DOCSLIB.ORG

Discovery and Protein Engineering of Baeyer-Villiger Monooxygenases

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Discovery and Protein Engineering of Baeyer-Villiger Monooxygenases Discovery and Protein Engineering of Baeyer-Villiger monooxygenases Inauguraldissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Andy Beier geboren am 11.10.1988 in Parchim Greifswald, den 02.08.2017 I Dekan: Prof. Dr. Werner Weitschies 1. Gutachter: Prof. Dr. Uwe T. Bornscheuer 2. Gutachter: Prof. Dr. Marko Mihovilovic Tag der Promotion: 24.10.2017 II We need to learn to want what we have, not to have what we want, in order to get stable and steady happiness. - The Dalai Lama - III List of abbreviations % Percent MPS Methyl phenyl sulfide % (v/v) % volume per volume MPSO Methyl phenyl sulfoxide % (w/v) % weight per volume MPSO2 Methyl phenyl sulfone °C Degrees Celsius MTS Methyl p-tolyl sulfide µM µmol/L MTSO Methyl p-tolyl sulfoxide aa Amino acids MTSO2 Methyl p-tolyl sulfone + AGE Agarose gel electrophoresis NAD Nicotinamide adenine dinucleotide, oxidized aq. dest. Distilled water NADH Nicotinamide adenine dinucleotide, reduced + BLAST Basic Local Alignment Search NADP Nicotinamide adenine dinucleotide Tool phosphate, oxidized bp Base pair(s) NADPH Nicotinamide adenine dinucleotide phosphate, reduced BVMO Baeyer-Villiger monooxyge- OD600 Optical density at 600 nm nase CHMO Cyclohexanone monooxyge- PAGE Polyacrylamide gel electrophoresis nase Da Dalton PAMO Phenylacetone monooxygenase DMF Dimethyl formamide PCR Polymerase chain reaction DMSO Dimethyl sulfoxide PDB Protein Data Bank DMSO2 Dimethyl sulfone rpm Revolutions per minute DNA Desoxyribonucleic acid rv Reverse dNTP Desoxynucleoside triphosphate SDS Sodium dodecyl sulfate E. coli Escherichia coli SOC Super Optimal broth with Catabolite repression ee Enantiomeric excess TAE TRIS-Acetate-EDTA FAD Flavin adenine dinucleotide TB Terrific broth Fig. Figure TCE 2,2,2-Trichloroethanol FMN Flavin adenine mononucleotide TCEP tris(2-carboxyethyl)phosphine FMO Flavoprotein monooxygenase TEMED Tetramethylethylenediamine fw Forward TRIS Tris(hydroxymethyl)aminomethane GC Gas chromatography UV Ultraviolet h Hours x g Times gravity of Earth HAPMO 4-Hydroxyacetophenone mo- nooxygenase His(6) hexahistidine tag IPTG Isopropyl β-D-1- thiogalactopyranoside Furthermore, SI units (base, derived and prefixes) and L Liter the common notation for amino acids and nucleic ac- LB Lysogenic broth ids are used. M mol/L min Minutes 1 U is defined as the amount of enzyme that catalyzes the depletion of 1 µmol NADPH per minute in the NADPH depletion assay. IV Table of contents 1 Introduction ....................................................................................................................... 1 1.1 White Biotechnology ............................................................................................................... 1 1.2 Protein Engineering ................................................................................................................. 2 1.3 Flavin-dependent monooxygenases......................................................................................... 6 1.3.1 Baeyer-Villiger-Monooxygenases ................................................................................. 10 2 Scope of this thesis .......................................................................................................... 34 3 Results .............................................................................................................................. 35 3.1 Baeyer-Villiger monooxygenases participating in the metabolism of ketones in yeasts ...... 35 3.1.1 Determination of metabolites from Candida maltosa and other yeasts from 2-dodecanone and 1-dodecene ...................................................................................... 35 3.1.2 Investigations of novel BVMOs from yeasts ................................................................ 37 3.2 Switch of the cofactor specificity of the cyclohexanone monooxygenase from Acinetobacter calcoaceticus NCIMB 9871 .................................................................................................. 61 3.2.1 Mutations of the phosphate recognition site .................................................................. 62 3.2.2 Investigating residues in proximity of NAD(P)H .......................................................... 68 3.2.3 Determination of kinetic parameters ............................................................................. 71 3.2.4 Biocatalysis with variants of CHMOAcineto .................................................................... 72 4 Discussion ......................................................................................................................... 73 4.1 Baeyer-Villiger monooxygenases participating in the metabolism of ketones in yeasts ...... 73 4.1.1 Determination of metabolites from yeasts from 2-dodecanone and 1-dodecene........... 73 4.1.2 Investigations of novel BVMOs from yeasts ................................................................ 78 4.2 Switch of the cofactor specificity of CHMOAcineto ............................................................... 101 4.2.1 Mutation of the phosphate recognition site ................................................................. 101 4.2.2 Mutation of residues in proximity of NADPH ............................................................ 106 4.2.3 Kinetics and uncoupling of CHMOAcineto ..................................................................... 109 4.2.4 Biocatalysis with CHMOAcineto .................................................................................... 110 4.2.5 Structural investigation of S186_S208E_K326H ........................................................ 112 4.2.6 Outlook ........................................................................................................................ 114 5 Summary ........................................................................................................................ 116 V 6 Material and Methods .................................................................................................. 118 6.1 Equipment ........................................................................................................................... 118 6.2 Chemicals ............................................................................................................................ 119 6.3 Buffers, growth media and solutions ................................................................................... 119 6.4 Kits / markers / enzymes ..................................................................................................... 123 6.5 Strains, plasmids and primers .............................................................................................. 124 6.6 Microbiological methods ..................................................................................................... 131 6.6.1 Strain maintenance ...................................................................................................... 131 6.6.2 Cultivation and expression Pichia pastoris X-33 ........................................................ 131 6.6.3 Cultivation of Yarrowia lipolytica ............................................................................... 131 6.6.4 Cultivation and expression in E. coli BL21(DE3) ....................................................... 132 6.7 Molecular biological methods ............................................................................................. 135 6.7.1 Determination of DNA concentration ......................................................................... 135 6.7.2 Isolation of genomic DNA from yeasts ....................................................................... 135 6.7.3 Plasmid preparation ..................................................................................................... 135 6.7.4 Agarose gel electrophoresis ......................................................................................... 136 6.7.5 Sequencing .................................................................................................................. 136 6.7.6 Cloning ........................................................................................................................ 136 6.7.7 Transformation ............................................................................................................ 147 6.7.8 Colony PCR ................................................................................................................. 150 6.7.9 Site-directed mutagenesis ............................................................................................ 152 6.7.10 DpnI digestion ............................................................................................................. 153 6.8 Biochemical methods .......................................................................................................... 153 6.8.1 Cell disruption ............................................................................................................. 153 6.8.2 Enzyme purification .................................................................................................... 154 6.8.3 Determination of protein concentration ....................................................................... 155 6.8.4 SDS-PAGE .................................................................................................................
Load more

Recommended publications
  • Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents
    International Journal of Molecular Sciences Article Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents 1, 2,3, 1 2, Elena V. Eremeeva y , Tianyu Jiang y , Natalia P. Malikova , Minyong Li * and Eugene S. Vysotski 1,* 1 Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, Krasnoyarsk 660036, Russia; [email protected] (E.V.E.); [email protected] (N.P.M.) 2 Key Laboratory of Chemical Biology (MOE), Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China; [email protected] 3 State Key Laboratory of Microbial Technology, Shandong University–Helmholtz Institute of Biotechnology, Shandong University, Qingdao 266237, China * Correspondence: [email protected] (M.L.); [email protected] (E.S.V.); Tel.: +86-531-8838-2076 (M.L.); +7-(391)-249-44-30 (E.S.V.); Fax: +86-531-8838-2076 (M.L.); +7-(391)-290-54-90 (E.S.V.) These authors contributed equally to this work. y Received: 23 June 2020; Accepted: 27 July 2020; Published: 30 July 2020 Abstract: Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial.
    [Show full text]
  • Cloning of Firefly Luciferase Cdna and the Expression of Active
    Proc. Natl. Acad. Sci. USA Vol. 82, pp. 7870-7873, December 1985 Biochemistry Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli (bioluminescence/Photinus pyralis/antibody screening/expression vector/recombinant DNA) JEFFREY R. DE WET*, KEITH V. WOODt, DONALD R. HELINSKI*, AND MARLENE DELUCAt Departments of *Biology and tChemistry, University of California, San Diego, La Jolla, CA 92093 Communicated by W. D. McElroy, July 26, 1985 ABSTRACT A cDNA library was constructed from firefly library was screened with anti-P. pyralis luciferase antibody, (Photinuspyralis) lantern poly(A)I RNA, using the Escherichia using a chromogenic detection technique (8), and several coli expression vector Xgtll. The library was screened with cDNA clones were isolated and characterized. These clones anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidore- were found to be homologous to the mRNA that encodes ductase, EC 1.13.12.7) antibody, and several cDNA clones luciferase. The largest luciferase cDNA clone that was expressing luciferase antigens were isolated. One clone, ALucl, isolated was able to direct the synthesis of active luciferase contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- in E. coli. to 2.0-kilobase band on a nitrocellulose blot of electrophoreti- cally fractionated lantern RNA. Hybridization of the cloned MATERIALS AND METHODS cDNA to lantern poly(A)I RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide Enzymes and Strains. Restriction endonucleases and E. coli comigrated with luciferase on NaDodSO4/PAGE and produced DNA polymerase I were purchased from New England bioluminescence upon the addition of luciferin and ATP.
    [Show full text]
  • Bioluminescence Is Produced by a Firefly-Like Luciferase but an Entirely
    www.nature.com/scientificreports OPEN New Zealand glowworm (Arachnocampa luminosa) bioluminescence is produced by a Received: 8 November 2017 Accepted: 1 February 2018 frefy-like luciferase but an entirely Published: xx xx xxxx new luciferin Oliver C. Watkins1,2, Miriam L. Sharpe 1, Nigel B. Perry 2 & Kurt L. Krause 1 The New Zealand glowworm, Arachnocampa luminosa, is well-known for displays of blue-green bioluminescence, but details of its bioluminescent chemistry have been elusive. The glowworm is evolutionarily distant from other bioluminescent creatures studied in detail, including the frefy. We have isolated and characterised the molecular components of the glowworm luciferase-luciferin system using chromatography, mass spectrometry and 1H NMR spectroscopy. The purifed luciferase enzyme is in the same protein family as frefy luciferase (31% sequence identity). However, the luciferin substrate of this enzyme is produced from xanthurenic acid and tyrosine, and is entirely diferent to that of the frefy and known luciferins of other glowing creatures. A candidate luciferin structure is proposed, which needs to be confrmed by chemical synthesis and bioluminescence assays. These fndings show that luciferases can evolve independently from the same family of enzymes to produce light using structurally diferent luciferins. Glowworms are found in New Zealand and Australia, and are a major tourist attraction at sites located across both countries. In contrast to luminescent beetles such as the frefy (Coleoptera), whose bioluminescence has been well characterised (reviewed by ref.1), the molecular details of glowworm bioluminescence have remained elusive. Tese glowworms are the larvae of fungus gnats of the genus Arachnocampa, with eight species endemic to Australia and a single species found only in New Zealand2.
    [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]
  • 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]
  • Antidepressant/Anxiolytic and Anti-Nociceptive Effects of Novel 2-Substituted 1,4-Benzodiazepine-2-Ones
    Sci Pharm www.scipharm.at Research article Open Access Antidepressant/Anxiolytic and Anti-Nociceptive Effects of Novel 2-Substituted 1,4-Benzodiazepine-2-ones 1 2 1 Harjit SINGH , Jintana SATTAYASAI , Pornthip LATTMANN , 2 1 Yodchai BOONPRAKOB , Eric LATTMANN * 1 School of Life & Health Sciences, Pharmacy, Aston University, Aston Triangle, Birmingham B4 7ET, England. 2 Department of Pharmacology, Faculty of Medicine, Khon Kaen University, 40002 Khon Kaen, Thailand. * Corresponding author. E-mail: [email protected] (E. Lattmann) Sci Pharm. 2010; 78: 155–169 doi:10.3797/scipharm.1004-12 Published: May 17th 2010 Received: April 15th 2010 Accepted: May 17th 2010 This article is available from: http://dx.doi.org/10.3797/scipharm.1004-12 © Singh et al.; licensee Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Oxazepam (4a) has been used as overall starting material in the synthesis of novel 2-substituted 1,4-benzodiazepines. By reacting Oxazepam 4a with commercially available hydrazines, hydrazides, semicarbazide, aminoguanidine and N,N-dimethylamino aniline in ethanol under acetic conditions, a series of diazenyl-1,4-benzodiazepines 5a–5i and 2-amino- 1,4-benzodiazepine 5k were obtained in good yields. These novel compounds served as new chemical entities (NCE) for testing in mice. The diazo-benzodiazepine 5d has shown a promising antidepressant effect in initial experiments in vivo at a dose of 5 mg/kg.
    [Show full text]
  • Design of Potent, Orally Effective, Nonpeptidal Antagonists of the Peptide Hormone Cholecystokinin (Neuropeptlde/Benzodiazepine) BEN E
    Proc. Nati. Acad. Sci. USA Vol. 83, pp. 4918-4922, July 1986 Medical Sciences Design of potent, orally effective, nonpeptidal antagonists of the peptide hormone cholecystokinin (neuropeptlde/benzodiazepine) BEN E. EVANS, MARK G. BOCK, KENNETH E. RITTLE, ROBERT M. DIPARDO, WILLIE L. WHITTER, DANIEL F. VEBER, PAUL S. ANDERSON, AND ROGER M. FREIDINGER Merck Sharp & Dohme Research Laboratories, West Point, PA 19486 Communicated by Edward M. Scolnick, March 12, 1986 ABSTRACT We describe the design and synthesis of selective nonpeptidal antagonist of CCK in vitro and in vivo nonpeptidal antagonists of the peptide hormone cholecystoki- (7). However, asperlicin has liabilities as a pharmacological nin. Several of these compounds have high specificity and or potential therapeutic agent, including lack oforal bioavail- nanomolar binding affinity and are active after oral adminis- ability, modest potency, and poor water solubility (7, 42). tration. To our knowledge, the design of such agents has not Many important drugs such as ivermectin (18) and cefoxitin previously been accomplished for any peptide hormone. The (19) are semisynthetic derivatives of natural products, sug- structural similarities between these synthetic compounds and gesting that derivatization of asperlicin might generate im- the anxiolytic 1,4-benzodiazepines are noted, and the potential of this structural feature for future design of ligands for other peptide hormone receptors is discussed. Long-acting orally effective agents that interact competitive- ly at peptide receptors are essential for the optimal develop- ment ofimportant discoveries in the neuropeptide field. Such compounds represent unique biological reagents for deter- mining unambiguously the role ofthe parent neurotransmitter or neurohormone in normal physiology and for assessing its contribution to pathophysiology.
    [Show full text]
  • Functions and Applications of Nmos
    Functions and applications of NMOs Willem Dijkman (1606212), June 20 2009 Assisted by drs. Anette Riebel and prof. dr. ir. Marco W. Fraaije Abstract The hydroxylation of amino groups is performed by different types of enzymes. Some of these types are metal dependent whereas others need a flavin cofactor. These flavoproteins (NMOs, for N-hydroxylating monooxygenases) can be assigned to subclass B of the external flavoproteins, an enzyme class consisting of enzymes with a broad range of monooxygenating activity. NMOs are mostly found in the biosynthesis of siderophores of different bacteria. Recently a NMO has been identified in the biosynthesis of a kutzneride. NMOs have a narrow substrate range, making them less applicable in industry. NMOs in siderophore biosynthesis can however be targeted to slow down bacterial growth. Contents enzymes which perform these monooxygenations often contain metal Introduction 1 atoms. Different types of enzymes are Classification of flavoproteins 2 known. P450 enzymes as well as non-heme 2 The catalytic cycle of flavins 3 monooxygenases contain iron and copper- N-hydroxylation in nature 4 dependent enzymes also perform Characteristics of NMOs 4 monooxygenations. Not all enzymes depend - NMOs in siderophore synthesis 5 on a (metallic) cofactor for their activity: - A NMO in kutzneride synthesis 7 flavin-dependent monooxygenases make use - N-hydroxylation in valanimycin synthesis 7 of a flavin group instead of a metal ion and Recombinant NMOs 8 recently enzymes without any cofactor have 3,4 Pharmaceutical applications 8 shown monooxygenase activity . Industrial applications 9 In this article one type of monooxygenation Conclusion 10 will be described, focusing on one group of Literature 10 monooxygenating enzymes.
    [Show full text]
  • Nitric Oxide Prevents a Pathogen-Permissive Granulocytic Inflammation During Tuberculosis
    ARTICLES PUBLISHED: 15 MAY 2017 | VOLUME: 2 | ARTICLE NUMBER: 17072 Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis Bibhuti B. Mishra1, Rustin R. Lovewell1,AndrewJ.Olive1, Guoliang Zhang2, Wenfei Wang2, Eliseo Eugenin3, Clare M. Smith1,JiaYaoPhuah1, Jarukit E. Long1, Michelle L. Dubuke4, Samantha G. Palace1, Jon D. Goguen1,RichardE.Baker1, Subhalaxmi Nambi1, Rabinarayan Mishra5, Matthew G. Booty1,ChristinaE.Baer1, Scott A. Shaffer4, Veronique Dartois3,BethA.McCormick1, Xinchun Chen2,6* and Christopher M. Sassetti1* Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen’s environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis
    [Show full text]
  • Supplementary Table S1 List of Proteins Identified with LC-MS/MS in the Exudates of Ustilaginoidea Virens Mol
    Supplementary Table S1 List of proteins identified with LC-MS/MS in the exudates of Ustilaginoidea virens Mol. weight NO a Protein IDs b Protein names c Score d Cov f MS/MS Peptide sequence g [kDa] e Succinate dehydrogenase [ubiquinone] 1 KDB17818.1 6.282 30.486 4.1 TGPMILDALVR iron-sulfur subunit, mitochondrial 2 KDB18023.1 3-ketoacyl-CoA thiolase, peroxisomal 6.2998 43.626 2.1 ALDLAGISR 3 KDB12646.1 ATP phosphoribosyltransferase 25.709 34.047 17.6 AIDTVVQSTAVLVQSR EIALVMDELSR SSTNTDMVDLIASR VGASDILVLDIHNTR 4 KDB11684.1 Bifunctional purine biosynthetic protein ADE1 22.54 86.534 4.5 GLAHITGGGLIENVPR SLLPVLGEIK TVGESLLTPTR 5 KDB16707.1 Proteasomal ubiquitin receptor ADRM1 12.204 42.367 4.3 GSGSGGAGPDATGGDVR 6 KDB15928.1 Cytochrome b2, mitochondrial 34.9 58.379 9.4 EFDPVHPSDTLR GVQTVEDVLR MLTGADVAQHSDAK SGIEVLAETMPVLR 7 KDB12275.1 Aspartate 1-decarboxylase 11.724 112.62 3.6 GLILTLSEIPEASK TAAIAGLGSGNIIGIPVDNAAR 8 KDB15972.1 Glucosidase 2 subunit beta 7.3902 64.984 3.2 IDPLSPQQLLPASGLAPGR AAGLALGALDDRPLDGR AIPIEVLPLAAPDVLAR AVDDHLLPSYR GGGACLLQEK 9 KDB15004.1 Ribose-5-phosphate isomerase 70.089 32.491 32.6 GPAFHAR KLIAVADSR LIAVADSR MTFFPTGSQSK YVGIGSGSTVVHVVDAIASK 10 KDB18474.1 D-arabinitol dehydrogenase 1 19.425 25.025 19.2 ENPEAQFDQLKK ILEDAIHYVR NLNWVDATLLEPASCACHGLEK 11 KDB18473.1 D-arabinitol dehydrogenase 1 11.481 10.294 36.6 FPLIPGHETVGVIAAVGK VAADNSELCNECFYCR 12 KDB15780.1 Cyanovirin-N homolog 85.42 11.188 31.7 QVINLDER TASNVQLQGSQLTAELATLSGEPR GAATAAHEAYK IELELEK KEEGDSTEKPAEETK LGGELTVDER NATDVAQTDLTPTHPIR 13 KDB14501.1 14-3-3
    [Show full text]
  • Zendah El Euch
    ZENDAH EL EUCH ___________________________________________ Isolation, Purification and Structure Elucidation of New Secondary Metabolites from Terrestrial, Marine, and Ruminal Microorganisms OH S N N N H H Dissertation Isolation, Purification and Structure Elucidation of New Secondary Metabolites from Terrestrial, Marine and Ruminal Microorganisms Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von Imène ZENDAH EL EUCH aus Tunesien Göttingen 2012 D7 Referent: Prof. Dr. H. Laatsch Referent: Prof. Aly Raies Korreferent: Prof. Dr. A. Zeeck Tag der mündlichen Prüfung: 13. Juli 2012 Die vorliegende Arbeit wurde in der Zeit von Oktober 2005 bis März 2007 in der Faculté des Sciences de Tunis (Laboratoire des Microorganismes et des Bio- molécules Actives) unter der Leitung von Herrn Prof. RAIES Aly und von April 2007 bis Juli 2012 im Institut für Organische und Biomolekulare Chemie der Georg- August-Universität zu Göttingen unter der Leitung von Herrn Prof. Dr. H. Laatsch angefertigt. Herrn Prof. Dr. H. Laatsch danke ich für die Möglichkeit zur Durchführung dieser Arbeit sowie die ständige Bereitschaft, auftretende Probleme zu diskutieren. Für meine Eltern, meine Geschwister und meinen Ehemann Content I TABLE OF CONTENTS 1 INTRODUCTION ...................................................................................................................... 1 1.1 NATURE AS A SOURCE OF NATURAL PRODUCTS ...................................................................
    [Show full text]
  • Flavoprotein Monooxygenases: Versatile Biocatalysts
    Biotechnology Advances xxx (xxxx) xxx Contents lists available at ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv Research review paper Flavoprotein monooxygenases: Versatile biocatalysts Caroline E. Paul a, Daniel Eggerichs b, Adrie H. Westphal c, Dirk Tischler b, Willem J. H. van Berkel d,* a Biocatalysis, Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands b Microbial Biotechnology, Faculty of Biology and Biotechnology, Ruhr-Universitat¨ Bochum, Universitatsstrasse¨ 150, 44780 Bochum, Germany c Laboratory of Biochemistry, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands d Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands ARTICLE INFO ABSTRACT Keywords: Flavoprotein monooxygenases (FPMOs) are single- or two-component enzymes that catalyze a diverse set of Baeyer-Villiger oxidation chemo-, regio- and enantioselective oxyfunctionalization reactions. In this review, we describe how FPMOs have biocatalysis evolved from model enzymes in mechanistic flavoprotein research to biotechnologically relevant catalysts that dearomatization can be applied for the sustainable production of valuable chemicals. After a historical account of the develop­ epoxidation ment of the FPMO field, we explain the FPMO classification system, which is primarily based on protein flavin halogenation structural properties and electron donor specificities.We then summarize the most appealing reactions catalyzed hydroxylation by each group with a focus on the different types of oxygenation chemistries. Wherever relevant, we report oxygenation engineering strategies that have been used to improve the robustness and applicability of FPMOs. (hydro)peroxide microbial degradation 1. Introduction the reduced flavin in most flavoenzymes has a lower pKa than that of free reduced FMN (pKa = 6.7) (Müller, 2014).
    [Show full text]