A Two-Component Flavin-Dependent Monooxygenase Involved In
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Biochemical and Biophysical Characterisation of Anopheles Gambiae Nadph-Cytochrome P450 Reductase
BIOCHEMICAL AND BIOPHYSICAL CHARACTERISATION OF ANOPHELES GAMBIAE NADPH-CYTOCHROME P450 REDUCTASE Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by PHILIP WIDDOWSON SEPTEMBER 2010 0 ACKNOWLEDGEMENTS There are a number of people whom I would like to thank for their support during the completion of this thesis. Firstly, I would like to thank my family; Mum, Louise and Chris for putting up with me over the years and for their love, patience and, in particular to Chris, technical support throughout– I could not have coped on my own without all your help. I would like to extend special thanks to Professor Lu-Yun Lian for her constant supervision throughout my Ph.D. She always kept me on the right path and was always available for support and advice which was especially useful when things were not going to plan. Thank you for all your help. In addition to Professor Lian I would like to thank all the members of the Structural Biology Group and everybody, past and present, whom I worked alongside in Lab C over the past four years. I would also like to thank the University of Liverpool for the funding that made all this possible. I would like to make particular mention to a few people in the School of Biological Sciences who were of particular help during my time at university. Dr. Mark Wilkinson was a constant support, not only during my Ph.D, but as my undergraduate tutor and honours project supervisor. Dr. Dan Rigden and Dr. -
Single-Molecule Spectroscopy Reveals How Calmodulin Activates NO Synthase by Controlling Its Conformational Fluctuation Dynamics
Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics Yufan Hea,1, Mohammad Mahfuzul Haqueb,1, Dennis J. Stuehrb,2, and H. Peter Lua,2 aCenter for Photochemical Sciences, Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403; and bDepartment of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195 Edited by Louis J. Ignarro, University of California, Los Angeles School of Medicine, Beverly Hills, CA, and approved July 31, 2015 (received for review May 5, 2015) Mechanisms that regulate the nitric oxide synthase enzymes (NOS) this model, the FMN domain is suggested to be highly dynamic are of interest in biology and medicine. Although NOS catalysis and flexible due to a connecting hinge that allows it to alternate relies on domain motions, and is activated by calmodulin binding, between its electron-accepting (FAD→FMN) or closed confor- the relationships are unclear. We used single-molecule fluores- mation and electron-donating (FMN→heme) or open conforma- cence resonance energy transfer (FRET) spectroscopy to elucidate tion (Fig. 1 A and B)(28,30–36). In the electron-accepting closed the conformational states distribution and associated conforma- conformation, the FMN domain interacts with the NADPH/FAD tional fluctuation dynamics of the two electron transfer domains domain (FNR domain) to receive electrons, whereas in the elec- in a FRET dye-labeled neuronal NOS reductase domain, and to tron donating open conformation the FMN domain has moved understand how calmodulin affects the dynamics to regulate away to expose the bound FMN cofactor so that it may transfer catalysis. -
View, the Catalytic Center of Bnoss Is Almost Identical to Mnos Except That a Conserved Val Near Heme Iron in Mnos Is Substituted by Iie[25]
STUDY OF ELECTRON TRANSFER THROUGH THE REDUCTASE DOMAIN OF NEURONAL NITRIC OXIDE SYNTHASE AND DEVELOPMENT OF BACTERIAL NITRIC OXIDE SYNTHASE INHIBITORS YUE DAI Bachelor of Science in Chemistry Wuhan University June 2008 submitted in partial fulfillment of requirements for the degree DOCTOR OF PHILOSOPHY IN CLINICAL AND BIOANALYTICAL CHEMISTRY at the CLEVELAND STATE UNIVERSITY July 2016 We hereby approve this dissertation for Yue Dai Candidate for the Doctor of Philosophy in Clinical-Bioanalytical Chemistry Degree for the Department of Chemistry and CLEVELAND STATE UNIVERSITY’S College of Graduate Studies by Dennis J. Stuehr. PhD. Department of Pathobiology, Cleveland Clinic / July 8th 2016 Mekki Bayachou. PhD. Department of Chemistry / July 8th 2016 Thomas M. McIntyre. PhD. Department of Cellular and Molecular Medicine, Cleveland Clinic / July 8th 2016 Bin Su. PhD. Department of Chemistry / July 8th 2016 Jun Qin. PhD. Department of Molecular Cardiology, Cleveland Clinic / July 8th 2016 Student’s Date of Defense: July 8th 2016 ACKNOWLEDGEMENT First I would like to express my special appreciation and thanks to my Ph. D. mentor, Dr. Dennis Stuehr. You have been a tremendous mentor for me. It is your constant patience, encouraging and support that guided me on the road of becoming a research scientist. Your advices on both research and life have been priceless for me. I would like to thank my committee members - Professor Mekki Bayachou, Professor Bin Su, Dr. Thomas McIntyre, Dr. Jun Qin and my previous committee members - Dr. Donald Jacobsen and Dr. Saurav Misra for sharing brilliant comments and suggestions with me. I would like to thank all our lab members for their help ever since I joint our lab. -
Potential for and Distribution of Enzymatic Biodegradation of Polystyrene by Environmental Microorganisms
materials Communication Potential for and Distribution of Enzymatic Biodegradation of Polystyrene by Environmental Microorganisms Liyuan Hou and Erica L.-W. Majumder * Department of Chemistry, SUNY College of Environmental Science and Forestry, Syracuse, NY 13210, USA; [email protected] * Correspondence: [email protected] or [email protected]; Tel.: +1-3154706854 Abstract: Polystyrene (PS) is one of the main polymer types of plastic wastes and is known to be resistant to biodegradation, resulting in PS waste persistence in the environment. Although previous studies have reported that some microorganisms can degrade PS, enzymes and mechanisms of microorganism PS biodegradation are still unknown. In this study, we summarized microbial species that have been identified to degrade PS. By screening the available genome information of microorganisms that have been reported to degrade PS for enzymes with functional potential to depolymerize PS, we predicted target PS-degrading enzymes. We found that cytochrome P4500s, alkane hydroxylases and monooxygenases ranked as the top potential enzyme classes that can degrade PS since they can break C–C bonds. Ring-hydroxylating dioxygenases may be able to break the side-chain of PS and oxidize the aromatic ring compounds generated from the decomposition of PS. These target enzymes were distributed in Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes, suggesting a broad potential for PS biodegradation in various earth environments and microbiomes. Our results provide insight into the enzymatic degradation of PS and suggestions for realizing the biodegradation of this recalcitrant plastic. Citation: Hou, L.; Majumder, E.L. Keywords: plastics; polystyrene biodegradation; enzymatic biodegradation; monooxygenase; alkane Potential for and Distribution of hydroxylase; cytochrome P450 Enzymatic Biodegradation of Polystyrene by Environmental Microorganisms. -
A New Mode of B Binding and the Direct Participation of A
Research Article 997 A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase Naoki Shibata1, Jun Masuda1, Takamasa Tobimatsu2, Tetsuo Toraya2*, Kyoko Suto1, Yukio Morimoto1 and Noritake Yasuoka1* Background: Diol dehydratase is an enzyme that catalyzes the adenosylcobalamin Addresses: 1Department of Life Science, Himeji (coenzyme B ) dependent conversion of 1,2-diols to the corresponding Institute of Technology, 1475-2 Kanaji, Kamigori, 12 Ako-gun, Hyogo 678-1297, Japan and 2Department aldehydes. The reaction initiated by homolytic cleavage of the cobalt–carbon bond of Bioscience and Biotechnology, Faculty of α β γ of the coenzyme proceeds by a radical mechanism. The enzyme is an 2 2 2 Engineering, Okayama University, Tsushima-Naka, heterooligomer and has an absolute requirement for a potassium ion for catalytic Okayama 700-8530, Japan. activity. The crystal structure analysis of a diol dehydratase–cyanocobalamin *Corresponding authors. complex was carried out in order to help understand the mechanism of action of E-mail: [email protected] this enzyme. [email protected] Results: The three-dimensional structure of diol dehydratase in complex with Key words: B12 enzyme, diol dehydratase, radicals, cyanocobalamin was determined at 2.2 Å resolution. The enzyme exists as a reaction mechanism, TIM barrel αβγ dimer of heterotrimers ( )2. The cobalamin molecule is bound between the Received: 16 March 1999 α and β subunits in the ‘base-on’ mode, that is, 5,6-dimethylbenzimidazole of Revisions requested: 7 April 1999 the nucleotide moiety coordinates to the cobalt atom in the lower axial position. -
Flavin-Containing Monooxygenases: Mutations, Disease and Drug Response Phillips, IR; Shephard, EA
Flavin-containing monooxygenases: mutations, disease and drug response Phillips, IR; Shephard, EA For additional information about this publication click this link. http://qmro.qmul.ac.uk/jspui/handle/123456789/1015 Information about this research object was correct at the time of download; we occasionally make corrections to records, please therefore check the published record when citing. For more information contact [email protected] Flavin-containing monooxygenases: mutations, disease and drug response Ian R. Phillips1 and Elizabeth A. Shephard2 1School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK 2Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK Corresponding author: Shephard, E.A. ([email protected]). and, thus, contribute to drug development. This review Flavin-containing monooxygenases (FMOs) metabolize considers the role of FMOs and their genetic variants in numerous foreign chemicals, including drugs, pesticides disease and drug response. and dietary components and, thus, mediate interactions between humans and their chemical environment. We Mechanism and structure describe the mechanism of action of FMOs and insights For catalysis FMOs require flavin adenine dinucleotide gained from the structure of yeast FMO. We then (FAD) as a prosthetic group, NADPH as a cofactor and concentrate on the three FMOs (FMOs 1, 2 and 3) that are molecular oxygen as a cosubstrate [5,6]. In contrast to most important for metabolism of foreign chemicals in CYPs FMOs accept reducing equivalents directly from humans, focusing on the role of the FMOs and their genetic NADPH and, thus, do not require accessory proteins. -
Chemistry and Functions of Proteins
TVER STATE MEDICAL UNIVERSITY BIOCHEMISTRY DEPARTMENT CHEMISTRY AND FUNCTIONS OF PROTEINS ILLUSTRATED BIOCHEMISTRY Schemes, formulas, terms and algorithm of preparation The manual for making notes of lectures and preparation for classes Tver, 2018 AMINO ACIDS -amino acids -These are organic acids with at least a minimum of one of its hydrogen atoms in the carbon chains substituted by an amino group.( Show the radical, amino and carboxyl groups) NH2 R | C – H | COOH Proteinogenous and Nonproteinogenous Amino acids - Major proteinogenous (standard) amino acids. (Give the names of each amino acid) R R 1 H – 2 CH3 – 12 HO – CH2 – (CH3)2 CH – 3 CH3 – CH – (CH3)2 CH – CH2 – 4 13 | CH3 – CH2 – CH – OH 5 | CH 3 6 14 HS – CH2 – HOOC – CH2 – 15 CH3 – S – CH2 – CH2 – 7 HOOC – CH2 – CH2 – NH2 | – C – H - | COOH 8 16 NH2 – CO – CH2 – 9 NH2 – CO – CH2 – CH2 – 17 10 NH2 – (CH2)3 – CH2 – 18 NH2 – C – NH – (CH2)3 – CH2 – 11 || NH 19 20 СООН NH -Glycine -Arginine Alanine -Serine -Valine -Threonine Leucine -Cysteine Isoleucine -Methionine Aspartic acid -Phenylalanine Glutamic acid -Tyrosine Asparagine -Tryptophan Glutamine -Histidine Lysine -Proline •Rare proteinogenous (standard) amino acids. ( Derivatives of lysine, proline and tyrosine) NH2 | H2N – CH2 – CH – (CH2)2 – CH | | OH COOH •Nonproteinogenous amino acids. (Name and show them) NH2 NH2 | | H2N – (CH2)3 – CH HS – (CH2)2 – CH | | COOH COOH NH2 | NH2 – C – HN – (CH2)3 – CH || | O COOH Ornithine Homocysteine Citrulline 2 CLASSIFICATION OF PROTEINOGENOUS (STANDARD) AMINO ACIDS Amino acids are classified by: *The structure of the radical (show); - aliphatic amino acids -monoaminodicarboxylic amino acids -amides of amino acids -diaminomonocarboxylic amino acids -hydroxy amino acids -sulfur-containing amino acids -cyclic (aromatic and heterolytic) amino acids *the polarity of the radical (show); -Non-polar (hydrophobic –Ala, Val, Leu, Ile, Trp, Pro.) -Polar (hydrophilic). -
Chapter 7. "Coenzymes and Vitamins" Reading Assignment
Chapter 7. "Coenzymes and Vitamins" Reading Assignment: pp. 192-202, 207-208, 212-214 Problem Assignment: 3, 4, & 7 I. Introduction Many complex metabolic reactions cannot be carried out using only the chemical mechanisms available to the side-chains of the 20 standard amino acids. To perform these reactions, enzymes must rely on other chemical species known broadly as cofactors that bind to the active site and assist in the reaction mechanism. An enzyme lacking its cofactor is referred to as an apoenzyme whereas the enzyme with its cofactor is referred to as a holoenzyme. Cofactors are subdivided into essential ions and organic molecules known as coenzymes (Fig. 7.1). Essential ions, commonly metal ions, may participate in substrate binding or directly in the catalytic mechanism. Coenzymes typically act as group transfer agents, carrying electrons and chemical groups such as acyl groups, methyl groups, etc., depending on the coenzyme. Many of the coenzymes are derived from vitamins which are essential for metabolism, growth, and development. We will use this chapter to introduce all of the vitamins and coenzymes. In a few cases--NAD+, FAD, coenzyme A--the mechanisms of action will be covered. For the remainder of the water-soluble vitamins, discussion of function will be delayed until we encounter them in metabolism. We also will discuss the biochemistry of the fat-soluble vitamins here. II. Inorganic cation cofactors Many enzymes require metal cations for activity. Metal-activated enzymes require or are stimulated by cations such as K+, Ca2+, or Mg2+. Often the metal ion is not tightly bound and may even be carried into the active site attached to a substrate, as occurs in the case of kinases whose actual substrate is a magnesium-ATP complex. -
Cbic.202000100Taverne
Delft University of Technology A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications Phonbuppha, Jittima; Tinikul, Ruchanok; Wongnate, Thanyaporn; Intasian, Pattarawan; Hollmann, Frank; Paul, Caroline E.; Chaiyen, Pimchai DOI 10.1002/cbic.202000100 Publication date 2020 Document Version Final published version Published in ChemBioChem Citation (APA) Phonbuppha, J., Tinikul, R., Wongnate, T., Intasian, P., Hollmann, F., Paul, C. E., & Chaiyen, P. (2020). A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications. ChemBioChem, 21(14), 2073-2079. https://doi.org/10.1002/cbic.202000100 Important note To cite this publication, please use the final published version (if applicable). Please check the document version above. Copyright Other than for strictly personal use, it is not permitted to download, forward or distribute the text or part of it, without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license such as Creative Commons. Takedown policy Please contact us and provide details if you believe this document breaches copyrights. We will remove access to the work immediately and investigate your claim. This work is downloaded from Delft University of Technology. For technical reasons the number of authors shown on this cover page is limited to a maximum of 10. Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public. -
Flavoprotein Hydroxylases and Epoxidases
277 Flavins on the Move: Flavoprotein Hydroxylases and Epoxidases Willem J.H. van Berkel1, Stefania Montersino1, Dirk Tischler2, Stefan Kaschabek2, Michael Schlömann2, George T. Gassner3 1Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands 2Environmental Microbiology, TU Bergakademie Freiberg, Germany 3Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, USA Introduction Flavoprotein monooxygenases perform chemo-, regio- and/or enantioselective oxygenations of organic substrates under mild reaction conditions [1]. These properties along with effective preparation methods turn flavoprotein monooxygenases in focus of industrial biocatalysis. Here we describe two biocatalytically relevant subclasses of flavoprotein monooxygenases with a close evolutionary relation: class A represented by p-hydroxybenzoate hydroxylase (PHBH) and class E formed by styrene monooxygenases (SMOs). PHBH family members perform highly regioselective hydroxylations on a wide variety of aromatic compounds. A rapid increase in available crystal structures and detailed mechanistic studies of such enzymes [2] are opening a new season of research in the field. SMOs catalyze a number of stereoselective epoxidation and sulfoxidation reactions [3]. Mechanistic and structural studies expose distinct characteristics, which provide a promising source for future biocatalyst development [4]. Nearly all bacterial SMOs are two- component proteins comprising a reductase and a monooxygenase. Remarkably, in few cases, the reductase is fused to the monooxygenase [5]. Such a self-sufficient enzyme can also cooperate with a single monooxygenase, resulting in a novel type of two-component SMO [6]. Results & Discussion Flavoprotein monooxygenases can be divided in six different subclasses based on structural features and oxygenation chemistry [1]. Table 1 gives an overview of the crystal structures of single-component flavoprotein aromatic hydroxylases (class A) and two-component styrene monooxygenases (SMO; class E). -
TRANSLATIONALLY by AMPK a Dissertation
CHOLESTEROL 7 ALPHA-HYDROXYLASE IS REGULATED POST- TRANSLATIONALLY BY AMPK A dissertation submitted to Kent State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy By Mauris E.C. Nnamani May 2009 Dissertation written by Mauris E. C. Nnamani B.S, Kent State University, 2006 Ph.D., Kent State University, 2009 Approved by Diane Stroup Advisor Gail Fraizer Members, Doctoral Dissertation Committee S. Vijayaraghavan Arne Gericke Jennifer Marcinkiewicz Accepted by Robert Dorman , Director, School of Biomedical Science John Stalvey , Dean, Collage of Arts and Sciences ii TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………..vi ACKNOWLEDGMENTS……………………………………………………..viii CHAPTER I: INTRODUCTION……………………………………….…........1 a. Bile Acid Synthesis…………………………………………….……….2 i. Importance of Bile Acid Synthesis Pathway………………….….....2 ii. Bile Acid Transport..…………………………………...…...………...3 iii. Bile Acid Synthesis Pathway………………………………………...…4 iv. Classical Bile Acid Synthesis Pathway…..……………………..…..8 Cholesterol 7 -hydroxylase (CYP7A1)……..........………….....8 Transcriptional Regulation of Cholesterol 7 -hydroxylase by Bile Acid-activated FXR…………………………….....…10 CYP7A1 Transcriptional Repression by SHP-dependant Mechanism…………………………………………………...10 CYP7A1 Transcriptional Repression by SHP-independent Mechanism……………………………………..…………….…….11 CYP7A1 Transcriptional Repression by Activated Cellular Kinase…….…………………………...…………………….……12 v. Alternative/ Acidic Bile Acid Synthesis Pathway…………......…….12 Sterol 27-hydroxylase (CYP27A1)……………….…………….12 -
Supplementary Materials
Supplementary Materials COMPARATIVE ANALYSIS OF THE TRANSCRIPTOME, PROTEOME AND miRNA PROFILE OF KUPFFER CELLS AND MONOCYTES Andrey Elchaninov1,3*, Anastasiya Lokhonina1,3, Maria Nikitina2, Polina Vishnyakova1,3, Andrey Makarov1, Irina Arutyunyan1, Anastasiya Poltavets1, Evgeniya Kananykhina2, Sergey Kovalchuk4, Evgeny Karpulevich5,6, Galina Bolshakova2, Gennady Sukhikh1, Timur Fatkhudinov2,3 1 Laboratory of Regenerative Medicine, National Medical Research Center for Obstetrics, Gynecology and Perinatology Named after Academician V.I. Kulakov of Ministry of Healthcare of Russian Federation, Moscow, Russia 2 Laboratory of Growth and Development, Scientific Research Institute of Human Morphology, Moscow, Russia 3 Histology Department, Medical Institute, Peoples' Friendship University of Russia, Moscow, Russia 4 Laboratory of Bioinformatic methods for Combinatorial Chemistry and Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia 5 Information Systems Department, Ivannikov Institute for System Programming of the Russian Academy of Sciences, Moscow, Russia 6 Genome Engineering Laboratory, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia Figure S1. Flow cytometry analysis of unsorted blood sample. Representative forward, side scattering and histogram are shown. The proportions of negative cells were determined in relation to the isotype controls. The percentages of positive cells are indicated. The blue curve corresponds to the isotype control. Figure S2. Flow cytometry analysis of unsorted liver stromal cells. Representative forward, side scattering and histogram are shown. The proportions of negative cells were determined in relation to the isotype controls. The percentages of positive cells are indicated. The blue curve corresponds to the isotype control. Figure S3. MiRNAs expression analysis in monocytes and Kupffer cells. Full-length of heatmaps are presented.