DOCSLIB.ORG

Fluoroacetate Resistance in Streptomyces Cattleya 3.1 Introduction 41 3.2 Materials and Methods 42 3.3 Results and Discussion 47 3.4 Conclusions 66 3.5 References 67

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Fluoroacetate Resistance in Streptomyces Cattleya 3.1 Introduction 41 3.2 Materials and Methods 42 3.3 Results and Discussion 47 3.4 Conclusions 66 3.5 References 67 UC Berkeley UC Berkeley Electronic Theses and Dissertations Title Expanding the scope of organofluorine biochemistry through the study of natural and engineered systems Permalink https://escholarship.org/uc/item/9n89c8zv Author Walker, Mark Chalfant Publication Date 2013 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Expanding the scope of organofluorine biochemistry through the study of natural and engineered systems by Mark Chalfant Walker A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Michelle C. Y. Chang, Chair Professor Judith P. Klinman Professor Susan Marqusee Professor Mathew B. Francis Spring 2013 Expanding the scope of organofluorine biochemistry through the study of natural and engineered systems © 2013 by Mark Chalfant Walker Abstract Expanding the scope of organofluorine biochemistry through the study of natural and engineered systems by Mark Chalfant Walker Doctor of Philosophy in Molecular and Cell Biology University of California, Berkeley Professor Michelle C. Y. Chang, Chair Fluorination has become a very useful tool in the design and optimization of bioactive small molecules ranging from pesticides to pharmaceuticals. Its small size allows a sterically conservative substitution for a hydrogen or hydroxyl, thus maintaining the overall size and shape of a molecule. However, the extreme electronegativity of fluorine can dramatically alter other properties of the molecule. As a result, the development of new methods for fluorine incorporation is currently a major focus in synthetic chemistry. It is our goal to use a complementary biosynthetic approach to use enzymes for the regio-selective incorporation of fluorine into complex natural product scaffolds through the fluoroacetate building block. Towards this goal, we initiated a study of the only known genetic host of a carbon-fluorine bond forming enzyme, the fluoroacetate- and fluorothreonine-producing bacterium Streptomyces cattleya. Sequencing and analysis of the genome identified several paralogs of enzymes predicted to be on the fluoroacetate and fluorothreonine biosynthetic pathway and whose function were probed by in vitro biochemistry and genetic knock-out studies. We sought to further explore how S. cattleya manages fluoroacetate, which is a potential biosynthetic building block but is also a potent toxin that operates by shutting down the tricarboxylic acid (TCA) cycle. Coordinated transcriptional changes of genes involved in central metabolism and organofluorine metabolism suggest transcriptional control may serve as the major mechanism for management of fluoroacetate toxicity. This hypothesis is further supported by biochemical analysis of S. cattleya enzymes involved in fluoroacetate toxicity, which showed that they were no more selective against fluorine than orthologs from non-producing bacteria. To explore the possibility of incorporating fluorinated building blocks into more complex natural products, we turned to type I polyketide synthases as their modular assembly line nature would make them ideal candidates for the engineering of site-selective incorporation of fluorinated subunits. We first developed methods to enzymatically synthesize fluoromalonyl- CoA, which is a fluorinated congener of the malonyl-CoA extender unit. With this building block in hand, we were able to observe incorporation of fluorine into a triketide polyketide. We then showed that it is possible to site-selectively incorporate fluorine into tetraketide products. These results suggest that the production of complex fluorinated natural products is possible and may allow us to explore the medicinal chemistry of these compounds using site-selective fluorination. 1 Table of Contents Table of Contents i List of Figures, Schemes, and Tables iii List of Abbreviations vi Acknowledgments vii Chapter 1: Introduction 1.1 Fluorine in pharmaceuticals 2 1.2 Halogens in biology 5 1.3 Polyketide synthases 8 1.4 Thesis motivation and organization 10 1.5 References 11 Chapter 2: Assembly and analysis of the Streptomyces cattleya draft genome 2.1 Introduction 17 2.2 Materials and methods 18 2.3 Results and discussion 24 2.4 Conclusions 36 2.5 References 37 Chapter 3: Fluoroacetate resistance in Streptomyces cattleya 3.1 Introduction 41 3.2 Materials and methods 42 3.3 Results and discussion 47 3.4 Conclusions 66 3.5 References 67 i Chapter 4: Engineering fluorinated polyketide biosynthesis 4.1 Introduction 71 4.2 Materials and methods 72 4.3 Results and discussion 83 4.4 Conclusions 97 4.5 References 97 Appendices Appendix 1: Plasmids and oligonucleotides 102 ii List of Figures, Schemes, and Tables Chapter 1 Figure 1.1 Potential impacts of fluorination on small molecules 2 Figure 1.2 Blockage of P450 degradation by fluorination 2 Figure 1.3 Tuning of pKa by fluorination 3 Figure 1.4 Fluorine in the design of a mechanism based inhibitor 3 Figure 1.5 Fluorination of the complex natural product erythronolide A 4 Figure 1.6 Naturally-occurring organochlorines and organobromines 4 Figure 1.7 Active sites of halogenases 5 Figure 1.8 Naturally-occurring organofluorines 5 Figure 1.9 Lethal synthesis of 4-hydroxy-transaconitate from fluoroacetate 6 Figure 1.10 Proposed biosynthetic pathway of fluoroacetate and fluorothreonine 7 in Streptomyces cattleya Figure 1.11 Pharmacologically important natural products produced by Type I 8 polyketide synthases Figure 1.12 A schematic diagram of the well studied DEBS polyketide synthase 9 Figure 1.13 Sequence alignment of malonyl-CoA and methylmalonyl-CoA 9 selective acyltransferase domains Figure 1.14 Proposed mechanism of acyl transfer in AT domains 10 Chapter 2 Scheme 2.1 The proposed biosynthetic pathway for fluoroacetate and 17 fluorothreonine in S. cattleya Figure 2.1 Electropherograms of paired-end libraries for Illumina sequencing 24 Table 2.1 Analysis of steps in draft genome assembly 24 Figure 2.2 Comparison of steps in draft genome assembly to the S. cattleya 25 closed genome Figure 2.3 Comparison of draft genomes produced with different assemblers 26 Table 2.2 Identity of gaps in draft genomes 27 Table 2.3 Comparison of fluoroacetate pathway paralogs in S. cattleya and 28 other sequenced streptomycetes iii Figure 2.4 Genomic contexts of methylthioribose-1-phosphate isomerases 29 in S. cattleya Figure 2.5 Genomic contexts of MRI2 orthologs in Nocardia farnicia 30 Figure 2.6 Expanded view of genomic context of MRI2 in S. cattleya 31 Figure 2.7 Characterization of MRI2 reaction product by LC/MS 32 Figure 2.8 Characterization of MRI knockout strains by 19F NMR 33 Figure 2.9 Growth and fluoride uptake by MRI knockout strains 33 Table 2.4 Prediction of PKS AT domain specificity in S. cattleya 34 Table 2.5 Prediction of NRPS A domain specificity in S. cattleya 35 Chapter 3 Scheme 3.1 Lethal synthesis of 4-hydroxy-transaconitate from fluoroacetate 41 Figure 3.1 Electropherograms of single-end library for Illumina sequencing 47 Figure 3.2 Comparison of RNA-seq and custom microarray results 48 Figure 3.3 Analysis of fluoride response by S. cattleya 49 Figure 3.4 Time- and fluoride-dependent response of predicted pathway 50 genes and their paralogs Figure 3.5 Transcription patterns of selected TCA cycle genes with respect 51 to fluoride and time Figure 3.6 Transcription patterns of all predicted TCA cycle genes with 52 respect to fluoride and time Table 3.1 Gene IDs and annotations for all predicted TCA cycle genes 53 Figure 3.7 COG categories of differentially-expressed genes 54 Figure 3.8 Transcriptional response of the crcB locus to the presence of 55 fluoride Figure 3.9 Sequence alignment of selectivity filter regions of selected eriC 56 orthologs Figure 3.10 Characterization of ∆flK strain 57 Figure 3.11 Heterologous expression and purification of acetate assimilation 58 enzymes Table 3.2 Kinetic parameters of acetate assimilation enzymes 59 Figure 3.12 Phylogenetic tree of selected citrate synthases 60 Figure 3.13 Heterologous expression of citrate synthases 61 Table 3.3 Kinetic parameters of citrate synthases 61 iv Figure 3.14 Dose response curves for citrate synthases 62 Figure 3.15 Production of citrate and fluorocitrate by citrate synthases 63 Figure 3.16 Sequence alignment of selected citrate synthases 63 Figure 3.17 Activation of citrate synthases by potassium chloride 64 Figure 3.18 Activation of streptomyces citrate synthases by AMP 65 Figure 3.19 Inhibition of the S. cattleya aconitase by fluorocitrate 65 Chapter 4 Figure 4.1 Synthetic biology of fluorine 71 Figure 4.2 Enzymes used in extender unit biosynthesis 84 Figure 4.3 Enzymatic synthesis of extender units from acetate and 85 fluoroacetate Figure 4.4 Kinetic parameters for malonate activation 86 Figure 4.5 Heterologous expression of NphT7 86 Figure 4.6 Chain extension and keto reduction with a fluorinated extender 87 Figure 4.7 Structural alignment of NphT7 and a DEBS ketosynthase domain 88 Figure 4.8 1H-19F HMBC NMR analysis of enzymatically produced 2-fluoro- 89 3-hydroxy-butyryl-CoA Figure 4.9 Enzymes used in the production of model polyketides 90 Figure 4.10 Production of a fluorinated polyketide using DEBSMod6+TE 91 Figure 4.11 GC-MS and 19F NMR analysis of F-TKL 92 Scheme 4.1 Hydrolysis and regeneration reactions for F-TKL synthesis 93 Figure 4.12 Test for covalent inhibition of DEBSMod6+TE by fluoromalonyl-CoA 93 Table
Load more

Recommended publications
  • Basic Aspects of Fluorine in Chemistry and Biology
    Introduction: Basic Aspects of Fluorine in Chemistry and Biology COPYRIGHTED MATERIAL 1 Unique Properties of Fluorine and Their Relevance to Medicinal Chemistry and Chemical Biology Takashi Yamazaki , Takeo Taguchi , and Iwao Ojima 1.1 Fluorine - Substituent Effects on the Chemical, Physical and Pharmacological Properties of Biologically Active Compounds The natural abundance of fl uorine as fl uorite, fl uoroapatite, and cryolite is considered to be at the same level as that of nitrogen on the basis of the Clarke number of 0.03. However, only 12 organic compounds possessing this special atom have been found in nature to date (see Figure 1.1) [1] . Moreover, this number goes down to just fi ve different types of com- pounds when taking into account that eight ω - fl uorinated fatty acids are from the same plant [1] . [Note: Although it was claimed that naturally occurring fl uoroacetone was trapped as its 2,4 - dinitrohydrazone, it is very likely that this compound was fl uoroacetal- dehyde derived from fl uoroacetic acid [1] . Thus, fl uoroacetone is not included here.] In spite of such scarcity, enormous numbers of synthetic fl uorine - containing com- pounds have been widely used in a variety of fi elds because the incorporation of fl uorine atom(s) or fl uorinated group(s) often furnishes molecules with quite unique properties that cannot be attained using any other element. Two of the most notable examples in the fi eld of medicinal chemistry are 9α - fl uorohydrocortisone (an anti - infl ammatory drug) [2] and 5 - fl uorouracil (an anticancer drug) [3], discovered and developed in 1950s, in which the introduction of just a single fl uorine atom to the corresponding natural products brought about remarkable pharmacological properties.
    [Show full text]
  • Differentiating Two Closely Related Alexandrium Species Using Comparative Quantitative Proteomics
    toxins Article Differentiating Two Closely Related Alexandrium Species Using Comparative Quantitative Proteomics Bryan John J. Subong 1,2,* , Arturo O. Lluisma 1, Rhodora V. Azanza 1 and Lilibeth A. Salvador-Reyes 1,* 1 Marine Science Institute, University of the Philippines- Diliman, Velasquez Street, Quezon City 1101, Philippines; [email protected] (A.O.L.); [email protected] (R.V.A.) 2 Department of Chemistry, The University of Tokyo, 7-3-1 Hongo, Bunkyo City, Tokyo 113-8654, Japan * Correspondence: [email protected] (B.J.J.S.); [email protected] (L.A.S.-R.) Abstract: Alexandrium minutum and Alexandrium tamutum are two closely related harmful algal bloom (HAB)-causing species with different toxicity. Using isobaric tags for relative and absolute quantita- tion (iTRAQ)-based quantitative proteomics and two-dimensional differential gel electrophoresis (2D-DIGE), a comprehensive characterization of the proteomes of A. minutum and A. tamutum was performed to identify the cellular and molecular underpinnings for the dissimilarity between these two species. A total of 1436 proteins and 420 protein spots were identified using iTRAQ-based proteomics and 2D-DIGE, respectively. Both methods revealed little difference (10–12%) between the proteomes of A. minutum and A. tamutum, highlighting that these organisms follow similar cellular and biological processes at the exponential stage. Toxin biosynthetic enzymes were present in both organisms. However, the gonyautoxin-producing A. minutum showed higher levels of osmotic growth proteins, Zn-dependent alcohol dehydrogenase and type-I polyketide synthase compared to the non-toxic A. tamutum. Further, A. tamutum had increased S-adenosylmethionine transferase that may potentially have a negative feedback mechanism to toxin biosynthesis.
    [Show full text]
  • N-Carbamoylation of 2,4-Diaminobutyrate Reroutes the Outcome in Padanamide Biosynthesis
    Chemistry & Biology Article N-Carbamoylation of 2,4-Diaminobutyrate Reroutes the Outcome in Padanamide Biosynthesis Yi-Ling Du,1 Doralyn S. Dalisay,1 Raymond J. Andersen,1,2 and Katherine S. Ryan1,* 1Department of Chemistry 2Department of Earth, Ocean and Atmospheric Sciences University of British Columbia, Vancouver, BC V6T 1Z1, Canada *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2013.06.013 SUMMARY literature. It is interesting that a compound identical to padana- mide A, named actinoramide A (Nam et al., 2011), was indepen- Padanamides are linear tetrapeptides notable for the dently reported from Streptomyces sp. CNQ-027. This actinomy- absence of proteinogenic amino acids in their struc- cete strain was isolated from sediment on the opposite side of the tures. In particular, two unusual heterocycles, (S)- Pacific Ocean, near San Diego, CA, suggesting a potentially wide 3-amino-2-oxopyrrolidine-1-carboxamide (S-Aopc) distribution of the padanamides/actinoramides. It is intriguing and (S)-3-aminopiperidine-2,6-dione (S-Apd), are that, whereas padanamide A and actinoramide A are identical, found at the C-termini of padanamides A and B, the minor compounds (actinoramides B and C) co-isolated from Streptomyces sp. CNQ-027 are unique (Figure 1A). Total respectively. Here we identify the padanamide synthesis of padanamides A and B was recently reported (Long biosynthetic gene cluster and carry out systematic et al., 2013), confirming the previous structural elucidations. gene inactivation studies. Our results show that The padanamides attracted our attention for their many padanamides are synthesized by highly dissociated unusual chemical features.
    [Show full text]
  • Polyketide Biosynthesis Beyond the Type I, II and III Polyketide Synthase Paradigms Ben Shen
    285 Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms Ben Shen Recent literature on polyketide biosynthesis suggests that Three types of bacterial PKSs are known to date. First, polyketide synthases have much greater diversity in both type I PKSs are multifunctional enzymes that are orga- mechanism and structure than the current type I, II and III nized into modules, each of which harbors a set of distinct, paradigms. These examples serve as an inspiration for searching non-iteratively acting activities responsible for the cata- novel polyketide synthases to give new insights into polyketide lysis of one cycle of polyketide chain elongation, as biosynthesis and to provide new opportunities for combinatorial exemplified by the 6-deoxyerythromycin B synthase biosynthesis. (DEBS) for the biosynthesis of reduced polyketides (i.e. macrolides, polyethers and polyene) such as erythro- Addresses mycin A (1)(Figure 1a) [1]. Second, type II PKSs are Division of Pharmaceutical Sciences and Department of Chemistry, multienzyme complexes that carry a single set of iteratively University of Wisconsin, Madison, WI 53705, USA acting activities, as exemplified by the tetracenomycin e-mail: [email protected] PKS for the biosynthesis of aromatic polyketides (often polycyclic) such as tetracenomycin C (2)(Figure 1b) [2]. Current Opinion in Chemical Biology 2003, 7:285–295 Third, type III PKSs, also known as chalcone synthase- like PKSs, are homodimeric enzymes that essentially are This review comes from a themed section on iteratively acting condensing enzymes, as exemplified by Biocatalysis and biotransformation Edited by Tadhg Begley and Ming-Daw Tsai the RppA synthase for the biosynthesis of aromatic poly- ketides (often monocyclic or bicyclic), such as flavolin (3) 1367-5931/03/$ – see front matter (Figure 1c) [3].
    [Show full text]
  • "Fluorine Compounds, Organic," In: Ullmann's Encyclopedia Of
    Article No : a11_349 Fluorine Compounds, Organic GU¨ NTER SIEGEMUND, Hoechst Aktiengesellschaft, Frankfurt, Federal Republic of Germany WERNER SCHWERTFEGER, Hoechst Aktiengesellschaft, Frankfurt, Federal Republic of Germany ANDREW FEIRING, E. I. DuPont de Nemours & Co., Wilmington, Delaware, United States BRUCE SMART, E. I. DuPont de Nemours & Co., Wilmington, Delaware, United States FRED BEHR, Minnesota Mining and Manufacturing Company, St. Paul, Minnesota, United States HERWARD VOGEL, Minnesota Mining and Manufacturing Company, St. Paul, Minnesota, United States BLAINE MCKUSICK, E. I. DuPont de Nemours & Co., Wilmington, Delaware, United States 1. Introduction....................... 444 8. Fluorinated Carboxylic Acids and 2. Production Processes ................ 445 Fluorinated Alkanesulfonic Acids ...... 470 2.1. Substitution of Hydrogen............. 445 8.1. Fluorinated Carboxylic Acids ......... 470 2.2. Halogen – Fluorine Exchange ......... 446 8.1.1. Fluorinated Acetic Acids .............. 470 2.3. Synthesis from Fluorinated Synthons ... 447 8.1.2. Long-Chain Perfluorocarboxylic Acids .... 470 2.4. Addition of Hydrogen Fluoride to 8.1.3. Fluorinated Dicarboxylic Acids ......... 472 Unsaturated Bonds ................. 447 8.1.4. Tetrafluoroethylene – Perfluorovinyl Ether 2.5. Miscellaneous Methods .............. 447 Copolymers with Carboxylic Acid Groups . 472 2.6. Purification and Analysis ............. 447 8.2. Fluorinated Alkanesulfonic Acids ...... 472 3. Fluorinated Alkanes................. 448 8.2.1. Perfluoroalkanesulfonic Acids
    [Show full text]
  • Letters to Nature
    letters to nature Received 7 July; accepted 21 September 1998. 26. Tronrud, D. E. Conjugate-direction minimization: an improved method for the re®nement of macromolecules. Acta Crystallogr. A 48, 912±916 (1992). 1. Dalbey, R. E., Lively, M. O., Bron, S. & van Dijl, J. M. The chemistry and enzymology of the type 1 27. Wolfe, P. B., Wickner, W. & Goodman, J. M. Sequence of the leader peptidase gene of Escherichia coli signal peptidases. Protein Sci. 6, 1129±1138 (1997). and the orientation of leader peptidase in the bacterial envelope. J. Biol. Chem. 258, 12073±12080 2. Kuo, D. W. et al. Escherichia coli leader peptidase: production of an active form lacking a requirement (1983). for detergent and development of peptide substrates. Arch. Biochem. Biophys. 303, 274±280 (1993). 28. Kraulis, P.G. Molscript: a program to produce both detailed and schematic plots of protein structures. 3. Tschantz, W. R. et al. Characterization of a soluble, catalytically active form of Escherichia coli leader J. Appl. Crystallogr. 24, 946±950 (1991). peptidase: requirement of detergent or phospholipid for optimal activity. Biochemistry 34, 3935±3941 29. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and (1995). the thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281±296 (1991). 4. Allsop, A. E. et al.inAnti-Infectives, Recent Advances in Chemistry and Structure-Activity Relationships 30. Meritt, E. A. & Bacon, D. J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505± (eds Bently, P. H. & O'Hanlon, P. J.) 61±72 (R. Soc. Chem., Cambridge, 1997).
    [Show full text]
  • Compound 1080), Its Pharmacology and Toxicology Ernest Kun
    MONOFLUOROACETIC ACID (COMPOUND 1080), ITS PHARMACOLOGY AND TOXICOLOGY ERNEST KUN. Department of Pharmacology, The University of California San Francisco. San Francisco. California 94143 ABSTRACT: The molecular mechanism of toxic action of fluoroacetate is analyzed in the perspective of scientific developments of the past 30 years. Stereospecific enzymatic conversion of fluoroacetate via fluoroacetyl-CoA + oxalacetate to (-)-erythrofluorocitrate in mitochondria is the metabolic pathway that converts the nontoxic fluoroacetate to the toxic intracellular effector molecule. The mode of toxic effect of (-)-erythrofluorocitrate cannot be equated with its reversible inhibitory effect on a mitochondrial enzyme (aconitase) as had been originally thought by Peters (1963) and is still propa~ated in textbooks. Instead, the chemical modification of inner mitochondrial membrane proteins by (-}-erythrofluorocitrate, comprising a novel, as yet incompletely understood biochemical mechanism, is the molecular basis of toxicity. Research in this new area may eventually explain selective {species­ dependent) toxicity, the development of resistance to this poison and can lead to the scientific basis of the development of antidotes. INTRO DU CTI ON The discovery of detailed mechanism of action of highly toxic agents inevitably reveals new areas of cellular physiology, as first pointed out by Claude Bernard (1). It is evident that if an agent in minute quantities is capable of tenninating life processes, the site of action of the toxic substance has to represent a vitally important biological system. Despite the plausibility of this axiom, only relatively few poisons have been studied in sufficient depth to allow the precise identification of their critical target systems, and their proposed mechanisms of action are frequently borrowed from concepts of a branch of biochemistry that happens to be fashionable at that time.
    [Show full text]
  • Investigation and Engineering of Polyketide Biosynthetic Pathways
    Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 12-2017 Investigation and Engineering of Polyketide Biosynthetic Pathways Lei Sun Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biological Engineering Commons Recommended Citation Sun, Lei, "Investigation and Engineering of Polyketide Biosynthetic Pathways" (2017). All Graduate Theses and Dissertations. 6903. https://digitalcommons.usu.edu/etd/6903 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. INVESTIGATION AND ENGINEERING OF POLYKETIDE BIOSYNTHETIC PATHWAYS by Lei Sun A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSPHY in Biological Engineering Approved: ______________________ ____________________ Jixun Zhan, Ph.D. David W. Britt, Ph.D. Major Professor Committee Member ______________________ ____________________ Dong Chen, Ph.D. Jon Takemoto, Ph.D. Committee Member Committee Member ______________________ ____________________ Elizabeth Vargis, Ph.D. Mark R. McLellan, Ph.D. Committee Member Vice President for Research and Dean of the School of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2017 ii Copyright© Lei Sun 2017 All Rights Reserved iii ABSTRACT Investigation and engineering of polyketide biosynthetic pathways by Lei Sun, Doctor of Philosophy Utah State University, 2017 Major Professor: Jixun Zhan Department: Biological Engineering Polyketides are a large family of natural products widely found in bacteria, fungi and plants, which include many clinically important drugs such as tetracycline, chromomycin, spirolaxine, endocrocin and emodin.
    [Show full text]
  • Enzymes Quinate Dehydrogenase
    THE ABSOLUTTE STEREOCHEMICAL COUtRSE OF CITRIC ACID BIOSYNTHESIS* BY KENNETH R. HANSON AND IRWIN A. RoSEt DEPARTMENTS OF BIOCHEMISTRY OF THE CONNECTICUT AGRICULTURAL EXPERIMENT STATION AND OF YALE UNIVERSITY SCHOOL OF MEDICINE, NEW HAVEN Communicated by H. B. Vickery, September 3, 1963 In 1948 Ogston noted that when such symmetrical molecules as citric (or amino- malonic) acid are acted upon by asymmetric enzyme surfaces, pairs of identical groups may well be differentiated.1 Shortly thereafter it was shown, by using labeled substrates, that the two -CH2COOH groups of citric acid could, in fact, be distinguished.2 When citrate is formed from oxaloacetate and acetyl-CoA by citrate synthase (E.C.4.1.3.7, condensing enzyme), and is then treated with aco- nitate hydratase (E.C.4.2.1.3, aconitase) to form cis-aconitate and threo-Dr-iso- citrate, the -CH2COOH ends of these compounds are derived from acetyl-CoA, while the other atoms are derived either from oxaloacetate or water.3-6 The acetyl-CoA-derived group yields acetate when citrate is cleaved by citrate-lyase (E.C.4.1.3.6, citratase),7'8 and acetyl-CoA when citrate is attacked by the citrate cleavage enzyme.9 The nonequivalence of the two -CH2COOH groups is apparent when citric acid is represented as I (heavy line above, dotted lines below the plane of the paper). Carbon atoms 1 and 2 are to 5 and 4 as right arm is to left arm:'0 they cannot be superimposed except by reflection. The same molecule is shown in Emil Fischer projection as II (vertical lines below and horizontal lines above the plane of the paper).
    [Show full text]
  • Rewiring Yarrowia Lipolytica Toward Triacetic Acid Lactone for Materials Generation
    Rewiring Yarrowia lipolytica toward triacetic acid lactone for materials generation Kelly A. Markhama,1, Claire M. Palmerb,1, Malgorzata Chwatkoa, James M. Wagnera, Clare Murraya, Sofia Vazqueza, Arvind Swaminathana, Ishani Chakravartya, Nathaniel A. Lynda, and Hal S. Alpera,b,2 aMcKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712; and bInstitute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712 Edited by Sang Yup Lee, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea, and approved January 22, 2018 (received for review December 6, 2017) Polyketides represent an extremely diverse class of secondary me- or challenging syntheses, this is not an option for any larger-scale tabolites often explored for their bioactive traits. These molecules are chemistry application. also attractive building blocks for chemical catalysis and polymeriza- Here, we focus on the interesting, yet simple, polyketide, tri- tion. However, the use of polyketides in larger scale chemistry acetic acid lactone (TAL) as it is derived from two common applications is stymied by limited titers and yields from both microbial polyketide precursors, acetyl–CoA and malonyl–CoA. TAL has and chemical production. Here, we demonstrate that an oleaginous been demonstrated as a platform chemical that can be converted organism (specifically, Yarrowia lipolytica) can overcome such produc- into a variety of valuable products traditionally derived from tion limitations owing to a natural propensity for high flux through fossil fuels including sorbic acid, a common food preservative acetyl–CoA. By exploring three distinct metabolic engineering strate- with a global demand of 100,000 t (1, 15–18).
    [Show full text]
  • Substrate Channeling in Proline Metabolism Benjamin W
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by DigitalCommons@University of Nebraska University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Biochemistry -- Faculty Publications Biochemistry, Department of 2012 Substrate channeling in proline metabolism Benjamin W. Arentson University of Nebraska-Lincoln, [email protected] Nikhilesh Sanyal University of Nebraska-Lincoln, [email protected] Donald F. Becker University of Nebraska-Lincoln, [email protected] Follow this and additional works at: http://digitalcommons.unl.edu/biochemfacpub Part of the Biochemistry Commons, Biotechnology Commons, and the Other Biochemistry, Biophysics, and Structural Biology Commons Arentson, Benjamin W.; Sanyal, Nikhilesh; and Becker, Donald F., "Substrate channeling in proline metabolism" (2012). Biochemistry -- Faculty Publications. 303. http://digitalcommons.unl.edu/biochemfacpub/303 This Article is brought to you for free and open access by the Biochemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Biochemistry -- Faculty Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. NIH Public Access Author Manuscript Front Biosci. Author manuscript; available in PMC 2013 January 01. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Front Biosci. ; 17: 375–388. Substrate channeling in proline metabolism Benjamin W. Arentson1, Nikhilesh Sanyal1, and Donald F. Becker1 1Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA Abstract Proline metabolism is an important pathway that has relevance in several cellular functions such as redox balance, apoptosis, and cell survival. Results from different groups have indicated that substrate channeling of proline metabolic intermediates may be a critical mechanism.
    [Show full text]
  • Long-Chain F-18 Fatty Acids for the Study of Regional Metabolism in Heart and Liver; Odd-Even Effects of Metabolism in Mice
    BASIC SCIENCES RADIOCHEMISTRY AND RADIOPHARMACEUTICALS Long-Chain F-18 Fatty Acids for the Study of Regional Metabolism in Heart and Liver; Odd-Even Effects of Metabolism in Mice E. J. Knust, Ch. Kupfernagel, and G. Stöcklin Institut fur Chemie 1 (Nuklearchemie) der KFA Julich GmbH, D-5170 Julich, FRG ( West Germany) In view of the potential usefulness of fluorine-tagged fatty acids in the study of regional metabolism in the heart and liver, the time courses of uptake and release of 9,10-[18F)fluorostearic acid, 2-{18F]fluorostearic acid, 16-{18F]fluorohexadec- anoic acid, 17-[18F]fluoroheptadecanoic acid have been investigated in several organs of NMRI mice. Whereas 2-[18F]fluorostearic acid shows very little uptake in the heart muscle but an increasing accumulation In the fiver, the fatty acids with the F-18 label in the middle or at the end of the carbon chain exhibit uptake and elimination behavior similar to that of the analogous C-11 -labeled compounds. After rapid concentration in the heart within 1 min, clearance takes place with fast and slow components. 16-| 18F|fluorohexadecanoic acid and 17-|18F|fluorohepta- decanoic acid have different half-times of elimination. These differences are also reflected by the fact that nearly all the activity present in the heart can be recov ered as fluoride(F-18) in the case of 17-|18F|fluoroheptadecanoic acid, whereas practically no fluoride was found among the metabolites of 16-[18F]fluorohexadec- anoic acid. Similar differences were observed for the F-18 activity in bone. The results can be interpreted in terms of the odd-even rule: ßoxidation of even-num bered fatty acids ends up with 118F|fluoroacetic acid, whereas the odd-numbered fatty acids give rise to ß-|18F|fluoropropionic acid.
    [Show full text]