DOCSLIB.ORG

ROOPALI ROY Tungsten-Containing Aldehyde Oxidoreductases: a Novel Family of Enzymes from Hyperthermophilic Archaea (Under the Direction of MICHAEL W

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
ROOPALI ROY Tungsten-Containing Aldehyde Oxidoreductases: a Novel Family of Enzymes from Hyperthermophilic Archaea (Under the Direction of MICHAEL W ROOPALI ROY Tungsten-Containing Aldehyde Oxidoreductases: A Novel family of Enzymes from Hyperthermophilic Archaea (Under the Direction of MICHAEL W. W. ADAMS) The main objective of this research was the molecular, biochemical and kinetic characterization of the members of the AOR family of enzymes and attempt to elucidate the role these enzymes play in the physiology of Pyrococcus furiosus. Three different types of tungsten-containing aldehyde oxidizing enzymes aldehyde ferredoxin oxidoreductase (AOR), formaldehyde ferredoxin oxidoreductase (FOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) have previously been isolated from this microorganism. Although P. furiosus AOR had been extensively characterized, relatively little was known about the related enzyme FOR. The molecular catalytic and structural properties of this enzyme are presented. Detailed kinetic analyses of FOR indicate that C4 – C6 dialdehyde or acid-substituted aldehyde may serve as the physiological substrate for this enzyme. Since such semialdehydes are involved in the metabolic pathways of certain amino acids, FOR is proposed to have a role in amino acid metabolism of P. furiosus. The oxygen-inactivated forms of FOR, AOR and GAPOR from P. furiosus could be reactivated to 100% of original specific activity by incubation with sodium sulfide under reducing conditions. Loss of sulfide from the W coordination sphere presumably accounts for the loss in specific activity of these enzymes on exposure to oxygen. However, the exact nature of this ‘restored’ sulfide is not clear at this point. Although, P. furiosus cells grown with S° have 3-5 fold higher intracellular concentrations of acid- labile sulfide and approximately 2-fold higher polysulfide than those grown without S°, these are associated with the high molecular weight fraction of the cell-free extract and not available in the free form. Based on these results, sulfide-activation does not appear to be a physiological reaction. A survey of the complete genome sequences of numerous microorganisms reveals that tungstoenzymes of the AOR family appear to be widespread among the hyperthermophilic archaea and some bacteria. Two hitherto unknown members have also been identified in the P. furiosus genome. Termed wor4 and wor5, these genes are proposed to encode putative tungstoenzymes. One of these tungstoproteins WOR 4 has been isolated from P. furiosus cell-free extract and characterized. INDEX WORDS: Hyperthermophilic Archaea, Pyrococcus furiosus, Aldehyde Ferredoxin Oxidoreductase, Formaldehyde Ferredoxin Oxidoreductase, Glyceraldehyde-3-phosphate Ferredoxin Oxidoreductase, Aldehyde Oxidoreductase WOR 4, Tungsten, Tungstopterin, Thermal stability, Oxygen-inactivation, Sulfide- activation, Selenide-activation, Intracellular Sulfide. TUNGSTEN-CONTAINING ALDEHYDE OXIDOREDUCTASES: A NOVEL FAMILY OF ENZYMES FROM HYPERTHERMOPHILIC ARCHAEA by ROOPALI ROY B.S., Banaras Hindu University, India, 1988 M.S., Bombay University, India, 1991 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2001 Ó 2001 Roopali Roy All Rights Reserved TUNGSTEN-CONTAINING ALDEHYDE OXIDOREDUCTASES: A NOVEL FAMILY OF ENZYMES FROM HYPERTHERMOPHILIC ARCHAEA by ROOPALI ROY Approved: Major Professor: Michael W.W. Adams Committee: Harry Dailey Michael Johnson Lars Ljungdahl Robert Scott Electronic Version Approved: Gordhan L. Patel Dean of the Graduate School The University of Georgia December 2001 ACKNOWLEDGEMENTS I would like to express my deepest gratitude to Dr. Michael Adams for his excellent guidance, continued encouragement, enthusiasm and support through my graduate studies. I have learnt a lot in the process and I appreciate the knowledge and experience gained during the years. I would like to thank my reading and examining committee members Dr. Harry Dailey, Dr. Michael Johnson, Dr. Lars Ljungdahl and Dr. Robert Scott for their effort and insightful suggestions in the completion of this work. This work would not have been possible without the excellent technical expertise of numerous people. I would like to thank Dr. Douglas Rees and Dr. Yonglin Hu for x-ray crystallography; Dr. Michael Johnson for EPR; Dr. Robert Weiss for sequencing and the Riverbend Chemical Analysis Laboratory for metal analysis. The support of colleagues both past and present at the Adams Lab especially Lucy Feingold, Angeli Menon, Gerti Schut, and Marc Verhagen has been invaluable. This work would not have been possible without the constant support and encouragement of my husband Nilay Roy and I am deeply grateful to him. Special thanks to my daughter for her love and sacrifices when I could not be there for her. I would like to thank my parents Deepali and Ranjit for teaching me that anything is possible and taking pride in my accomplishments. I acknowledge and appreciate my mother and father-in–law Smita and Pronab Roy for their tremendous support this past year. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS............................................................................................ iv LIST OF TABLES ........................................................................................................ vii LIST OF FIGURES...................................................................................................... viii LIST OF ABBREVIATIONS......................................................................................... xi CHAPTER 1 INTRODUCTION...........................................................................................................1 CHAPTER 2 THREE ALDEHYDE OXIDOREDUCTASES FROM Pyrococcus furiosus .................52 CHAPTER 3 PURIFICATION AND MOLECULAR CHARACTERIZATION OF THE TUNGSTEN-CONTAINING FORMALDEHYDE FERREDOXIN OXIDOREDUCTASE FROM THE HYPERTHERMOPHILIC ARCHAEON Pyrococcus furiosus ......................................................................................................77 CHAPTER 4 EFFECTS ON FOR AND AOR ACTIVITY OF THE GROWTH CONDITIONS OF Pyrococcus furiosus.............................................................................................. 119 v vi CHAPTER 5 PHYSIOLOGICAL SIGNIFICANCE OF SULFIDE-ACTIVATION OF TUNGSTEN-CONTAINING ALDEHYDE OXIDOREDUCTASES FROM THE HYPERTHERMOPHILIC ARCHAEON Pyrococcus furiosus ........................... 144 CHAPTER 6 PHYLOGENETIC ANALYSIS OF KNOWN AND PUTATIVE TUNGSTOENZYMES OF THE AOR FAMILY ........................................................ 179 CHAPTER 7 A FOURTH TUNGSTEN-CONTAINING ENZYME FROM THE HYPERTHERMOPHILIC ARCHAEON Pyrococcus furiosus.................................... 194 CHAPTER 8 SUMMARY AND CONCLUSIONS........................................................................... 217 REFERENCES............................................................................................................ 232 LIST OF TABLES Page 1.1 The hyperthermophilic genera: organisms that grow at 90 °C.................................4 1.2 Tungsten-containing enzymes .................................................................................23 1.3 Catalytic properties of tungstoenzymes ...................................................................30 1.4 Substrate specificity of AOR ...................................................................................39 2.1 Molecular properties of tungstoenzymes from P. furiosus........................................64 2.2 Substrate specificities of AOR and FOR ..................................................................69 3.1 Purification of FOR from P. furiosus .......................................................................85 3.2 Substrate specificity of P. furiosus FOR ..................................................................94 3.3 Kinetic constants for the purified and sulfide-activated forms of P. furiosus FOR.........................................................................................96 4.1 Various combinations of substrates used for P. furiosus cultures............................ 133 5.1 Kinetic constants for various forms of FOR ........................................................... 167 5.2 Acid-labile sulfide concentrations inside and outside cells of various types ..................................................................................................... 170 5.3 Acid-labile sulfide concentration in low (< 30 kDa) and high (> 30 kDa) molecular weight fractions of P. furiosus cell-free extract...................................... 172 6.1 List of putative relatives of tungsten-containing enzymes of P. furiosus in the genomic databases of various microorganisms ............................................ 189 7.1 Purification of WOR 4 from P. furiosus cell-free extract........................................ 204 vii LIST OF FIGURES Page 1.1 Universal phylogenetic tree in rooted form, showing the three domains.....................6 1.2 Electron micrograph of Pyrococcus furiosus............................................................10 1.3 Proposed glycolytic pathway in Pyrococcus furiosus ...............................................13 1.4 Proposed pathway for peptide fermentation from Pyrococcus furiosus.....................17 1.5 Structure of the pterin cofactor.................................................................................25 1.6 Proposed physiological role of AOR in the catabolism of glucose and amino acids ..........................................................................................40
Load more

Recommended publications
  • Characterization of a Microsomal Retinol Dehydrogenase Gene from Amphioxus: Retinoid Metabolism Before Vertebrates
    Chemico-Biological Interactions 130–132 (2001) 359–370 www.elsevier.com/locate/chembiont Characterization of a microsomal retinol dehydrogenase gene from amphioxus: retinoid metabolism before vertebrates Diana Dalfo´, Cristian Can˜estro, Ricard Albalat, Roser Gonza`lez-Duarte * Departament de Gene`tica, Facultat de Biologia, Uni6ersitat de Barcelona, A6. Diagonal, 645, E-08028, Barcelona, Spain Abstract Amphioxus, a member of the subphylum Cephalochordata, is thought to be the closest living relative to vertebrates. Although these animals have a vertebrate-like response to retinoic acid, the pathway of retinoid metabolism remains unknown. Two different enzyme systems — the short chain dehydrogenase/reductases and the cytosolic medium-chain alcohol dehydrogenases (ADHs) — have been postulated in vertebrates. Nevertheless, recent data show that the vertebrate-ADH1 and ADH4 retinol-active forms originated after the divergence of cephalochordates and vertebrates. Moreover, no data has been gathered in support of medium-chain retinol active forms in amphioxus. Then, if the cytosolic ADH system is absent and these animals use retinol, the microsomal retinol dehydrogenases could be involved in retinol oxidation. We have identified the genomic region and cDNA of an amphioxus Rdh gene as a preliminary step for functional characterization. Besides, phyloge- netic analysis supports the ancestral position of amphioxus Rdh in relation to the vertebrate forms. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Retinol dehydrogenase; Retinoid metabolism; Amphioxus * Corresponding author. Fax: +34-93-4110969. E-mail address: [email protected] (R. Gonza`lez-Duarte). 0009-2797/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0009-2797(00)00261-1 360 D.
    [Show full text]
  • Methionine Sulfoxide Reductase a Is a Stereospecific Methionine Oxidase
    Methionine sulfoxide reductase A is a stereospecific methionine oxidase Jung Chae Lim, Zheng You, Geumsoo Kim, and Rodney L. Levine1 Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, MD 20892-8012 Edited by Irwin Fridovich, Duke University Medical Center, Durham, NC, and approved May 10, 2011 (received for review February 10, 2011) Methionine sulfoxide reductase A (MsrA) catalyzes the reduction Results of methionine sulfoxide to methionine and is specific for the S epi- Stoichiometry. Branlant and coworkers have studied in careful mer of methionine sulfoxide. The enzyme participates in defense detail the mechanism of the MsrA reaction in bacteria (17, 18). against oxidative stresses by reducing methionine sulfoxide resi- In the absence of reducing agents, each molecule of MsrA dues in proteins back to methionine. Because oxidation of methio- reduces two molecules of MetO. Reduction of the first MetO nine residues is reversible, this covalent modification could also generates a sulfenic acid at the active site cysteine, and it is function as a mechanism for cellular regulation, provided there reduced back to the thiol by a fast reaction, which generates a exists a stereospecific methionine oxidase. We show that MsrA disulfide bond in the resolving domain of the protein. The second itself is a stereospecific methionine oxidase, producing S-methio- MetO is then reduced and again generates a sulfenic acid at the nine sulfoxide as its product. MsrA catalyzes its own autooxidation active site. Because the resolving domain cysteines have already as well as oxidation of free methionine and methionine residues formed a disulfide, no further reaction forms.
    [Show full text]
  • A Brief Guide to Enzyme Classification and Nomenclature Rev AM
    A Brief Guide to Enzyme Nomenclature and Classification Keith Tipton and Andrew McDonald 1) Introduction NC-IUBMB Enzyme List, or, to give it its full title, “Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes by the Reactions they Catalyse,1 is a functional system, based solely on the substrates transformed and products formed by an enzyme. The basic layout of the classification for each enzyme is described below with some indication of the guidelines followed. More detailed rules for enzyme nomenclature and classification are available online.2 Further details of the principles governing the nomenclature of individual enzyme classes are given in the following sections. 2. Basic Concepts 2.1. EC numbers Enzymes are identified by EC (Enzyme Commission) numbers. These are also valuable for relating the information to other databases. They were divided into 6 major classes according to the type of reaction catalysed and a seventh, the translocases, was added in 2018.3 These are shown in Table 1. Table 1. Enzyme classes Name Reaction catalysed 1 Oxidoreductases *AH2 + B = A +BH2 2 Transferases AX + B = BX + A 3 Hydrolases A-B + H2O = AH + BOH 4 Lyases A=B + X-Y = A-B ç ç X Y 5 Isomerases A = B 6 LiGases †A + B + NTP = A-B + NDP + P (or NMP + PP) 7 Translocases AX + B çç = A + X + ççB (side 1) (side 2) *Where nicotinamide-adenine dinucleotides are the acceptors, NAD+ and NADH + H+ are used, by convention. †NTP = nucleoside triphosphate. The EC number is made up of four components separated by full stops.
    [Show full text]
  • SELENOF) with Retinol Dehydrogenase 11 (RDH11
    Tian et al. Nutrition & Metabolism (2018) 15:7 DOI 10.1186/s12986-017-0235-x RESEARCH Open Access The interaction of selenoprotein F (SELENOF) with retinol dehydrogenase 11 (RDH11) implied a role of SELENOF in vitamin A metabolism Jing Tian1* , Jiapan Liu1, Jieqiong Li2, Jingxin Zheng3, Lifang Chen4, Yujuan Wang1, Qiong Liu1 and Jiazuan Ni1 Abstract Background: Selenoprotein F (SELENOF, was named as 15-kDa selenoprotein) has been reported to play important roles in oxidative stress, endoplasmic reticulum (ER) stress and carcinogenesis. However, the biological function of SELENOF is still unclear. Methods: A yeast two-hybrid system was used to screen the interactive protein of SELENOF in a human fetal brain cDNA library. The interaction between SELENOF and interactive protein was validated by fluorescence resonance energy transfer (FRET), co-immunoprecipitation (co-IP) and pull-down assays. The production of retinol was detected by high performance liquid chromatograph (HPLC). Results: Retinol dehydrogenase 11 (RDH11) was found to interact with SELENOF. RDH11 is an enzyme for the reduction of all-trans-retinaldehyde to all-trans-retinol (vitamin A). The production of retinol was decreased by SELENOF overexpression, resulting in more retinaldehyde. Conclusions: SELENOF interacts with RDH11 and blocks its enzyme activity to reduce all-trans-retinaldehyde. Keywords: SELENOF (Seleonoprotein F) , Yeast two hybrid system, Protein-protein interaction, Retinol dehydrogenase 11 (RDH11), Fluorescence resonance energy transfer (FRET), Co-immunoprecipitation (co-IP), Pull- down, Retinol (vitamin a), Retinaldehyde Background SELENOF shows that the protein contains a Selenium (Se) is a necessary trace element for human thioredoxin-like motif. The redox potential of this motif health.
    [Show full text]
  • Pyruvate Ferredoxin Oxidoreductase from the Hyperthermophilic
    Proc. Natl. Acad. Sci. USA Vol. 94, pp. 9608–9613, September 1997 Biochemistry Pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon, Pyrococcus furiosus, functions as a CoA-dependent pyruvate decarboxylase (archaeayaldehydeydecarboxylationy2-keto acidythiamine pyrophosphate) KESEN MA*, ANDREA HUTCHINS*, SHI-JEAN S. SUNG†, AND MICHAEL W. W. ADAMS*‡ *Center for Metalloenzyme Studies, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602; and †Institute of Tree Root Biology, U.S. Department of Agriculture–Forest Service, Athens, GA 30602 Communicated by Gregory A. Petsko, Brandeis University, Waltham, MA, June 17, 1997 (received for review June 1, 1996) ABSTRACT Pyruvate ferredoxin oxidoreductase (POR) hyperthermophilic archaea and these are involved in peptide has been previously purified from the hyperthermophilic fermentation. They use 2-ketoglutarate (KGOR) (11), in- archaeon, Pyrococcus furiosus, an organism that grows opti- dolepyruvate (IOR) (12), and 2-ketoisovalerate (VOR) (13) as mally at 100°C by fermenting carbohydrates and peptides. The substrates, and function to oxidatively decarboxylate the 2- enzyme contains thiamine pyrophosphate and catalyzes the keto acids generated by the transamination of glutamate, oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 aromatic amino acids, and branched chain amino acids, re- and reduces P. furiosus ferredoxin. Here we show that this spectively, to the corresponding CoA derivative (13). enzyme also catalyzes the formation of acetaldehyde from The growth of hyperthermophilic archaea such as P. furiosus pyruvate in a CoA-dependent reaction. Desulfocoenzyme A is also unusual in that it is dependent upon tungsten (14), a substituted for CoA showing that the cofactor plays a struc- metal seldom used in biological systems (15).
    [Show full text]
  • A Nitrogenase-Like Methylthio-Alkane Reductase Complex Catalyzes Anaerobic Methane, Ethylene, and Methionine Biosynthesis Justin A
    A Nitrogenase-like Methylthio-alkane Reductase Complex Catalyzes Anaerobic Methane, Ethylene, and Methionine Biosynthesis Justin A. North,1 Srividya Murali1* ([email protected]), Adrienne B. Narrowe,3 Weili Xiong,4 Kathryn M. Byerly,1 Sarah J. Young,1 Yasuo Yoshikuni,5 Sean McSweeney,6 Dale Kreitler,6 William R. Cannon,2 Kelly C. Wrighton,3 Robert L. Hettich,4 and F. Robert Tabita1 (former PI, deceased) 1Department of Microbiology, The Ohio State University, Columbus, OH; 2Pacific Northwest National Laboratory, Richland, WA. 3Department of Soil and Crop Sciences, Colorado State University, Fort Collins, CO; 4Chemical Sciences Division, ORNL, Oak Ridge, TN; 5DOE Joint Genome Institute, Berkeley, CA; 6NSLS-II, Brookhaven National Laboratory, Upton, NY. Project Goals: The goal of this project is to identify and characterize the specific enzyme(s) that catalyze anaerobic ethylene synthesis. This is part of a larger project to develop an industrially compatible microbial process to synthesize ethylene in high yields. The specific goals are: 1. Identify the genes and gene products responsible for anaerobic ethylene synthesis. 2. Probe the substrate specificity and metagenomic functional diversity of methylthio-alkane reductases to identify optimal bioproduct generating systems. 3. Characterize the enzymes and the reactions that directly generate anaerobic ethylene. Abstract Text: Our previous work identified a novel anaerobic microbial pathway (DHAP- Ethylene Shunt) [1] that recycled 5’-methylthioadenosine (MTA) back to methionine with stoichiometric amounts of ethylene produced as a surprising side-product. MTA is a metabolic byproduct of methionine utilization in a multitude of cellular processes. The initial steps of the DHAP-ethylene sequentially converts MTA to dihydroxyacetone phosphate (DHAP) and ethylene precursor (2-methylthio)ethanol (Fig.
    [Show full text]
  • Methylenetetrahydrofolate Reductase: a Common Human Polymorphism and Its Biochemical Implications
    THE CHEMICAL RECORD Methylenetetrahydrofolate Reductase: THE CHEMICAL A Common Human Polymorphism and RECORD Its Biochemical Implications ROWENA G. MATTHEWS1,2 1Biophysics Research Division, The University of Michigan, 930 N. University Avenue, Ann Arbor, Michigan 48109-1055 2Department of Biological Chemistry, The University of Michigan, 930 N. University Avenue, Ann Arbor, Michigan 48109-1055 Received 6 June 2001; accepted 7 September 2001 ABSTRACT: Methlenetetrahydrofolate (CH2-H4folate) is required for the conversion of homocys- teine to methionine and of dUMP to dTMP in support of DNA synthesis, and also serves as a major source of one carbon unit for purine biosynthesis. This review presents biochemical studies of a human polymorphism in methylenetetrahydrofolate reductase, which catalyzes the reaction shown below. The mutation decreases the flux of CH2-H4folate into CH3-H4folate, and is associated with both beneficial and deleterious effects that can be traced to the molecular effect of the substitution of alanine 222 by valine. © 2002 The Japan Chemical Journal Forum and John Wiley & Sons, Inc. Chem Rec 2: 4–12, 2002 Key words: flavoprotein; homocysteine; methionine Introduction One of the more remarkable chemical syntheses carried out by transferase, as shown in Equation 1. Alternate sources of the biological organisms is the de novo biosynthesis of methyl methylene group include formate, which is converted to 10- groups. Du Vigneaud and Bennett are credited with the initial formyltetrahydrofolate, and thence to methenyl- and finally observations that rats could synthesize methionine from ho- methylenetetrahydrofolate by the action of formyltetra- mocysteine in the absence of a source of preformed methyl hydrofolate synthetase, methenyltetrahydrofolate cyclohydro- groups, and this synthesis was later shown to require the pres- lase, and methylenetetrahydrofolate dehydrogenase.2 ence of folate and cobalamin in the diet.
    [Show full text]
  • Ecdysone Oxidase and 3-Oxoecdysteroid Reductases in Manduca Sexta Midgut: Kinet I C Parameters
    Archives of Insect Biochemistry and Physiology 12:201-218 (1 989) Ecdysone Oxidase and 3-Oxoecdysteroid Reductases in Manduca sexta Midgut: Kinet i c Parameters Gunter F. Weirich, Malcolm J. Thompson, and James A. Svoboda Insect Hormone Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland Ecdysone and 20-hydroxyecdysone are converted to their 3-epimers by enzymes in the midgut cytosol of Manduca sexta larvae. A partially purified cytosol preparation has been used to analyze the nature of and the interaction between these enzymes. The cytosol was shown to contain ecdysone oxidase, one or more 3-oxoecdysteroid 3wreductase(s), and one or more 3-oxoecdysteroid 3P-reductase(s). The reductases reacted at different velocities with NADH and NADPH. With NADH, 3a-reduction was the major reaction; with NADPH, 3P-reduction was the major reaction. The apparent kinetic parameters for the enzymes support the assumed two-step mechanism for the 3-epimerization with a 3-oxoecdysteroid as intermediate. Key words: 3-epimerizatior1, 3-dehydroecdysonef 3-epiecdysonef 3a-hydroxyecdysteroids, 3P-hydroxyecdysteroidsf NADH, NADPH, molting hormone inactivation INTRODUCTION 3-Epiecdysteroids (3a-hydroxyecdysteroids)have been isolated from several insect species [1,2]. Because of their low molting hormone activity [l-31 they are assumed to be inactivation products [2,4]. In vitro ecdysone 3-epimerization was first observed by Nigg et al. in incubations of ecdysone with midgut homog- enates of the tobacco hornworm, Manduca sexta L. [5]. The enzyme system for Acknowledgments: We thank Rosemary E. Hennessey and Lynda J. Liska for their dedicated technical assistance; and Drs. Govindan Bhaskaran, David J.
    [Show full text]
  • Deep Learning Enables High-Quality and High-Throughput Prediction Of
    Deep learning enables high-quality and high- throughput prediction of enzyme commission numbers Jae Yong Ryua,b,c,d,1, Hyun Uk Kimc,d,e,f,2, and Sang Yup Leea,b,c,d,f,2 aMetabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 34141 Daejeon, Republic of Korea; bInstitute for the BioCentury, Korea Advanced Institute of Science and Technology, 34141 Daejeon, Republic of Korea; cBioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, 34141 Daejeon, Republic of Korea; dBioInformatics Research Center, Korea Advanced Institute of Science and Technology, 34141 Daejeon, Republic of Korea; eSystems Biology and Medicine Laboratory, Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 34141 Daejeon, Republic of Korea; and fSystems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Korea Advanced Institute of Science and Technology, 34141 Daejeon, Republic of Korea Contributed by Sang Yup Lee, May 21, 2019 (sent for review December 24, 2018; reviewed by Nathan E. Lewis and Costas D. Maranas) High-quality and high-throughput prediction of enzyme commission been developed: PRIAM (11), EzyPred (12), CatFam (13), (EC) numbers is essential for accurate understanding of enzyme EnzML (14), EFICAz2.5 (15), EnzDP (16), SVM-prot (17), functions, which have many implications in pathologies and indus- DEEPre (18), DETECT v2 (19), and ECPred (20). However, trial biotechnology. Several EC number prediction tools are currently prediction performances of these tools have room for further available, but their prediction performance needs to be further improvement with respect to computation time, precision, and improved to precisely and efficiently process an ever-increasing coverage for the prediction of EC numbers.
    [Show full text]
  • A Flavin-Dependent Sulfhydryl Oxidase With
    AUGMENTER OF LIVER REGENERATION: A FLAVIN-DEPENDENT SULFHYDRYL OXIDASE WITH CYTOCHROME C REDUCTASE ACTIVITY by Scott Farrell A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for Master of Science in Chemistry and Biochemistry Spring 2013 © 2013 Scott Farrell All Rights Reserved AUGMENTER OF LIVER REGENERATION A FLAVIN-DEPENDENT SULFHYDRYL OXIDASE WITH CYTOCHROME C REDUCTASE ACTIVITY by Scott Farrell Approved: __________________________________________________________ Colin Thorpe, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: __________________________________________________________ Murray V. Johnston, Ph.D. Chair of the Department of Department Chemistry and Biochemistry Approved: __________________________________________________________ George H. Watson, Ph.D Dean of the College of Arts and Science Approved: __________________________________________________________ James G. Richards, Ph.D. Vice Provost for Graduate and Professional Education ACKNOWLEDGMENTS I would first like to thank my advisor, Dr. Colin Thorpe, for giving me an unique perspective on not only biochemistry but on life. His patience, persistence and dedication has helped me see the project through. I will always remember the days that we had to stare at the enzyme cross-eyed in order for it to behave. I would also like to thank the faculty and staff of University of Delaware for without whom none of this would be possible. A special acknowledgement is for my daughter, Gracie. Even before the first time I held her in my arms, she has been my diving force to succeed. She is my inspiration and when I look at her, I feel the desire to improve upon myself and become better not only as a scientist but as person overall.
    [Show full text]
  • Equine 5Α-Reductase Activity and Expression in Epididymis
    231 1 C J CORBIN and others Equine 5 -reductase activity 231:1 23–33 Research α Equine 5α-reductase activity and expression in epididymis C J Corbin1, E L Legacki1, B A Ball2, K E Scoggin2, S D Stanley3 and A J Conley1 1Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, California, USA 2Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, Correspondence Kentucky, USA should be addressed 3Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, to A J Conley California, USA Email [email protected] Abstract The 5α-reductase enzymes play an important role during male sexual differentiation, Key Words and in pregnant females, especially equine species where maintenance relies on f progesterone 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses f testosterone 5α-reductases but was not studied elaborately in horses. Epididymis from younger and f steroidogenesis older postpubertal stallions was divided into caput, corpus and cauda and examined f epididymis for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real- f pregnancy Endocrinology time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone f equine of to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein f reductase was examined by thin-layer chromatography and verified by liquid chromatography Journal tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined.
    [Show full text]
  • Superoxide, Neuroleptics and the Ubiquinone and Cytochrome B5
    Molecular Psychiatry (1998) 3, 227–237 1998 Stockton Press All rights reserved 1359–4184/98 $12.00 ORIGINAL RESEARCH ARTICLE Superoxide, neuroleptics and the ubiquinone and cytochrome b5 reductases in brain and lymphocytes from normals and schizophrenic patients SA Whatley1, D Curti2, F Das Gupta1, IN Ferrier3, S Jones1, C Taylor4 and RM Marchbanks1,5 1Department of Neuroscience, Institute of Psychiatry, Denmark Hill, London SE5 8AF, UK; 2Istituto di Farmacologia, Universita di Pavia, 11 Piazza Botta, 27100 Pavia, Italy; 3MRC Neurochemical Pathology Unit, Newcastle General Hospital, NE4 6BE, UK; 4Department of Forensic Psychiatry, Institute of Psychiatry, Denmark Hill, London SE5 8AF, UK The effects of the neuroleptic flupenthixol on the expression of the genes coding for the mito- chondrial ubiquinone and cytochrome b5 reductases have been studied because of the impor- tance of these enzymes in energy metabolism, oxidative stress and also because similar but oppositely directed changes have been previously observed in the cerebral cortex from schizophrenics. The neuroleptic flupenthixol reduces the expression in rats of the gene coding for NADH-cytochrome b5 reductase as measured by in situ hybridisation and its enzy- mic manifestation. Flupenthixol also reduces the enzymic activity of the mitochondrial NADH- ubiquinone reductase, and it has been previously shown that mRNA from the mitochondrially coded parts of the enzyme are reduced by the drug. Both the cis- and therapeutically less active trans-flupenthixol were found to produce these changes in rats. Post-mortem brain tissue from schizophrenics who have received neuroleptic medication have reduced levels of both reductases as measured enzymically, Lymphocyte samples from schizophrenics also − have reduced levels of both reductases compared with normals.
    [Show full text]