recovery of added oxalate was 99 ± 4 in serum, and 98 ± 4 in urine (n = 10). Oxalate serum levels varied from 16.9 to 44.8 μηηοΙ/L Oxalate values can be determined within 20 minutes, without time consuming pretreatment of samples. The detection limit is 5 μπιοΐ/ΐ. Direkte spektrophotometrische Bestimmung von Serum- und Urin-Oxalat mit Oxalat-Oxidase Zusammenfassung: Es wird eine neue enzymatische Methode zur direkten photometrischen Bestimmung von Oxalat in Serum und Urin mittels Oxalatoxidase beschrieben. In einer gekoppelten Reaktion wird H202 enzymatisch durch Katalase und Aldehyddehydrogenase gemessen. Die Wiederfindung von zugesetztem Oxalat betrug 99 ± 4% in Serum und 98 ± 4% in Urin (n = 10). Die Oxalatkonzentrationen in Serum variierten von 16,9 bis 44,8 /imol/l. Oxalatwerte k nnen ohne zeitraubende Probenvorbehandlung innerhalb 20 Minuten ermittelt werden. Die Erfassungsgrenze liegt bei 5 μπιοΐ/ΐ. Introduction aldehyde dehydrogenase reaction for H2O2 determina- tion (12) is widely accepted in laboratory practice be- Reliable measurement of oxalate is required in the cause of its specifity.
    [Show full text]
  • Relationship Between Oxalate, Oxalate Oxidase Activity, Oxalate Sensitivity, and White Mold Susceptibility in Phaseolus Coccineus
    Genetics and Resistance Relationship Between Oxalate, Oxalate Oxidase Activity, Oxalate Sensitivity, and White Mold Susceptibility in Phaseolus coccineus Timothy J. Chipps, Barbara Gilmore, James R. Myers, and Henrik U. Stotz Department of Horticulture, Oregon State University, 4017 Agriculture and Life Science Building, Corvallis 97331. Current address of T. J. Chipps: Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland 97239. Current address of B. Gilmore: U.S. Department of Agriculture, National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97330. Accepted for publication 22 November 2004. ABSTRACT Chipps, T. J., Gilmore, B., Myers, J. R., and Stotz, H. U. 2005. Relation- Pole’. Sensitivity to oxalate ranked highest for Wolven Pole, lowest for PI ship between oxalate, oxalate oxidase activity, oxalate sensitivity, and white 255956, and intermediate for PI 535278. Oxalate concentrations were mold susceptibility in Phaseolus coccineus. Phytopathology 95:292-299. similar in infected stem tissues of the partially resistant lines and lower than Wolven Pole. Moreover, oxalate oxidase and superoxide dismutase Sclerotinia sclerotiorum is a necrotrophic pathogen that devastates the activities were absent in the more resistant lines but induced in Wolven yields of numerous crop species, including beans. The disease in common Pole. Collectively, these results suggest that genetic differences in bean and pea is referred to as white mold. We examined the relationship susceptibility to S. sclerotiorum among different P. coccineus lines are between oxalate, an established virulence factor of S. sclerotiorum, and partially dependent on oxalic acid. partial white mold resistance of scarlet runner bean (Phaseolus coc- cineus). P. coccineus genotypes PI 255956 (‘Mayan White Runner’) and Additional keywords: activity gels, oxalate-deficient mutant fungus, PI 535278 (Tars-046A) were more resistant than susceptible ‘Wolven Phaseolus vulgaris, straw test.
    [Show full text]
  • Banana Peel Oxalate Oxidase-Detection, Purification, Characterization and Physiological Role
    Innovare International Journal of Pharmacy and Pharmaceutical Sciences Academic Sciences ISSN- 0975-1491 Vol 7, Issue 1, 2015 Original Article BANANA PEEL OXALATE OXIDASE-DETECTION, PURIFICATION, CHARACTERIZATION AND PHYSIOLOGICAL ROLE SHADMA ANJUM ** , SHANTHY SUNDARAM *, G. K. RAI. ** *Centre for Biotechnology, University of Allahabad, Allahabad, ** Centre of Food Technology, University of Allahabad, Allahabad. Email: [email protected] Received: 13 Jul 2014 Revised and Accepted: 25 Aug 2014 ABSTRACT Objective: Enzymes like oxalate oxidase (EC 1.2.3.4) and superoxide dismutase (EC 1.15.1.1) from germin family are known to generate active oxygen species. In the mammalian system, excess accumulations of oxalate causes kidney stones. Oxalate oxidase, an H2O2-generating enzyme, used for detection of oxalate. The aim of the present work is to screen out the activity of enzymes from all three stages of banana (Musa paradisica L. Variety “Bhusawal”) peel and to isolate, purified and characterized oxalate oxidase from this. With that describe the physiological role of both oxalate oxidase and superoxide dismutase in the plant. Methods: Oxalate oxidase activity can be detected directly in SDS-PAGE gel. Purification was done by using ion-exchange chromatography and SDS- PAGE Gel. Results: Highest activity 5.99+0.021 unit /mg of oxalate oxidase were detected in leaky ripe stage of banana peel after purification. In crude extract of unripe banana peel activity of superoxide dismutase were found high (2.41unit/mg) compared to oxalate oxidase (0.269+ 0.020 unit/mg). Their occurence in different ripening stage of banana peel shows its role in plant defense mechanism. Conclusion: The purified enzyme of oxalate oxidase from banana peel is useful in the determination of oxalate content in common food, which is necessary for the prescription of the low oxalate diet for a patient with urinary and kidney stone where as superoxide dismutase work against ageing.
    [Show full text]
  • Abstracts from the 50Th European Society of Human Genetics Conference: Electronic Posters
    European Journal of Human Genetics (2019) 26:820–1023 https://doi.org/10.1038/s41431-018-0248-6 ABSTRACT Abstracts from the 50th European Society of Human Genetics Conference: Electronic Posters Copenhagen, Denmark, May 27–30, 2017 Published online: 1 October 2018 © European Society of Human Genetics 2018 The ESHG 2017 marks the 50th Anniversary of the first ESHG Conference which took place in Copenhagen in 1967. Additional information about the event may be found on the conference website: https://2017.eshg.org/ Sponsorship: Publication of this supplement is sponsored by the European Society of Human Genetics. All authors were asked to address any potential bias in their abstract and to declare any competing financial interests. These disclosures are listed at the end of each abstract. Contributions of up to EUR 10 000 (ten thousand euros, or equivalent value in kind) per year per company are considered "modest". Contributions above EUR 10 000 per year are considered "significant". 1234567890();,: 1234567890();,: E-P01 Reproductive Genetics/Prenatal and fetal echocardiography. The molecular karyotyping Genetics revealed a gain in 8p11.22-p23.1 region with a size of 27.2 Mb containing 122 OMIM gene and a loss in 8p23.1- E-P01.02 p23.3 region with a size of 6.8 Mb containing 15 OMIM Prenatal diagnosis in a case of 8p inverted gene. The findings were correlated with 8p inverted dupli- duplication deletion syndrome cation deletion syndrome. Conclusion: Our study empha- sizes the importance of using additional molecular O¨. Kırbıyık, K. M. Erdog˘an, O¨.O¨zer Kaya, B. O¨zyılmaz, cytogenetic methods in clinical follow-up of complex Y.
    [Show full text]
  • Supplementary Table 9. Functional Annotation Clustering Results for the Union (GS3) of the Top Genes from the SNP-Level and Gene-Based Analyses (See ST4)
    Supplementary Table 9. Functional Annotation Clustering Results for the union (GS3) of the top genes from the SNP-level and Gene-based analyses (see ST4) Column Header Key Annotation Cluster Name of cluster, sorted by descending Enrichment score Enrichment Score EASE enrichment score for functional annotation cluster Category Pathway Database Term Pathway name/Identifier Count Number of genes in the submitted list in the specified term % Percentage of identified genes in the submitted list associated with the specified term PValue Significance level associated with the EASE enrichment score for the term Genes List of genes present in the term List Total Number of genes from the submitted list present in the category Pop Hits Number of genes involved in the specified term (category-specific) Pop Total Number of genes in the human genome background (category-specific) Fold Enrichment Ratio of the proportion of count to list total and population hits to population total Bonferroni Bonferroni adjustment of p-value Benjamini Benjamini adjustment of p-value FDR False Discovery Rate of p-value (percent form) Annotation Cluster 1 Enrichment Score: 3.8978262119731335 Category Term Count % PValue Genes List Total Pop Hits Pop Total Fold Enrichment Bonferroni Benjamini FDR GOTERM_CC_DIRECT GO:0005886~plasma membrane 383 24.33290978 5.74E-05 SLC9A9, XRCC5, HRAS, CHMP3, ATP1B2, EFNA1, OSMR, SLC9A3, EFNA3, UTRN, SYT6, ZNRF2, APP, AT1425 4121 18224 1.18857065 0.038655922 0.038655922 0.086284383 UP_KEYWORDS Membrane 626 39.77128335 1.53E-04 SLC9A9, HRAS,
    [Show full text]
  • Oxalate Decarboxylase from Bacillus Subtilis, Recombinant Cat
    Oxalate Decarboxylase from Bacillus subtilis, recombinant Cat. No. NATE-1688 Lot. No. (See product label) Introduction Description Oxalate decarboxylase (OxdC, EC4.1.1.2) is a manganese-containing enzyme, which decomposes oxalic acid and oxalate. With OxdC catalysis, oxalate is split into formate and CO2. This enzyme belongs to the family of lyases, specifically the carboxy-lyases, which cleave carbon-carbon bonds. The systematic name of this enzyme class is oxalate carboxy-lyase (formate-forming). This enzyme is also called oxalate carboxy-lyase. The enzyme is composed of two cupin domains, each of which contains a Mn (II) ion. This enzyme participates in glyoxylate and dicarboxylate metabolism. This enzyme has been recognized for diagnostics in diverse biotechnological applications such as the clinical assay of oxalate in blood and urine, therapeutics, process industry, and agriculture to lower oxalate levels in foods and the environment. The recombinant protein made from the Bacillus Subtilis sequence includes OxdC with N-terminal His-tag. Synonyms Oxalate carboxy-lyase; EC 4.1.1.2; Oxalate decarboxylase; OxdC Product Information Species Bacillus subtilis Source E. coli Form Liquid Formulation In 50 mM NaOAC, pH 5.5. The activation was stopped by addition of 10 mM MMTS, which can be removed under reducing conditions. EC Number EC 4.1.1.2 CAS No. 9024-97-9 Molecular Weight 45.9 kDa Purity > 98% by SDS-PAGE Activity 150U/mg Creative Enzymes. All rights reserved. 45-1 Ramsey Road, Shirley, NY 11967, USA Tel:1-631-562-8517 1-516-512-3133 Fax:1-631-938-8127 E-mail: [email protected] http://www.creative-enzymes.com Concentration 2 mg/mL Unit Definition One unit is the amount of enzyme that generates 1.0 µmole of NADH at 37°C.
    [Show full text]