DOCSLIB.ORG

One-Carbon Chemistry of Oxalate Oxidoreductase Captured by X-Ray Crystallography

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
One-Carbon Chemistry of Oxalate Oxidoreductase Captured by X-Ray Crystallography One-carbon chemistry of oxalate oxidoreductase captured by X-ray crystallography Marcus I. Gibsona,1, Percival Yang-Ting Chena, Aileen C. Johnsona,2, Elizabeth Pierceb,3, Mehmet Canb, Stephen W. Ragsdaleb, and Catherine L. Drennana,c,d,4 aDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; bDepartment of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109; cHoward Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139; and dDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved November 30, 2015 (received for review September 21, 2015) Thiamine pyrophosphate (TPP)-dependent oxalate oxidoreductase (Fig. 1A) (10). This OOR, discovered in Moorella thermoacetica (OOR) metabolizes oxalate, generating two molecules of CO2 and (11, 12), feeds both carbon dioxide and low-potential electrons two low-potential electrons, thus providing both the carbon and into the Wood−Ljungdahl pathway for acetogenesis, allowing reducing equivalents for operation of the Wood−Ljungdahl path- M. thermoacetica to grow exclusively on oxalate—a boon to this way of acetogenesis. Here we present structures of OOR in which anaerobic bacterium commonly found in soil, which is often rich two different reaction intermediate bound states have been trap- in oxalate (13). Like oxalyl-CoA decarboxylase, OOR uses TPP ped: the covalent adducts between TPP and oxalate and between as its catalytic cofactor, but differs in that it does not require TPP and CO2. These structures, along with the previously deter- CoA to activate oxalate, and it uses a chain of [4Fe-4S] clusters mined structure of substrate-free OOR, allow us to visualize how to store and transfer the electrons captured from the C−C bond. active site rearrangements can drive catalysis. Our results suggest OOR belongs to a larger superfamily of microbial enzymes, that OOR operates via a bait-and-switch mechanism, attracting known as 2-oxoacid:ferredoxin oxidoreductases (OFORs), which substrate into the active site through the presence of positively use TPP and [4Fe-4S] clusters to oxidize pyruvate and other charged and polar residues, and then altering the electrostatic cellular building blocks (14, 15). The best characterized family environment through loop and side chain movements to drive member is pyruvate:ferredoxin oxidoreductase (PFOR), for which catalysis. This simple but elegant mechanism explains how oxa- X-ray structures of substrate-free and substrate-bound forms (SI late, a molecule that humans and most animals cannot break Appendix,Fig.S1B) as well as some biochemical data are available down, can be used for growth by acetogenic bacteria. for the Desulfovibrio africanus enzyme (Da PFOR) (16–19), and more biochemical and spectroscopic data are available for the oxalate | oxidoreductase | carbon dioxide | thiamine pyrophosphate M. thermoacetica enzyme (20–23). PFOR catalyzes the reversible cleavage of pyruvate to form CO2, acetyl-CoA, and two electrons xalate is a difficult molecule to metabolize, so much so that that are capable of reducing low-potential ferredoxins (Fig. 1B). In Ohumans and most animals lack the capacity to break it this reaction, TPP acts as a nucleophile and forms a covalent down. This inability to metabolize oxalate is associated with adduct with pyruvate to initiate the subsequent decarboxylation. chronic renal disease in humans, some of the manifestations of Although we assume the same will be true for TPP-dependent which are painful kidney stones, crystalline arthritis, and total – OOR, oxalate is a very different substrate than pyruvate and the kidney failure (1 3). Plants, fungi, and some Gram-positive other 2-oxoacid substrates of the OFOR superfamily, all of which bacteria have evolved oxygen-dependent pathways for metabo- are characterized by a ketone immediately adjacent to a carboxylic lizing oxalate with the help of manganese-dependent enzymes in the cupin family of proteins (4, 5). These aerobic enzymes, ox- alate oxidase and oxalate decarboxylase, take advantage of the Significance propensity of oxalate to ligate metal ions to form a Mn oxalate complex, following which dioxygen is used to generate an oxalate- The microbial Wood−Ljungdahl pathway is the biological equiv- based radical species that causes rapid decarboxylation. The re- alent of the Monsanto process, responsible for converting duced products of these enzymes are hydrogen peroxide in the greenhouse gas CO2 into acetate. In addition to CO2, this microbial case of the oxidase and formate in the case of the decarboxylase. pathway requires low-potential electrons. The recently discovered Before 2010, only one enzyme pathway was known to catalyze the oxalate oxidoreductase produces both low-potential electrons degradation of oxalic acid in the absence of dioxygen: oxalyl-CoA and CO2 through the oxidation of oxalate. Here our structural decarboxylase (6). This enzyme is best known for its role in the data allow us to visualize intermediates along the reaction cycle metabolism of Oxalobacter formigenes, a bacterium that colonizes that have not been previously described, providing insight into the human gut and has been the subject of research into the role of the molecular mechanism by which oxalate is metabolized. the human gut microbiome in preventing, and possibly treating, – Author contributions: M.I.G., S.W.R., and C.L.D. designed research; M.I.G., P.Y.-T.C., A.C.J., human renal disease (7 9). As its name implies, however, oxalyl- E.P., and M.C. performed research; M.I.G., P.Y.-T.C., S.W.R., and C.L.D. analyzed data; and CoA decarboxylase first requires the activation of oxalate by CoA, M.I.G. and C.L.D. wrote the paper. a feat that is accomplished by a formyl-CoA transferase. This acti- The authors declare no conflict of interest. vated oxalate, featuring a thioester in place of a carboxylate, is This article is a PNAS Direct Submission. susceptible to nucleophilic attack by thiamin pyrophosphate (TPP; Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, SI Appendix,Fig.S1A), which is the catalytic cofactor in this an- www.pdb.org (PDB ID codes 5EXD and 5EXE). aerobic enzyme. The end product of this reaction is formate, which 1Present address: Department of Chemistry, Princeton University, Princeton, NJ 08540. can either enter cellular metabolism or be pumped out of the cell by 2Present address: Emory University School of Medicine, Atlanta, GA 30307. a formate:oxalate antiporter to generate ATP (8). 3Present address: Physical Science Department, Southern Utah University, Cedar City, Recently, an oxalate oxidoreductase (OOR) was reported that UT 84720. performs a previously unknown metabolic transformation of 4To whom correspondence should be addressed. Email: [email protected]. oxalate by splitting it into two molecules of carbon dioxide and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. recovering two reducing equivalents for use in cellular processes 1073/pnas.1518537113/-/DCSupplemental. 320–325 | PNAS | January 12, 2016 | vol. 113 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1518537113 Downloaded by guest on September 28, 2021 (25). Note that the Greek symbols after the residue number in- dicate the OOR chain ID (see SI Appendix, Fig. S1D). The structure presented here of OOR with oxalate covalently attached to TPP is consistent with this hypothesis. Arg31α occupies an analogous position to Thr31, and substrate interactions with Arg109α (OOR)/Arg114 (PFOR) and the imine group of the TPP iminopyr- imidine are also conserved (Fig. 3 B and E). Additionally, Asn143β (OOR)/Asn996 (PFOR) is in a similar position to hydrogen bond Fig. 1. OOR and PFOR catalyze the oxidation of oxalate and pyruvate, re- with the bound substrate, although this 3.1-Å interaction appears to spectively, reducing two ferredoxin equivalents in the process. (A) The re- action scheme for OOR. (B) The reaction scheme for PFOR. be with O2 of the bound oxalate, whereas, in PFOR, Asn996 inter- acts with the carboxylate group of the substrate (Fig. 3 B and E). To facilitate hydrogen-bonding interactions, rotation of the ethyl linker acid. Oxalate, on the other hand, is a diprotic acid and carries two of TPP (that links the thiazole ring to the pyrophosphate moiety) negative charges at neutral pH (24), and is therefore less ame- changes the angle of the thiazole plane, tilting the bound substrate nable to nucleophilic attack by a TPP cofactor. toward the various hydrogen bond donors. This ethyl linker rotation To probe how OOR was adapted to act on oxalate, we pre- is akin to previously described TPP flexibility in PFOR (19), and it is viously solved the structure of OOR from M. thermoacetica in its not observed in any active sites in which oxalate is not bound. resting state (25). Structural analysis revealed that OOR is a Unexpectedly, a close contact with the COOM intermediate is α – dimer of trimers, (αγβ)2, with each αγβ forming a catalytic unit also made by Asp116 , which is part of a loop from residues 107 that is composed of five domains (SI Appendix, Fig. S1 C and D). 121 of chain α that helps form the substrate-binding pocket. In the The active site, which houses the TPP and one of the three [4Fe- previously reported structure (Fig. 4A), Asp116α points away from 4S] clusters, appears to be more polar than that found in PFOR, theactivesiteandPhe117α points toward where substrate would consistent with OOR’s unique substrate (25). Here, we further bind (25) (an Asp-out conformation). However, in each of the four interrogate substrate binding to OOR through soaking and active sites of the oxalate-soaked structure, we see this situation cocrystallization experiments with oxalate. In addition to reversed, with Asp116α rotated about 150° to point toward the identifying residues responsible for activating oxalate, these carboxylate group of oxalate, Phe117α rotated about 90°, and the structures allow us to consider the molecular mechanism phenyl group swung away from the active site (Asp-in conforma- responsible for conversion of oxalate to CO2 and release of tion) (Fig.
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]