DOCSLIB.ORG

Viewing Angles of a Cartoon Representation Are Shown Representing Rotation by 75 Degrees About a Vertical Axis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Viewing Angles of a Cartoon Representation Are Shown Representing Rotation by 75 Degrees About a Vertical Axis A Dissertation Entitled Porcine Leukocyte 12-Lipoxygenase By Shu Xu Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry Dr. Max O. Funk, Jr., Committee Chair Dr. Douglas W. Leaman, Committee Member Dr. Timothy C. Mueser, Committee Member Dr. Steven J. Sucheck, Committee Member Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo May 2012 Copyright © 2012, Shu Xu This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Porcine Leukocyte 12-Lipoxygenase by Shu Xu Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry The University of Toledo May 2012 Lipoxygenases are a class of non-heme, non-sulfur iron dioxygenases in polyunsaturated fatty acid metabolism involved in the modulation of basic physiologic processes. The work described within this dissertation covers biochemical characterization of porcine leukocyte 12-lipoxygenase, and presents the crystal structure of its catalytic domain. For the first time, the iron content of the full length protein was obtained as high as 0.94 atom per molecule. The loss of this iron atom from the protein was evident, as the pH declined from 5 to 4 without losing the native protein folding, by electrospray ionization mass spectroscopy measurements. The full length protein failed to yield crystals in the screening conditions using numerous site-directed mutagenesis experiments and multiple crystallization techniques. As part of this study, the catalytic domain of porcine leukocyte 12-lipoxygenase was successfully expressed in E. coli cells. The crystal structure of porcine 12-lipoxygenase iii catalytic domain was determined to 1.89 Å as a complex with its specific inhibitor, 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) (PDB id: 3RDE). This represents the first structural description of the 12-lipoxygenase catalytic domain. The complex revealed a new one-open-end U-shaped channel for the natural substrate, arachidonic acid, which is remarkably different from the inhibitor (RS75091) binding pocket of rabbit 15-lipoxygenase. Crystallization of the 12-lipoxygenase catalytic domain with arachidonic acid was also performed. Only fragments of electron density in the active site were present in the structure of the substrate-enzyme complex, illustrating that the substrate is not conformationally fixed in the crystal and may be converted into its products and derivatives. In addition, an unbound form of the catalytic domain was observed in the crystal structure of a recombinant manganese reconstituted lipoxygenase catalytic domain. Almost no structural differences were observed between the bound and unbound forms suggesting that lipoxygenase catalytic domain has little structural fluctuation or conformational alteration upon the binding of the inhibitor or substrate. This research resulted in detailed biochemical insights for recombinant porcine leukocyte 12-lipoxygenase and its catalytic domain. The crystal structures of 12-lipoxygenase catalytic domain revealed key structural features of this enzyme, and also provided a basis for understanding enzymatic catalysis. iv To my wife, Hongyan Ma, for your endless love. To my parents and parents-in-law, for your support and understanding the importance of higher learning. To my lovely son, Shaun Xu, for the happiness you bring into my life. Acknowledgements First, I would like to address special thanks to my advisor, Dr. Max Funk, for all his support, advice, endless patience and continuous encouragement during these years. I would also like to thank Dr. Douglas Leaman, Dr. Timothy Mueser, and Dr. Steven Sucheck for their service in my advisory committee. I acknowledge Dr. Lawrence Marnett at Vanderbilt University for providing the cDNA of 12-lipoxygenase and the inhibitor used in this dissertation. Thanks go to Dr. Timothy Mueser, Dr. Alexander Pavlovsky and Dr. Leif Hanson for their assistances with X-ray crystallographic techniques; to Dr. Pannee Burckel for the measurements of atomic absorption; to Dr. Wendell Griffith and Dr. Dragan Isailovic for electrospay ionization measurements. I would like to thank the staff members at LS-CAT at the Advanced Photon Source of Argonne National Laboratory for their help with X-ray diffraction data collection. I would like to express thanks to former and current members of Dr. Funk’s group: Dr. Allan Sharp, Johanna Rapp, Ilka Decker, Maureen Gibbs, Marie Miniear, Kathryn Guinta, and Waqar Arif. Thanks to electronics specialists Thomas Kina and Youming Cao. I also extend appreciation to our neighbors at the University of Toledo, Dr. Viola’s, Dr. Mueser’s, and Dr. Leaman’s groups and all others for letting me using their equipment, as well as providing a wonderful work environment. vi Table of Contents Abstract…………………………………………………………………………………...iii Acknowledgements………………………………………………………………………vi Table of Contents………………………………………………………………………vii List of Tables……………………………………………………………………………xi List of Figures…………………………………………………………………...………xii List of Abbreviations……………………………………………………………………xv 1 Lipoxygenase: Structure, Biochemistry, and Biological Functions…………………1 1.1 Introduction…………………………………………………………………1 1.2 Structures of lipoxygenases…………………………………………………4 1.3 Lipoxygenase catalytic reactions……………………………………………7 1.3.1 Dioxygenase activity of lipoxygenases…………………………………7 1.3.2 Other enzymatic activities of lipoxygenases…………………………11 1.4 Metabolites from lipoxygenase reactions………………………………….12 1.4.1 Leukotriene pathway…………………………………………………15 1.4.2 Lipoxin pathway………………………………………………………17 1.4.3 Hepoxilin pathway…………………………….………………………18 1.4.4 Peroxide reduction pathway…………………………………………20 vii 1.5 Biological functions of lipoxygenase metabolites…………………………21 1.5.1 Roles of leukotrienes through 5-LOX pathway……………………….21 1.5.2 Roles of metabolites via 12/15-LOX pathway………………………22 1.5.2.1 Biological functions of lipoxins…………………………………23 1.5.2.2 Biological function of hepoxilins…………………………………23 1.5.3 Biological functions of HETEs and derivatives………………………24 1.6 Perspectives………………...………………………………………………25 2 Experimental Methods………………..……………………………………………..27 2.1 Mutagenesis of plasmids of porcine leukocyte 12-lipoxygenase………….27 2.2 Construction of plasmids of 12-lipoxygenase catalytic domain…………...28 2.3 Expression of 12-lipoxygenase and its mutants……………………………29 2.4 Expression of 12-lipoxygenase catalytic domain…………………….29 2.5 Expression of recombinant manganese 12-lipoxygenase catalytic domain……………………………………………………………………29 2.6 Purification of 12-lipoxygenase and 12-lipoxygenase catalytic domain…..30 2.7 Lipoxygenase enzymatic assay……………………………………………31 2.8 Protein concentration determination……………………………………….31 2.9 Sodium dodecyl sulfate polyacrylamide gel electrophoresis………………32 2.10 Atomic absorption measurements…………………………………………32 2.11 Electron ionization mass spectroscometry measurements…………………33 2.12 Ellman’s tritration…………………………………………………………33 viii 2.13 Dynamic light scattering measurements…………………………………34 2.14 Optimization of crystallization conditions…………………………………35 2.15 Crystallization of HLCDS-OPP and Mn-HLCDS-OPP……………………35 2.16 Crystallization of HLCDS-AA complex…………………………………...36 2.17 Diffraction data collection and structure determination…………………36 3 Biochemical Characterization of Porcine Leukocyte 12-Lipoxygenase………….…38 3.1 Introduction………………………………………………………………38 3.2 Results……………………………………………………………………39 3.2.1 Expression and purification of 12-lipoxygenase………………………39 3.2.2 Iron content of 12-lipoxygenase………………………………………41 3.2.3 ESI-MS measurements of 12-lipoxygenase…………………………...42 3.2.4 Crystallization experiments of 12-lipoxygenase………………………43 3.2.5 Protein engineering mutation to enhance crystallizability of PLL……45 3.2.6 Crystal screening results of PLL mutants……………………………48 3.3 Discussion…………………………………………………………………49 4 Crystal Structures of 12-Lipoxygenase Catalytic Domain………..……………52 4.1 Introduction………………………………………………………………52 4.2 Results……………………………………………………………………53 4.2.1 Expression of 12-lipoxygenae catalytic domain………………………53 4.2.2 Crystallization of 12-lipoxygenae catalytic domain…………………..55 4.2.3 Data collection and structure determination of HLCDS-OPP ix complex………………………………………………………………56 4.2.4 Specific inhibitor in the structure of 12-lipoxygenae catalytic domain…………………………………………………………………60 4.2.5 Expression of recombinant manganese 12-lipoxygenase catalytic domain…………………………………………………………………62 4.2.6 Crystallization of Mn-HLCDS-OPP and HLCDS–AA complexes……62 4.2.7 Data collection and structure determination of Mn-HLCDS-OPP and HLCDS-AA complexes……………………………………………….65 4.2.8 An inhibitor unbound form of 12-lipoxygenase catalytic domain from the crystal structure of Mn-HLCDS-OPP complex…………………65 4.2.9 The position of AA in HLCDS-AA complex………………………….67 4.3 Discussion…………………………………………………………………68 4.3.1 Crystallization conditions for 12-lipoxygenase catalytic domain……68 4.3.2 Variation of helix α2 in mammalian lipoxygenases…………………...69 4.3.3 The iron sites of mammalian lipoxygenases…………………………71 4.3.4 The inhibitor binding pockets of mammalian lipoxygenases…………72 4.3.5 Implication for inhibition and enzymatic catalysis……………………73 4.3.6 Substrate/intermediate binding site in 12-lipoxygenase………………76 5 Conclusions………………………………………………………………………….79 References………………………………………………………………………………..83 x List of Tables 1.1 Iron environment of lipoxygenases………………………………………………….6 2.1
Load more

Recommended publications
  • Bioinorganic Chemistry of Nickel
    inorganics Editorial Bioinorganic Chemistry of Nickel Michael J. Maroney 1,* and Stefano Ciurli 2,* 1 Department of Chemistry and Program in Molecular and Cellular Biology, University of Massachusetts Amherst, 240 Thatcher Rd. Life Sciences, Laboratory Rm N373, Amherst, MA 01003, USA 2 Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Viale G. Fanin 40, I-40127 Bologna, Italy * Correspondence: [email protected] (M.J.M.); [email protected] (S.C.) Received: 11 October 2019; Accepted: 11 October 2019; Published: 30 October 2019 Following the discovery of the first specific and essential role of nickel in biology in 1975 (the dinuclear active site of the enzyme urease) [1], nickel has become a major player in bioinorganic chemistry,particularly in microorganisms, having impacts on both environmental settings and human pathologies. At least nine classes of enzymes are now known to require nickel in their active sites, including catalysis of redox [(Ni,Fe) hydrogenases, carbon monoxide dehydrogenase, methyl coenzyme M reductase, acetyl coenzyme A synthase, superoxide dismutase] and nonredox (glyoxalase I, acireductone dioxygenase, lactate isomerase, urease) chemistries. In addition, the dark side of nickel has been illuminated in regard to its participation in microbial pathogenesis, cancer, and immune responses. Knowledge gleaned from the investigations of inorganic chemists into the coordination and redox chemistry of this element have boosted the understanding of these biological roles of nickel in each context. In this issue, eleven contributions, including four original research articles and seven critical reviews, will update the reader on the broad spectrum of the role of nickel in biology.
    [Show full text]
  • Cytochrome P450 Enzymes in Oxygenation of Prostaglandin Endoperoxides and Arachidonic Acid
    Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 231 _____________________________ _____________________________ Cytochrome P450 Enzymes in Oxygenation of Prostaglandin Endoperoxides and Arachidonic Acid Cloning, Expression and Catalytic Properties of CYP4F8 and CYP4F21 BY JOHAN BYLUND ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2000 Dissertation for the Degree of Doctor of Philosophy (Faculty of Pharmacy) in Pharmaceutical Pharmacology presented at Uppsala University in 2000 ABSTRACT Bylund, J. 2000. Cytochrome P450 Enzymes in Oxygenation of Prostaglandin Endoperoxides and Arachidonic Acid: Cloning, Expression and Catalytic Properties of CYP4F8 and CYP4F21. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from Faculty of Pharmacy 231 50 pp. Uppsala. ISBN 91-554-4784-8. Cytochrome P450 (P450 or CYP) is an enzyme system involved in the oxygenation of a wide range of endogenous compounds as well as foreign chemicals and drugs. This thesis describes investigations of P450-catalyzed oxygenation of prostaglandins, linoleic and arachidonic acids. The formation of bisallylic hydroxy metabolites of linoleic and arachidonic acids was studied with human recombinant P450s and with human liver microsomes. Several P450 enzymes catalyzed the formation of bisallylic hydroxy metabolites. Inhibition studies and stereochemical analysis of metabolites suggest that the enzyme CYP1A2 may contribute to the biosynthesis of bisallylic hydroxy fatty acid metabolites in adult human liver microsomes. 19R-Hydroxy-PGE and 20-hydroxy-PGE are major components of human and ovine semen, respectively. They are formed in the seminal vesicles, but the mechanism of their biosynthesis is unknown. Reverse transcription-polymerase chain reaction using degenerate primers for mammalian CYP4 family genes, revealed expression of two novel P450 genes in human and ovine seminal vesicles.
    [Show full text]
  • Eicosanoids Mediate Insect Nodulation Responses to Bacterial Infections (Cyclooxygenase/Lipoxygenase/Phospholipase A2/Manduca Sexta/Serratia Marcescens) JON S
    Proc. Natl. Acad. Sci. USA Vol. 91, pp. 12418-12422, December 1994 Physiology Eicosanoids mediate insect nodulation responses to bacterial infections (cyclooxygenase/lipoxygenase/phospholipase A2/Manduca sexta/Serratia marcescens) JON S. MILLER, TUANH NGUYEN, AND DAVID W. STANLEY-SAMUELSON* Insect Biochemistry/Physiology Laboratory, Department of Entomology, University of Nebraska, Lincoln, NE 68583-0816 Communicated by Wendell L. Roelofs, September 12, 1994 (receivedfor review August 11, 1993) ABSTRACT We propose that nodule formation is medi- inhibiting eicosanoid biosynthesis. On the basis of these ated by eicosanoids in insects. Nodulation is the temporally and findings, we proposed that eicosanoid products of the cyclo- quantitatively predominant cellular defense response to bac- oxygenase and lipoxygenase pathways are involved in insect terial infection in insects and other invertebrates. Inhibition of immune responses to bacterial infections. Because most of eicosanoid biosynthesis in larvae of the tobacco hornworn our experiments were done during the first hour postinfection Manduca sexta immediately prior to intrahemocoelic infections (PI), long before the appearance of antibacterial proteins in with the bacterium Serratia marcescens strongly reduced the insect hemolymph, we suggested that eicosanoids mediate nodulation response. Inhibition of eicosanoid biosynthesis also one or more hemocytic defense responses (6). reduced formation of cellular aggregates at 1 hr postinfection, This suggestion opens a crucial question: which
    [Show full text]
  • Cysteine Dioxygenase 1 Is a Metabolic Liability for Non-Small Cell Lung Cancer Authors: Yun Pyo Kang1, Laura Torrente1, Min Liu2, John M
    bioRxiv preprint doi: https://doi.org/10.1101/459602; this version posted November 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Cysteine dioxygenase 1 is a metabolic liability for non-small cell lung cancer Authors: Yun Pyo Kang1, Laura Torrente1, Min Liu2, John M. Asara3,4, Christian C. Dibble5,6 and Gina M. DeNicola1,* Affiliations: 1 Department of Cancer Physiology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA 2 Proteomics and Metabolomics Core Facility, Moffitt Cancer Center and Research Institute, Tampa, FL, USA 3 Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, USA 4 Department of Medicine, Harvard Medical School, Boston, MA, USA 5 Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Boston, MA, USA 6 Department of Pathology, Harvard Medical School, Boston, MA, USA *Correspondence to: [email protected]. Keywords: KEAP1, NRF2, cysteine, CDO1, sulfite Summary NRF2 is emerging as a major regulator of cellular metabolism. However, most studies have been performed in cancer cells, where co-occurring mutations and tumor selective pressures complicate the influence of NRF2 on metabolism. Here we use genetically engineered, non-transformed primary cells to isolate the most immediate effects of NRF2 on cellular metabolism. We find that NRF2 promotes the accumulation of intracellular cysteine and engages the cysteine homeostatic control mechanism mediated by cysteine dioxygenase 1 (CDO1), which catalyzes the irreversible metabolism of cysteine to cysteine sulfinic acid (CSA). Notably, CDO1 is preferentially silenced by promoter methylation in non-small cell lung cancers (NSCLC) harboring mutations in KEAP1, the negative regulator of NRF2.
    [Show full text]
  • Indoleamine 2,3-Dioxygenase and Its Therapeutic Inhibition in Cancer George C
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Scholarship, Research, and Creative Work at Bryn Mawr College | Bryn Mawr College... Bryn Mawr College Scholarship, Research, and Creative Work at Bryn Mawr College Chemistry Faculty Research and Scholarship Chemistry 2018 Indoleamine 2,3-Dioxygenase and Its Therapeutic Inhibition in Cancer George C. Prendergast William Paul Malachowski Bryn Mawr College, [email protected] Arpita Mondal Peggy Scherle Alexander J. Muller Let us know how access to this document benefits ouy . Follow this and additional works at: https://repository.brynmawr.edu/chem_pubs Part of the Chemistry Commons Custom Citation George C. Prendergast, William J. Malachowski, Arpita Mondal, Peggy Scherle, and Alexander J. Muller. 2018. "Indoleamine 2,3-Dioxygenase and Its Therapeutic Inhibition in Cancer." International Review of Cell and Molecular Biology 336: 175-203. This paper is posted at Scholarship, Research, and Creative Work at Bryn Mawr College. https://repository.brynmawr.edu/chem_pubs/25 For more information, please contact [email protected]. Indoleamine 2,3-Dioxygenase and Its Therapeutic Inhibition in Cancer George C. Prendergast, William, Malachowski, Arpita Mondal, Peggy Scherle, and Alexander J. Muller International Review of Cell and Molecular Biology 336: 175-203. http://doi.org/10.1016/bs.ircmb.2017.07.004 ABSTRACT The tryptophan catabolic enzyme indoleamine 2,3-dioxygenase-1 (IDO1) has attracted enormous attention in driving cancer immunosuppression, neovascularization, and metastasis. IDO1 suppresses local CD8+ T effector cells and natural killer cells and induces CD4+ T regulatory cells (iTreg) and myeloid-derived suppressor cells (MDSC). The structurally distinct enzyme tryptophan dioxygenase (TDO) also has been implicated recently in immune escape and metastatic progression.
    [Show full text]
  • Muscle Regeneration Controlled by a Designated DNA Dioxygenase
    Wang et al. Cell Death and Disease (2021) 12:535 https://doi.org/10.1038/s41419-021-03817-2 Cell Death & Disease ARTICLE Open Access Muscle regeneration controlled by a designated DNA dioxygenase Hongye Wang1, Yile Huang2,MingYu3,YangYu1, Sheng Li4, Huating Wang2,5,HaoSun2,5,BingLi 3, Guoliang Xu6,7 andPingHu4,8,9 Abstract Tet dioxygenases are responsible for the active DNA demethylation. The functions of Tet proteins in muscle regeneration have not been well characterized. Here we find that Tet2, but not Tet1 and Tet3, is specifically required for muscle regeneration in vivo. Loss of Tet2 leads to severe muscle regeneration defects. Further analysis indicates that Tet2 regulates myoblast differentiation and fusion. Tet2 activates transcription of the key differentiation modulator Myogenin (MyoG) by actively demethylating its enhancer region. Re-expressing of MyoG in Tet2 KO myoblasts rescues the differentiation and fusion defects. Further mechanistic analysis reveals that Tet2 enhances MyoD binding by demethylating the flanking CpG sites of E boxes to facilitate the recruitment of active histone modifications and increase chromatin accessibility and activate its transcription. These findings shed new lights on DNA methylation and pioneer transcription factor activity regulation. Introduction Ten-Eleven Translocation (Tet) family of DNA dioxy- 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Skeletal muscles can regenerate due to the existence of genases catalyze the active DNA demethylation and play muscle stem cells (MuSCs)1,2. The normally quiescent critical roles in embryonic development, neural regen- MuSCs are activated after muscle injury and further dif- eration, oncogenesis, aging, and many other important – ferentiate to support muscle regeneration3,4.
    [Show full text]
  • Montelukast, a Leukotriene Receptor Antagonist, Reduces the Concentration of Leukotrienes in the Respiratory Tract of Children with Persistent Asthma
    Montelukast, a leukotriene receptor antagonist, reduces the concentration of leukotrienes in the respiratory tract of children with persistent asthma Benjamin Volovitz, MD,a,b Elvan Tabachnik, MD,c Moshe Nussinovitch, MD,b Biana Shtaif, MSc,b Hanna Blau, MD,a Irit Gil-Ad, PhD,b Abraham Weizman, MD,b and Itzhak Varsano, MDa,b Petah Tikva, Tel Aviv, and Rehovot, Israel Background: Leukotrienes are bronchoactive mediators secreted by inflammatory cells in the respiratory mucosa on Abbreviations used exposure to asthma triggers. BAL: Bronchoalveolar lavage Objective: We investigated the effect of montelukast, a CysLT1: Cysteinyl leukotriene 1 (receptor) leukotriene receptor antagonist, on the release of leukotrienes ECP: Eosinophilic cationic protein in the respiratory mucosa of children with persistent asthma. LTC4: Leukotriene C4 Method: Twenty-three children aged 6 to 11 years with moder- LTD4: Leukotriene D4 ately severe asthma were treated in a cross-over design start- LTE4: Leukotriene E4 ing, after a 2-week run in period, with either montelukast (n = 12) or cromolyn (n = 11) for 4 weeks with a 2-week washout period between treatments. Twelve of them were then treated Cysteinyl leukotrienes are potent proinflammatory with either montelukast or beclomethasone for 6 months. The mediators produced from a variety of inflammatory use of β -agonists was recorded on a diary card. The concen- 2 cells, including mast cells, eosinophils, basophils and tration of leukotriene C4 (LTC4) was measured by HPLC in nasal washes obtained before and at the end of each treatment macrophages. Leukotriene C4 (LTC4) is metabolized period. Eosinophilic cationic protein (ECP) was measured in enzymatically to leukotriene D4 (LTD4) and subsequent- the nasal washes by RIA.
    [Show full text]
  • Relating Metatranscriptomic Profiles to the Micropollutant
    1 Relating Metatranscriptomic Profiles to the 2 Micropollutant Biotransformation Potential of 3 Complex Microbial Communities 4 5 Supporting Information 6 7 Stefan Achermann,1,2 Cresten B. Mansfeldt,1 Marcel Müller,1,3 David R. Johnson,1 Kathrin 8 Fenner*,1,2,4 9 1Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, 10 Switzerland. 2Institute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, 8092 11 Zürich, Switzerland. 3Institute of Atmospheric and Climate Science, ETH Zürich, 8092 12 Zürich, Switzerland. 4Department of Chemistry, University of Zürich, 8057 Zürich, 13 Switzerland. 14 *Corresponding author (email: [email protected] ) 15 S.A and C.B.M contributed equally to this work. 16 17 18 19 20 21 This supporting information (SI) is organized in 4 sections (S1-S4) with a total of 10 pages and 22 comprises 7 figures (Figure S1-S7) and 4 tables (Table S1-S4). 23 24 25 S1 26 S1 Data normalization 27 28 29 30 Figure S1. Relative fractions of gene transcripts originating from eukaryotes and bacteria. 31 32 33 Table S1. Relative standard deviation (RSD) for commonly used reference genes across all 34 samples (n=12). EC number mean fraction bacteria (%) RSD (%) RSD bacteria (%) RSD eukaryotes (%) 2.7.7.6 (RNAP) 80 16 6 nda 5.99.1.2 (DNA topoisomerase) 90 11 9 nda 5.99.1.3 (DNA gyrase) 92 16 10 nda 1.2.1.12 (GAPDH) 37 39 6 32 35 and indicates not determined. 36 37 38 39 S2 40 S2 Nitrile hydration 41 42 43 44 Figure S2: Pearson correlation coefficients r for rate constants of bromoxynil and acetamiprid with 45 gene transcripts of ECs describing nucleophilic reactions of water with nitriles.
    [Show full text]
  • Lipoxygenase Inhibition Activity of Coumarin Derivatives—QSAR and Molecular Docking Study
    pharmaceuticals Article Lipoxygenase Inhibition Activity of Coumarin Derivatives—QSAR and Molecular Docking Study Melita Lonˇcari´c 1 , Ivica Strelec 1 , Valentina Pavi´c 2 , Domagoj Šubari´c 3, Vesna Rastija 3 and Maja Molnar 1,* 1 Faculty of Food Faculty Osijek, Josip Juraj Strossmayer University, 31000 Osijek, Croatia; [email protected] (M.L.); [email protected] (I.S.) 2 Department of Biology, Josip Juraj Strossmayer University of Osijek, 31000 Osijek, Croatia; [email protected] 3 Department of Agroecology and Environmental Protection, Faculty of Agrobiotechnical Sciences Osijek, Josip Juraj Strossmayer University of Osijek, 31000 Osijek, Croatia; [email protected] (D.Š.); [email protected] (V.R.) * Correspondence: [email protected]; Tel.: +385-31-224-342 Received: 2 July 2020; Accepted: 15 July 2020; Published: 17 July 2020 Abstract: Lipoxygenases (LOXs) are a family of enzymes found in plants, mammals, and microorganisms. In animals and plants, the enzyme has the capability for the peroxidation of unsaturated fatty acids. Although LOXs participate in the plant defense system, the enzyme’s metabolites can have numerous negative effects on human health. Therefore, many types of research are searching for compounds that can inhibit LOXs. The best quantitative structure–activity relationship (QSAR) model was obtained using a Genetic Algorithm (GA). Molecular docking was performed with iGEMDOCK. The inhibition of lipoxygenase was in the range of 7.1 to 96.6%, and the inhibition of lipid peroxidation was 7.0–91.0%. Among the synthesized compounds, the strongest inhibitor of soybean LOX-3 (96.6%) was found to be 3-benzoyl- 7-(benzyloxy)-2H-chromen-2-one.
    [Show full text]
  • The Role of 5-Lipoxygenase in Pathophysiology and Management of Neuropathic Pain
    REVIEW ARTICLE THE ROLE OF 5-LIPOXYGENASE IN PATHOPHYSIOLOGY AND MANAGEMENT OF NEUROPATHIC PAIN Pascanus Lamsihar PT∗, Faldi Yaputra∗∗, Jimmy FA Barus4 and I Putu Eka Widyadharma∗∗,1 ∗Department of Neurology, Provincial Mental Hospital, West Borneo, Indonesia., ∗∗Department of Neurology, Faculty of Medicine, Udayana University-Sanglah General Hospital, Bali, Indonesia., 4Department of Neurology, Faculty of Medicine, Atma Jaya Catholic University, Jakarta-Indonesia. ABSTRACT Neuropathic pain (NP) is a pain caused by lesions in the nervous system. Several causes of NP are traumatic, metabolic disorders, ischemia, toxins, infections, immune-related, and hereditary. The pathophysiology of NP is very complicated and unknown entirely. Therefore the treatment of NP is still unsatisfactory. Recent studies believed the critical role of primary inflammatory mediators in the pathophysiology of NP especially leukotrienes (LTs). The 5- lipoxygenase enzyme (5-LOX) is an enzyme that plays a role in the metabolism of arachidonic acid into LTs. Leukotrienes (LTs) are the essential inflammatory mediators in the pathophysiology of NP. Leukotriene B4 (LTB4) can cause chemotaxis on neutrophils, lowering nociceptors threshold and may contribute to NP. Several studies believed the administration of 5-LOX inhibitors or LTs receptor antagonists could be useful in the management of NP. The purpose of this review is to summarize the involvement of 5-LOX enzyme as an essential role in the pathophysiology and management of NP. KEYWORDS 5-lipoxygenase, leukotrienes,
    [Show full text]
  • DMD # 35121 1 Human Cytochrome P450, CYP2S1, Metabolizes
    DMD Fast Forward. Published on November 10, 2010 as DOI: 10.1124/dmd.110.035121 DMD FastThis Forward. article has not Published been copyedited on andNovember formatted. The 10, final 2010 version as maydoi:10.1124/dmd.110.035121 differ from this version. DMD # 35121 Human Cytochrome P450, CYP2S1, Metabolizes Cyclooxygenase – and Lipoxygenase – Derived Eicosanoids Peter Bui, Satoshi Imaizumi, Sudheer Reddy Beedanagari, Srinivasa T. Reddy, and Oliver Hankinson Department of Pathology and Laboratory Medicine (P.B., S.R.B., and O.H.), Molecular Toxicology Interdepartmental Program (P.B., S.R.B., and O.H.) , Jonsson Comprehensive Cancer Center (P.B., S.R.B., S.T.R., and O.H.), Department of Medicine (S.I., and S.T.R.), and Downloaded from Department of Molecular and Medical Pharmacology (S.T.R.), University of California at Los Angeles, Los Angeles, California 90095 dmd.aspetjournals.org at ASPET Journals on September 26, 2021 1 Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics. DMD Fast Forward. Published on November 10, 2010 as DOI: 10.1124/dmd.110.035121 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 35121 Running title: CYP2S1 metabolizes eicosanoids To whom correspondence should be addressed: Oliver Hankinson, Ph.D. Departmental of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California at Los Angeles. 650 Charles E. Young Drive, Los Angeles, CA. 90095 Tel: 310-825-2936 Fax: 310-794-9272 Email: [email protected] Downloaded from Text: 36 pages Tables: 4 dmd.aspetjournals.org Figures: 10 References: 44 Abstract: 228 words at ASPET Journals on September 26, 2021 Introduction: 588 words Discussion: 1,146 words Abbreviations: CYP and P450, cytochrome P450; HPLC, high-performance liquid chromatography; LC-ESI-MS, liquid chromatography-electrospray ionization-mass spectrometry; MS/MS, tandem mass spectrometry; m/z, mass-to-charge ratio; M-H, molecular mass minus 1 2 DMD Fast Forward.
    [Show full text]
  • ALOX12 Antibody
    Efficient Professional Protein and Antibody Platforms ALOX12 Antibody Basic information: Catalog No.: UPA60470 Source: Rabbit Size: 50ul/100ul Clonality: polyclonal Concentration: 1mg/ml Isotype: Rabbit IgG Purification: affinity purified by Protein A Useful Information: Applications: WB:1:500-2000 Reactivity: Human, Mouse, Rat, Cow Specificity: This antibody recognizes ALOX12 protein. KLH conjugated synthetic peptide derived from human 12 Lipoxygenase Immunogen: 101-200/663 Non-heme iron-containing dioxygenase that catalyzes the stereo-specific peroxidation of free and esterified polyunsaturated fatty acids generating a spectrum of bioactive lipid mediators. Mainly converts arachidonic acid to (12S)-hydroperoxyeicosatetraenoic acid/(12S)-HPETE but can also metabo- lize linoleic acid. Has a dual activity since it also converts leukotriene A4/LTA4 into both the bioactive lipoxin A4/LXA4 and lipoxin B4/LXB4. Description: Through the production of specific bioactive lipids like (12S)-HPETE it regu- lates different biological processes including platelet activation. It also probably positively regulates angiogenesis through regulation of the expres- sion of the vascular endothelial growth factor. Plays a role in apoptotic pro- cess, promoting the survival of vascular smooth muscle cells for instance. May also play a role in the control of cell migration and proliferation. {ECO:0000269|PubMed:16638750, Uniprot: P18054 Human BiowMW: 76 KDa Buffer: 0.01M TBS(pH7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol. Storage: Store at 4°C short term and -20°C long term. Avoid freeze-thaw cycles. Note: For research use only, not for use in diagnostic procedure. Data: Gene Universal Technology Co. Ltd www.universalbiol.com Tel: 0550-3121009 E-mail: [email protected] Efficient Professional Protein and Antibody Platforms Sample: Raw264.7 Cell Lysate at 40 ug A431 Cell Lysate at 40 ug Primary: Anti-ALOX12 at 1/300 dilution Secondary: IRDye800CW Goat An- ti-Rabbit IgG at 1/20000 dilution Predicted band size: 73 kD Observed band size: 73 kD Gene Universal Technology Co.
    [Show full text]