CHARACTERISATION of a NOVEL NON-HENE DIOXYGENASE Mary

Total Page:16

File Type:pdf, Size:1020Kb

CHARACTERISATION of a NOVEL NON-HENE DIOXYGENASE Mary CHARACTERISATION OF A NOVEL NON-HENE DIOXYGENASE Mary G. Wallis Thesis presented for the Degree of Doctor of Philosophy University of Edinburgh r ACKNOWLEDGEMENTS I owe a great deal to my supervisor Dr. S. K. Chapman who has been unceasing in his help and enthusiasm throughout this study. I am deeply indepted to Drs. H.C. Baxter, R.L. Baxter, A. Cooper (University of Glasgow), J.G. Farmer, L.A. Gilmore, G.A. Reid, Miss H. Grant and Mr. J. Millar for help and expertise. Special thanks to Dr. N.C. McClure (University of Wales) for generous donations of bacteria and the use of unpublished material. My thanks are also due to Mrs. L. Marouf for the hectic typing of this thesis. I am very grateful to the Science and Engineering Research Council and also to Mr. and Mrs. R.G. Wallis for financial assistance over the last few years. I acknowledge the University of Edinburgh for the use of their facilities. - Finally, I would like to thank my family and friends for tremendous support, encouragement and many moments of madness, in particular during the preparation of this manuscript. (i) For my family, especially Jack Wallis and Campbell Beattie (ii) Inspiration did not come easily to Mary: 'I thought and pondered - vainly. I felt that blank incapability of invention which is the greatest misery of authorship, when dull Nothing replies to our anxious invocations. "Have you thought of a story?" I was asked each morning, and each morning I was forded to reply with a mortifying negative'. But revelation was at hand. Maurice Hindle on Mary Shelley (iii) DECLARATION Except where specific reference is made to other sources, the work presented in this thesis is the original work of the author. It has not been submitted, in whole or in part, for any other degree. Some of the results have already been published. Mary G. Wallis (iv) ABSTRACT A purification procedure has been developed for a novel extradiol dioxygenase, designated as 3-methyl- catechol 2,3-dioxygenase. The enzyme which is expressed in Escherichia coli, was originally derived from a Pseudomonas putida strain able to grow on toluidine. 3-Methylcatechol 2,3-dioxygenase was purified to homogeneity as judged by sodium dodecyl sulphate-poly- acrylamide gel electrophoresis (SDS-PAGE). Physical and kinetic properties of the purified enzyme were investigated. The enzyme consists of a single subunit type of Mr = 33,500 ± 2,000 by SDS-PAGE. Gel filtration indicated a molecular weight, under non-denaturing conditions, of 120,000 ± 20,000 consistent with the native enzyme existing as a tetramer of identical subunits. The NH 2 -terminal sequence (35 residues) has been determined and shows 50% identity with other extradiol dioxygenases. The structural charaqterisation of 3-methylcatechol 2,3-dioxygenase at the primary, secondary and quaternary levels indicates that the enzyme is typical of the extradiol dioxygenases. Evidence from several experiments, such as metal removal and replacement, oxidation and reduction, indicates that Fe (II) is essential for enzymatic function with the oxidised, ferric, form being inactive. No requirement for other cofactors was detected. (v) Thermal inactivation experiments demonstrated the complete stability of the enzyme up to 45°C for prolonged periods, however, above this temperature the enzymic activity was seen to decline. The kinetics of 3-methylcatechol 2,3-dioxygenase were investigated using UV/visible spectrophotometry and oxygen electrode polarography. Measurements were made under both standard and modified conditions. Typical saturation kinetics were observed for catechol, 3-methylcatechol and 4-methylcatechol as substrates. Data were analysed to give values of Vmax and Km. The substrate specificity for this enzyme was somewhat different from that seen for other catechol 2,3-dioxygenases, with 3-methylcatechol being cleaved at the highest rate. The Km values for the organic substrates were all around 0.3 AM, the lowest found for any dioxygenase to date. The Km for dioxygen was determined to be < 10 6 M. The enzyme consumed one mole of oxygen per mole of substrate in all three cases. The dependence of enzyme activity on pH follows a classic bell-shaped curve with a pH optimum of about 7.5 and pKa values of 6.9 (± 0.1) and 8.7 (± 0.1). The possibility that the lower pKa value might be due to an active site histidine was investigated by attempted modification of this residue. The implications of these results are discussed in relation to the structure and function of the enzyme. (vi) UNITS AND ABBREVIATIONS Abbreviations of units are of a standard form; dalton units Da grain(s) g litre(s) 1 second(s) s Other unit abbreviations include; molar absorption coefficient M- 1 cm' first order rate constant Both the single and three letter codes have been used to denote the amino acid residues; Amino Acid Three Letter One Letter Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Giutamine Gin Q Giutamic Acid Glu E Glycine Gly G Histidine His H (vii) Three Letter One Letter Amino Acid Abbreviation Symbol Isoleucine lie I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Vaiine Val V The following nomenclature has been adopted in referring to the dioxygenases; Enzyme Abbreviation Catechol 1,2-dioxygenase C1,2D Catechol 2,3-dioxygenase C2,3D 1, 2-Dihydroxybiphenyl 2, 3-dioxygenase 1, 2DHB2, 3D 1, 2-Dihydroxynaphthalene 2, 3-dioxygenase 1, 2DHN2, 3D 3, 4-Dihydroxyphenylacetate 2, 3-dioxygenase 3, 4DHPA2, 3D Gentisate 1,2-dioxygenase G1,2D 3-Methylcatechol 2, 3-dioxygenase 3MC2, 3D Protocatechuate 3, 4-dioxygenase 4D Protocatechuate 4, 5-dioxygenase 5D Steroid 4,5-dioxygenase 54,5D (viii) The following abbreviations have been employed in referring to the bioluimics; Chelating Liciand Abbreviation acetylacetonate acac - nitrilotriacetic acid NTA N,N'-(3, 3'dipropylamine)bis(salicylidene- amine) saldpt N, N' -ethylenebis (sal icyl ideneamine) salen N,N'-1, 2-benzenebis(salicylideneamine) saloph Other miscellaneous abbreviations include; Michaelis constant Km limiting value for reaction rate Vmax nicotinamide adenine dinucleotide NAD nicotinamide adenine dinucleotide phosphate NADP pounds per square inch p.s.i. revolutions per minute r.p.m. tris (hydroxymethyl) aminomethane tris (ix) LIST OF CONTENTS Page No. CHAPTER 1 : INTRODUCTION 1.1 Historical Perspective 1 1.2 Distribution of Oxygenases 3 1.3 Physiological Significance of Oxygenases 4 1.4 The Classification of Oxygenases 8 1.5 Structural Studies of Both the Intradiol and Extradiol Catechol Dioxygenases 16 1.6 Dioxygenase Biomimics 25 1.7 Mechanistic Studies of the Catechol Dioxy- genases 28 1.8 References 39 CHAPTER 2 : ISOLATION AND PURIFICATION OF 3- NETHYLCATECHOL 2, 3-DIOXYGENASE FROM ESCHERICHIA COLI 2.1 Introduction 50 2.2 Strains, Media and Growth of Bacteria 50 2.3 Enzyme Isolation 52 2.3.1 Step .1 : Crude Extract 52 2.3.2 Step 2 : Ammonium Sulphate Fractionation 54 2.3.3 Step 3 : Ion Exchange Chromatography 54 2.3.4 Step 4 : Gel Filtration 57 (x) 2 • 3 'd) Pace No. 2.3.5 Isolation and Purification Summary 57 2.4 Discussion of Isolation Techniques Employed and Comparison with Other Dioxygenase Preparations 60 2.4.1 Cell Lysis 60 2.4.2 Nucleic Acid Treatment 63 2.4.3 Acetone-Phosphate Buffer 64 2.4.4 Ammonium Sulphate Fractionation 65 2.4.5 Dialysis 66 2.4.6 Ion Exchange Chromatography 66 2.4.7 Pressure Dialysis 66 2.4.8 Gel Filtration 67 2.5 Discussion of a New Dioxygenase Purification Protocol 68 2.6 References 70 CHAPTER 3 : PRELIMINARY CHARACTERISATION OF 3- NETHYLCATECHOL 2, 3-DIOXYGENASE 3.1 Introduction 72 3.2 Experimental 72 3.2.1 Subunit Molecular Weight Determination 72 3.2.2 Native Molecular Weight Determination 72 3.2.3 NH 2 -Terminal Amino Acid Sequence Analysis 74 3.2.4 Thermal Stability 74 (xi) CHAPTER 3 (Cont'd) Page No. 3.3 Results and Discussion 78 3.3.1 Molecular Weight Determination 78 3.3.2 Amino Acid Sequence Analysis 81 3.3.3 Thermal Stability 106 3.4 Summary of Physical Properties 109 3.5 References 112 CHAPTER 4 : FURTHER CHARACTERISATION OF 3- METHYLCATEHOL 2, 3-DIOXYGENASE 4.1 Introduction 116 4.2 Experimental 116 4.2.1 Spectrophotometric Determination 116 4.2.2 Fe(II) Removal to Prepare the 3MC2,3D Apo-protein and Reconstruction of the Holo-enzyme by Fe(II) Replacement 117 4.2.3 Inactivation of 3NC2,3D by Oxidation and Reactivation of the Inactivated Enzyme by Re-reduction 117 4.2.4 Metal for Metal Replacement 118 4.3 Results and Discussion 118 4.3.1 Spectrophotometric Determination 119 4.3.2 Fe(II) Removal and Replacement ii 4.3.3 3MC2,3D Oxidation and Reduction 125 - (xii) 4.3 (Cont'd) Page No. 4.3.4 Metal for Metal Replacement, Metal Ions as probes to Obtain Comparative Information 127 4.4 Summary 131 4.5 References 133 CHAPTER 5 : KINETIC CHARACTERISATION OF 3-METHYL- CATECHOL 2, 3-DIOXYGENASE 5.1 Introduction 136 5.2 Experimental 137 5.2.1 Kinetic Studies 137 5.2.2 Attempted Modification of Histidine 139 5.2.3 Oxygen Consumption Studies 143 5.3 Results and Discussion 144 5.3.1 Kinetic Studies 144 5.3.2 pH Dependence of Enzyme Kinetics 153 5.3.3 Attempted Modification of Histidine 159 5.3.4 Oxygen Consumption Studies 161 5.3.5 Mechanistic Implications 163 5.4 Summary 164 5.5 References 166 CHAPTER 6 : METHODS AND MATERIALS 6.1 Growth Media 172 6.1.1 Luria Broth (L.B.) for the Growth of E.
Recommended publications
  • Mechanistic Study of Cysteine Dioxygenase, a Non-Heme
    MECHANISTIC STUDY OF CYSTEINE DIOXYGENASE, A NON-HEME MONONUCLEAR IRON ENZYME by WEI LI Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON August 2014 Copyright © by Student Name Wei Li All Rights Reserved Acknowledgements I would like to thank Dr. Pierce for your mentoring, guidance and patience over the five years. I cannot go all the way through this without your help. Your intelligence and determination has been and will always be an example for me. I would like to thank my committee members Dr. Dias, Dr. Heo and Dr. Jonhson- Winters for the directions and invaluable advice. I also would like to thank all my lab mates, Josh, Bishnu ,Andra, Priyanka, Eleanor, you all helped me so I could finish my projects. I would like to thank the Department of Chemistry and Biochemistry for the help with my academic and career. At Last, I would like to thank my lovely wife and beautiful daughter who made my life meaningful and full of joy. July 11, 2014 iii Abstract MECHANISTIC STUDY OF CYSTEINE DIOXYGENASE A NON-HEME MONONUCLEAR IRON ENZYME Wei Li, PhD The University of Texas at Arlington, 2014 Supervising Professor: Brad Pierce Cysteine dioxygenase (CDO) is an non-heme mononuclear iron enzymes that catalyzes the O2-dependent oxidation of L-cysteine (Cys) to produce cysteine sulfinic acid (CSA). CDO controls cysteine levels in cells and is a potential drug target for some diseases such as Parkinson’s and Alzhermer’s.
    [Show full text]
  • Catalytic Strategies of the Non-Heme Iron Dependent Oxygenases And
    Available online at www.sciencedirect.com ScienceDirect Catalytic strategies of the non-heme iron dependent oxygenases and their roles in plant biology Mark D White and Emily Flashman Non-heme iron-dependent oxygenases catalyse the these enzymes play in plant biology. Targeted manipula- incorporation of O2 into a wide range of biological molecules tion of these enzymes could have beneficial effects for and use diverse strategies to activate their substrates. Recent plant growth and/or stress tolerance, addressing the 21st kinetic studies, including in crystallo, have provided century issue of food security. We therefore highlight experimental support for some of the intermediates used by those of potential agricultural (and/or health) interest. different subclasses of this enzyme family. Plant non-heme iron-dependent oxygenases have diverse and important Resolving intermediates in non-heme iron biological roles, including in growth signalling, stress dependent oxygenase catalysis responses and secondary metabolism. Recently identified To activate O2, non-heme iron-dependent oxygenases use roles include in strigolactone biosynthesis, O-demethylation in a redox active iron, coordinated octahedrally at their active morphine biosynthesis and regulating the stability of hypoxia- site with water, usually in a vicinal facial triad arrangement responsive transcription factors. We discuss current structural [1]. During turnover, H2O is sequentially displaced by and mechanistic understanding of plant non-heme iron substrate/co-substrate and O2, which is reductively acti- oxygenases, and how their chemical/genetic manipulation vated enabling a specific substrate modification. Enzyme could have agricultural benefit, for example, for improved yield, subfamilies are proposed to employ different reactive stress tolerance or herbicide development.
    [Show full text]
  • Catalytic Triad' in Directing Promiscuous Substrate-Specificity
    Spectroscopic and mechanistic investigation of the enzyme mercaptopropionic acid dioxygenase (MDO), the role of the conserved outer-sphere 'catalytic triad' in directing promiscuous substrate-specificity A DISSERTATION Presented to the faculty of the Graduate School of The University of Texas at Arlington in Partial fulfillment Of the requirements of the Degree of Doctor in Philosophy in CHEMISTRY by SINJINEE SARDAR Under the guidance of Dr. Brad S. Pierce 2nd May, 2018 ACKNOWLEDGEMENT It gives me immense pleasure to thank my compassionate guide Dr. Brad S. Pierce, for his exemplary guidance to accomplish this work. It has been an extraordinary pleasure and privilege to work under his guidance for the past few years. I express my sincere gratitude to my doctoral committee members Prof. Frederick. MacDonnell, Dr. Rasika Dias, and Dr. Jongyun Heo for their valuable advice and constant help. I would also like to thank the Department of Chemistry and Biochemistry of UTA for bestowing me the opportunity to have access to all the departmental facilities required for my work. Here, I express my heartiest gratitude to the ardent and humane guidance of my senior lab mates Dr. Josh Crowell and Dr. Bishnu Subedi which was indispensable. I am immeasurably thankful to my lab mates Wei, Mike, Phil, Nick, Jared and Sydney for their constant support and help. It has been a pleasure to working with all of you. The jokes and laughter we shared have reduced the hardships and stress of graduate school and had made the stressful journey fun. To end with, I utter my heartfelt gratefulness to my parents and my sister for their lifelong support and encouragement which helped me chase my dreams and aspirations and sincere appreciation towards my friends who are my extended family for their lively and spirited encouragement.
    [Show full text]
  • Structural and Mechanistic Studies on 2-Oxoglutarate-Dependent Oxygenases and Related Enzymes Christopher J Schofield and Zhihong Zhang
    722 Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes Christopher J Schofield and Zhihong Zhang Mononuclear nonheme-Fe(II)-dependent oxygenases comprise The 2OG-dependent oxygenases have emerged as the an extended family of oxidising enzymes, of which the largest known family of nonheme oxidising enzymes [7,8] 2-oxoglutarate-dependent oxygenases and related enzymes are (Figure 1a). Their occurrence is ubiquitous, having been the largest known subgroup. Recent crystallographic and identified in many organisms ranging from prokaryotes to mechanistic studies have helped to define the overall fold of eukaryotes. Recent evidence also indicates that a 2OG- the 2-oxoglutarate-dependent enzymes and have led to the dependent oxygenase, prolyl 4-hydroxylase, is expressed by identification of coordination chemistry closely related to that of the P. bursaria Chlorella virus-1 [9]. Oxidative reactions catal- other nonheme-Fe(II)-dependent oxygenases, suggesting ysed by 2OG-dependent dioxygenases are steps in the related mechanisms for dioxygen activation that involve iron- biosynthesis of a variety of metabolites, including materials mediated electron transfer. of medicinal or agrochemical importance, such as plant ‘hor- mones’ (e.g. gibberellins) and antibiotics (e.g. cephalosporins Address and the β-lactamase inhibitor clavulanic acid). The Oxford Centre for Molecular Sciences and the Department of Chemistry, The Dyson Perrins Laboratory, South Parks Road, Oxford In mammals, the best-characterised 2OG-dependent oxy- OX1 3QY, UK genase is prolyl-4-hydroxylase, which catalyses the Current Opinion in Structural Biology 1999, 9:722–731 post-translational hydroxylation of proline residues in col- lagen. Mammalian prolyl-4-hydroxylase is an α β 0959-440X/99/$ — see front matter © 1999 Elsevier Science Ltd.
    [Show full text]
  • University of Groningen Exploring the Cofactor-Binding and Biocatalytic
    University of Groningen Exploring the cofactor-binding and biocatalytic properties of flavin-containing enzymes Kopacz, Malgorzata IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2014 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Kopacz, M. (2014). Exploring the cofactor-binding and biocatalytic properties of flavin-containing enzymes. Copyright Other than for strictly personal use, it is not permitted to download or to forward/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 (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 29-09-2021 Exploring the cofactor-binding and biocatalytic properties of flavin-containing enzymes Małgorzata M. Kopacz The research described in this thesis was carried out in the research group Molecular Enzymology of the Groningen Biomolecular Sciences and Biotechnology Institute (GBB), according to the requirements of the Graduate School of Science, Faculty of Mathematics and Natural Sciences.
    [Show full text]
  • Kinetic and Spectroscopic Studies on the Quercetin 2,3-Dioxygenase from Bacillus Subtilis† Matthew R
    Biochemistry 2006, 45, 1009-1016 1009 Kinetic and Spectroscopic Studies on the Quercetin 2,3-Dioxygenase from Bacillus subtilis† Matthew R. Schaab, Brett M. Barney,‡ and Wilson A. Francisco* Department of Chemistry and Biochemistry, Arizona State UniVersity, Tempe, Arizona 85287-1604 ReceiVed August 8, 2005; ReVised Manuscript ReceiVed NoVember 16, 2005 ABSTRACT: Quercetin 2,3-dioxygenase from Bacillus subtilis (QueD) converts the flavonol quercetin and molecular oxygen to 2-protocatechuoylphloroglucinolcarboxylic acid and carbon monoxide. QueD, the only known quercetin 2,3-dioxygenase from a prokaryotic organism, has been described as an Fe2+- dependent bicupin dioxygenase. Metal-substituted QueDs were generated by expressing the enzyme in Escherichia coli grown on minimal media in the presence of a number of divalent metals. The addition of Mn2+,Co2+, and Cu2+ generated active enzymes, but the addition of Zn2+,Fe2+, and Cd2+ did not increase quercetinase activity to any significant level over a control in which no divalent ions were added to the media. The Mn2+- and Co2+-containing QueDs were purified, characterized by metal analysis and EPR spectroscopy, and studied by steady-state kinetics. Mn2+ was found to be incorporated nearly stoichiometrically to the two cupin motifs. The hyperfine coupling constant of the g ) 2 signal in the EPR spectra of the Mn2+-containing enzyme showed that the two Mn2+ ions are ligated in an octahedral coordination. The turnover number of this enzyme was found to be in the order of 25 s-1, nearly 40-fold higher than that of the Fe2+-containing enzyme and similar in magnitude to that of the Cu2+-containing quercertin 2,3-dioxygenase from Aspergillus japonicus.
    [Show full text]
  • Spectroscopic and Thermodynamic Characterization of Human 3-Hydroxyanthranilate-3,4-Dioxygenase
    Georgia State University ScholarWorks @ Georgia State University Chemistry Theses Department of Chemistry 5-7-2016 Spectroscopic and Thermodynamic Characterization of Human 3-Hydroxyanthranilate-3,4-Dioxygenase Calmour Henry [email protected] Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_theses Recommended Citation Henry, Calmour, "Spectroscopic and Thermodynamic Characterization of Human 3-Hydroxyanthranilate-3,4-Dioxygenase." Thesis, Georgia State University, 2016. https://scholarworks.gsu.edu/chemistry_theses/88 This Thesis is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Theses by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. SPECTROSCOPIC AND THERMODYNAMIC CHARACTERIZATION OF HUMAN 3- HYDROXYANTHRANILATE-3,4-DIOXYGENASE by CALMOUR HENRY Under the direction of Aimin Liu, Ph.D. ABSTRACT 3-Hydroxyanthranilate-3,4-dioxygenase (HAO) is a non-heme iron dependent enzyme that catalyzes the oxidative ring opening of 3-hydroxyanthranilate (HAA) which is an intermediate in the kynurenine pathway and its ring opening is the final enzymatic step from tryptophan to quinolinic acid (QUIN). QUIN functions as an N-methyl-D-aspartate (NMDA) receptor agonist and elevated brain levels of QUIN have been observed in neurodegenerative diseases. Reducing QUIN levels is of pharmacological importance. Kinetic and calorimetric studies were performed on human HAO using UV/Vis spectroscopy and isothermal titration calorimetry to understand its stability and behavior using its natural substrate (3-HAA) and neurological inhibitors such as acetylsalicylic acid (aspirin). This study reveals the first analysis of human HAO and facilitates understanding of its binding dynamics and enzymatic activity which will later support discovering suitable pharmacological compounds.
    [Show full text]
  • Iron Complexes Mimicking the Catechol Dioxygenases
    materials Review Biological Inspirations: Iron Complexes Mimicking the Catechol Dioxygenases Karolina Kałdu ´nska 1,* , Anna Kozakiewicz 1 , Magdalena Wujak 2 and Andrzej Wojtczak 1,* 1 Faculty of Chemistry, Nicolaus Copernicus University in Toru´n,Gagarina 7, 87-100 Toru´n,Poland; [email protected] 2 Faculty of Pharmacy, Nicolaus Copernicus University in Toru´n,Collegium Medicum in Bydgoszcz, Jurasza 2, 85-089 Bydgoszcz, Poland; [email protected] * Correspondence: [email protected] (K.K.); [email protected] (A.W.) Abstract: Within the broad group of Fe non-heme oxidases, our attention was focused on the catechol 1,2- and 2,3-dioxygenases, which catalyze the oxidative cleavage of aromatic rings. A large group of Fe complexes with N/O ligands, ranging from N3 to N2O2S, was developed to mimic the activity of these enzymes. The Fe complexes discussed in this work can mimic the intradiol/extradiol catechol dioxygenase reaction mechanism. Electronic effects of the substituents in the ligand affect the Lewis acidity of the Fe center, increasing the ability to activate dioxygen and enhancing the catalytic activity of the discussed biomimetic complexes. The ligand architecture, the geometric isomers of the complexes, and the substituent steric effects significantly affect the ability to bind the substrate in a monodentate and bidentate manner. The substrate binding mode determines the preferred mechanism and, consequently, the main conversion products. The preferred mechanism of action can also be affected by the solvents and their ability to form the stable complexes with the Fe center. The electrostatic interactions of micellar media, similar to SDS, also control the intradiol/extradiol Citation: Kałdu´nska,K.; mechanisms of the catechol conversion by discussed biomimetics.
    [Show full text]
  • Puriwcation and Characterization of a Catechol 1,2-Dioxygenase from a Phenol Degrading Candida Albicans TL3
    Arch Microbiol (2007) 187:199–206 DOI 10.1007/s00203-006-0187-4 ORIGINAL PAPER PuriWcation and characterization of a catechol 1,2-dioxygenase from a phenol degrading Candida albicans TL3 San-Chin Tsai · Yaw-Kuen Li Received: 31 May 2006 / Revised: 29 September 2006 / Accepted: 13 October 2006 / Published online: 7 November 2006 © Springer-Verlag 2006 Abstract A eukaryotic catechol 1,2-dioxygenase (1,2- Introduction CTD) was produced from a Candida albicans TL3 that possesses high tolerance for phenol and strong phenol Catechols are formed during biodegradation of a degrading activity. The 1,2-CTD was puriWed via variety of aromatic compounds by aerobic microor- ammonium sulfate precipitation, Sephadex G-75 gel ganisms. The aromatic rings of catechols may be Wltration, and HiTrap Q Sepharose column chromatog- cleaved by intradiol dioxygenase via an ortho-cleavage raphy. The enzyme was puriWed to homogeneity and pathway to form cis,cis-muconate or by extradiol found to be a homodimer with a subunit molecular dioxygenase via a meta-cleavage pathway to form weight of 32,000. Each subunit contained one iron. The 2-hydroxymuconic semialdehyde. These metabolites optimal temperature and pH were 25°C and 8.0, are degraded in the tricarboxylic acid cycle (Ngai et al. respectively. Substrate analysis showed that the puri- 1990; Chen and Lovell 1990; Aoki et al. 1990). Both Wed enzyme was a type I catechol 1,2-dioxygenase. This types of catechol dioxygenase use nonheme iron as a is the Wrst time that a 1,2-CTD from a eukaryote (Can- cofactor (Nozaki 1979). Intradiol dioxygenases are dida albicans) has been characterized.
    [Show full text]
  • The Structure and Function Study of Three Metalloenzymes That Utilize Three Histidines As Metal Ligands
    Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry Fall 11-19-2013 The Structure and Function Study of Three Metalloenzymes That Utilize Three Histidines as Metal Ligands Yan Chen Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Recommended Citation Chen, Yan, "The Structure and Function Study of Three Metalloenzymes That Utilize Three Histidines as Metal Ligands." Dissertation, Georgia State University, 2013. https://scholarworks.gsu.edu/chemistry_diss/86 This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES THAT UTILIZE THREE HISTIDINES AS METAL LIGANDS by Yan Chen Under the Direction of Dr. Aimin Liu ABSTRACT The function of the metalloenzymes is mainly determined by four structural features: the metal core, the metal binding motif, the second sphere residues in the active site and the electron- ic statistics. Cysteamine dioxygenase (ADO) and cysteine dioxygenase (CDO) are the only known enzymes that oxidize free thiol containing molecules in mammals by inserting of a dioxygen molecue. Both ADO and CDO are known as non-heme iron dependent enzymes with 3-His metal binding motif. However, the mechanistic understanding of both enzymes is obscure. The understanding of the mechanistic features of the two thiol dioxygenases is approached through spectroscopic and metal substitution in this dissertation. Another focus of the disserta- tion is the understanding of the function of a second sphere residue His228 in a 3-His-1-carboxyl zinc binding decarboxylase α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD).
    [Show full text]
  • Identifying and Creating Pathways to Improve Biological Lignin Valorization T ⁎ Zhi-Hua Liua,B,1, Rosemary K
    Renewable and Sustainable Energy Reviews 105 (2019) 349–362 Contents lists available at ScienceDirect Renewable and Sustainable Energy Reviews journal homepage: www.elsevier.com/locate/rser Identifying and creating pathways to improve biological lignin valorization T ⁎ Zhi-Hua Liua,b,1, Rosemary K. Lec,1, Matyas Kosad, Bin Yange, Joshua Yuana,b, , ⁎⁎ Arthur J. Ragauskasc,f,g, a Synthetic and Systems Biology Innovation Hub (SSBiH), Texas A&M University, College Station, TX 77843, United States b Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, United States c Department of Chemical & Biomolecular Engineering, University of Tennessee Knoxville, Knoxville, TN 37996, United States d Fortress Advanced Bioproducts Inc., Vancouver, BC, Canada V6T 1W5 e Bioproducts, Sciences, and Engineering Laboratory, Department of Biological Systems Engineering, Washington State University, Richland, WA 99354, United States f Joint Institute for Biological Sciences, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States g Center for Renewable Carbon, Department of Forestry, Wildlife, and Fisheries, University Tennessee, Knoxville, TN 37996, United States ARTICLE INFO ABSTRACT Keywords: Biological lignin valorization to fuels and value-added chemicals enables sustainable and economic biorefineries. Biological lignin valorization While significant progress has been made, several major challenges arose due to high recalcitrance and het- Ligninolytic microorganism erogeneity of lignin, which needs to be addressed to improve lignin processing. This work provides an overview Bioprospecting of biological lignin conversion and its regulation from a metabolic engineering and systems biology viewpoint. Metabolic engineering Biological lignin valorization includes three stages: lignin depolymerization, aromatics degradation, and target Systems biology product biosynthesis.
    [Show full text]
  • Dioxygenases
    KINETIC AND SPECTROSCOPIC STUDIES OF COBALT- AND MANGANESE- SUBSTITUTED EXTRADIOL-CLEAVING HOMOPROTOCATECHUATE 2,3- DIOXYGENASES A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Andrew J. Fielding IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Professor Lawrence Que, Jr. February 2013 © Andrew Jay Fielding 2013 Acknowledgements I would like to thank all those who have helped me in my efforts here at graduate school at the University of Minnesota including: My advisors Professors Larry Que and John Lipscomb for their instruction and advice. I would also like to thank my comrades in the Que and Lipscomb labs, specifically Drs. Erik Farquhar, Van Vu, and Mike Mbughuni, and fellow graduate students Rahul Banerjee, Brent Rivard, and Anna Komor for their friendship and the time they spent helping me. I would also like to thank all the many postdoctoral research associates from the Que and Lipscomb labs who have taken time to talk to me about my chemistry including: Ass’t Prof. Joseph Emerson, Dr. Elena Kovaleva, Ass’t Prof. Tom Makris, Dr. Yuming Zhou, Ass’t Prof. Aidan McDonald, Ass’t Prof. Matthew Cranswick, Ass’t Prof. Kathy Van Heuvelen, Dr. Williamson Oloo and Dr. Lisa Engstrom. I am also grateful for the generous support of the University of Minnesota Department of Chemistry, the University of Minnesota Chemical Biology Initiative and the National Institutes of Health. I would also like to thank my parents Kurt and Patti Fielding who encouraged my interests in Science while growing up, where dinner table discussions regularly lead to referencing our copy of the Encyclopedia Britannica .
    [Show full text]