DOCSLIB.ORG

Weeks Thesis 20130726

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Weeks Thesis 20130726 Molecular insights into fluorine chemistry in living systems by Amy Weeks A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Michelle C. Y. Chang, Chair Professor Matthew B. Francis Professor J. Christopher Anderson Fall 2013 Molecular insights into fluorine chemistry in living systems © 2013 by Amy Weeks Abstract Molecular insights into fluorine chemistry in living systems by Amy Weeks Doctor of Philosophy in Chemistry University of California, Berkeley Professor Michelle C. Y. Chang, Chair Based on its unique elemental properties, fluorine has emerged as an important design element in synthetic pharmaceuticals, but also a difficult substituent to incorporate into small molecules using synthetic chemistry. Synthetic biology approaches represent an attractive alternative that could allow us to harness the catalytic power and specificity of enzymes for fluorine incorporation into complex small molecules. However, fluorinated natural products are rare and derive from a single pathway for biosynthesis of fluoroacetate, an antimetabolite poison. The fluoroacetate-producing bacterium Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that is involved in fluoroacetate resistance. Using FlK as a model system, we have focused on understanding enzymatic fluorine selectivity with the goal of defining principles for the design of fluorine-specific enzymes that can be applied in the development of new methods for preparation of fluorinated molecules. We have demonstrated that FlK exhibits a remarkable 106-fold selectivity for hydrolysis of fluoroacetyl-CoA compared to acetyl-CoA, an abundant central metabolite and cellular competitor. 2 4 This selectivity is based on a decrease in KM (10 ) and an increase in kcat (10 ) for the fluorinated substrate. Through x-ray crystallographic studies, we have identified a unique hydrophobic ‘lid’ in our crystal structure of FlK that shields the active site from water. Mutation of a key lid residue (Phe 36) led to a loss of fluorine-based binding specificity, which was correlated with the appearance of additional ordered water molecules at the active site in the mutant crystal structure. A crystal structure of the FlK product complex revealed that Phe 36 is dynamic, undergoing a conformational change when the active site is occupied with product. By studying the binding of a series of non- hydrolyzable substrate analogs, we have shown that the lid confers the fluorinated substrate with an entropic binding advantage that is lost in lid mutants, which may be related to water release from the ‘polar hydrophobic’ C-F unit. Kinetic studies revealed that Phe 36 controls the substrate off rate, providing the fluorinated substrate with a kinetic as well as a thermodynamic advantage. We have also shown that catalytic specificity in FlK is based on utilization of an unusual reaction pathway initiated by deprotonation at the α-carbon for hydrolysis of fluoroacetyl-CoA but not acetyl-CoA. The enolate that is formed can then breakdown through a putative ketene intermediate to give the same product that would be produced by a canonical hydrolysis mechanism. FlK therefore represents a rare example in which the existence of two reaction pathways in the same active site controls substrate selectivity rather than product outcome. Although catalytic fluorine discrimination occurs mainly in one step of the reaction mechanism, we have demonstrated that fluorine is specifically recognized by the enzyme in both the acylation and deacylation steps of the reaction mechanism. These studies have defined the basis of fluorine selectivity in a naturally occurring enzyme-substrate pair, with implications for drug design and development of fluorine- selective biocatalysts. 1 Table of Contents Table of Contents i List of Figures, Schemes, and Tables iv List of Abbreviations ix Acknowledgments xi Chapter 1: Introduction 1.1 Organohalogen natural products 2 1.2 Fluorine in drug design 4 1.3 Fluorinated secondary metabolites 8 1.4 Organofluorine biosynthesis in Streptomyces cattleya 11 1.5 Mechanisms of fluoroacetate toxicity and resistance 15 1.6 Specific aims and thesis organization 21 1.7 References 22 Chapter 2: Structural and biochemical characterization of FlK 2.1 Introduction 33 2.2 Materials and methods 34 2.3 Results and discussion 37 2.4 Conclusion 56 2.5 Acknowledgments 56 2.6 References 57 Chapter 3: Catalytic control of enzymatic fluorine specificity 3.1 Introduction 62 3.2 Materials and methods 63 3.3 Results and discussion 71 3.4 Conclusion 89 3.5 Acknowledgments 90 i 3.6 References 90 Chapter 4: Molecular recognition and kinetic discrimination of the C-F bond in FlK 4.1 Introduction 96 4.2 Materials and methods 97 4.3 Results and discussion 106 4.4 Conclusion 121 4.5 Acknowledgments 121 4.6 References 121 Chapter 5: Determinants of specificity in the acylation and deacylation steps of FlK-catalyzed thioester hydrolysis 5.1 Introduction 126 5.2 Materials and methods 127 5.3 Results and discussion 133 5.4 Conclusion 146 5.5 Acknowledgments 146 5.6 References 146 Chapter 6: Exploring organofluorine resistance, biosynthesis, and regulation in Streptomyces cattleya 6.1 Introduction 149 6.2 Materials and methods 151 6.3 Results and discussion 153 6.4 Conclusion 159 6.5 References 160 ii Appendices Appendix 1: Published plasmids and oligonucleotides 163 Appendix 2: Unpublished plasmids, strains, and oligonucleotides 168 Appendix 3: 2D NMR spectra and HPLC chromatograms for synthetic acyl- 176 CoAs and analogs Appendix 4: Protein purification gels 190 iii List of Figures, Schemes, and Tables Chapter 1 Figure 1.1 Organohalogen natural products 2 Figure 1.2 Enzymatic halogenation strategies observed in nature 3 Figure 1.3 Fluorine in drug design 5 Figure 1.4 Fluorine-protein interactions 6 Figure 1.5 Erythromycin and flurithomycin 7 Figure 1.6 Mechanism-based inhibition of thymidylate synthase by trifluridine 8 Figure 1.7 Fluorinated secondary metabolites 9 Figure 1.8 5-Fluorouracil derivatives isolated from Phakellia fusca 10 Figure 1.9 Fluorometabolite biosynthesis in Streptomyces cattleya 11 Figure 1.10 Structure and mechanism of the fluorinase 13 Figure 1.11 Organization of the fluorometabolite biosynthesis gene cluster 14 Table 1.1 Proposed functions of genes encoded in the fluorometabolite 15 biosynthesis cluster of S. cattleya Figure 1.12 Lethal synthesis of fluorocitrate 16 Figure 1.13 Fluoroacetate dehalogenase structure and mechanism 17 Figure 1.14 Fluoroacetyl-CoA hydrolysis as a fluoroacetate resistance 18 mechanism Figure 1.15 Hotdog-fold superfamily structure and mechanism 20 Chapter 2 Scheme 2.1 Biogenic organofluorines found in the environment 33 Figure 2.1 FlK confers fluoroacetate resistance in vivo 38 Figure 2.2 Purification of wild-type FlK 39 Figure 2.3 Uncatalyzed rates of fluoroacetyl-CoA and acetyl-CoA hydrolysis 40 Figure 2.4 Rates of FlK-catalyzed hydrolysis of fluoroacetyl-CoA in the 40 presence of varying concentrations of acetyl-CoA Figure 2.5 Comparison of catalyzed and uncatalyzed rates of hydrolysis 41 Figure 2.6 Crystal structure of FlK 42 iv Table 2.1 Data collection and refinement statistics for wild-type FlK 43 structures Figure 2.7 Multiple sequence alignment of FlK and homologs identified 44 by BLAST Figure 2.8 Structural alignments of FlK, 4-hydroxybenzoyl-CoA thioesterase, 45 hTHEM2, and PaaI Table 2.2 Kinetic constants measured for fluoroacetyl-CoA and acetyl-CoA 47 hydrolysis by wild-type and mutant FlKs Figure 2.9 Superdex 75 chromatogram of FlK-E50Q after purification and 48 concentration Figure 2.10 Model of the FlK-fluoroacetyl-CoA complex 49 Figure 2.11 Conformational changes in the fluoroacetate complex of FlK 51 Table 2.3 Data collection and refinement statistics for FlK-F36A structures 52 Figure 2.12 Accessibility of the active site to water in FlK and FlK-F36A 54 Figure 2.13 FlK-F36A product complex 55 Chapter 3 Figure 3.1 Molecular basis of fluoroacetate toxicity 62 Figure 3.2 Alternative mechanisms for discrimination of fluoroacetyl-CoA 72 and acetyl-CoA Figure 3.3 pH dependence of non-enzymatic acyl-CoA hydrolysis 72 Figure 3.4 Steady state and pre-steady state kinetics of FlK-catalyzed 73 acyl-CoA hydrolysis Figure 3.5 Michaelis-Menten curves for substrates used in Taft analysis 74 Figure 3.6 1H/13C HMBC spectrum of fluoroacetyl-CoA under FlK assay 75 conditions Figure 3.7 Characterization of FlK burst-phase kinetics 77 Figure 3.8 Phylogenetic analysis of FlK homologs 80 Figure 3.9 Multiple sequence alignment of FlK and its closest homologs 81 Figure 3.10 Structural comparison of FlK to PhaJ 82 Figure 3.11 Breakdown of the fluoroacetyl-enzyme intermediate is accelerated 83 by Cα-deprotonation Figure 3.12 3,3,3-Trifluoropropionyl-CoA hydrolysis, fluoride release, and FlK 84 inactivation 2 Figure 3.13 Pre-steady-state kinetics of [ H2]-fluoroacetyl-CoA hydrolysis 85 v by FlK Figure 3.14 A change in catalytic mechanism accounts for FlK’s specificity 86 for fluoroacetyl-CoA Figure 3.15 Characterization of FlK-H76A 87 Figure 3.16 Alternative mechanism for enolate breakdown 88 Figure 3.17 Quantification of exchange of α-protons with D2O 88 Chapter 4 Figure 4.1 Sequence alignment of FlK and homologs 107 Figure 4.2 Conformational change observed in FlK upon soaking with 108 substrate or products Figure 4.3 Pre-steady state kinetics of FlK-F36A 108 Scheme 4.1 Synthesis of pantethiene precursors
Load more

Recommended publications
  • Enzyme-Catalyzed C–F Bond Formation and Cleavage
    Tong et al. Bioresour. Bioprocess. (2019) 6:46 https://doi.org/10.1186/s40643-019-0280-6 REVIEW Open Access Enzyme-catalyzed C–F bond formation and cleavage Wei Tong1,2 , Qun Huang1,2, Min Li1,2 and Jian‑bo Wang1,2* Abstract Organofuorines are widely used in a variety of applications, ranging from pharmaceuticals to pesticides and advanced materials. The widespread use of organofuorines also leads to its accumulation in the environment, and two major questions arise: how to synthesize and how to degrade this type of compound efectively? In contrast to a considerable number of easy‑access chemical methods, milder and more efective enzymatic methods remain to be developed. In this review, we present recent progress on enzyme‑catalyzed C–F bond formation and cleav‑ age, focused on describing C–F bond formation enabled by fuorinase and C–F bond cleavage catalyzed by oxidase, reductase, deaminase, and dehalogenase. Keywords: Organofuorines, C–F bonds, Enzyme‑catalyzed, Degradation, Formation Introduction usually require harsh conditions and are not environ- Incorporation of fuorine into organic compounds usu- ment friendly (Dillert et al. 2007; Lin et al. 2012; Sulbaek ally endows organofuorines with unique chemical and Andersen et al. 2005). To solve these problems, devel- physical properties, a strategy that has been successfully opment of mild and green methods is urgently needed. applied in agrochemicals, materials science, and pharma- Biocatalysis has been playing an increasingly more ceutical chemistry (Phelps 2004; Müller et al. 2007; Shah important role in modern chemistry due to its high ef- and Westwell 2007; Hagmann 2008; Nenajdenko et al.
    [Show full text]
  • Nouchali Bandaranayaka Phd Thesis
    Studies of enzymes relevant to the biotransformation of fluorinated natural products Nouchali Bandaranayaka Supervisor: Prof. David O’Hagan This thesis is submitted in partial fulfilment for the degree of PhD at the University of St Andrews NOVEMBER 2016 ii Declarations 1. Candidate’s declarations: I, Nouchali Bandaranayaka hereby certify that this thesis, which is approximately 50,000 words in length, has been written by me, and that it is the record of work carried out by me, or principally by myself in collaboration with others as acknowledged, and that it has not been submitted in any previous application for a higher degree. I was admitted as a research student in November 2009 and as a candidate for the degree of Doctor of Philosophy in November 2011; the higher study for which this is a record was carried out in the University of St Andrews between 2009 and 2016. Date Signature of candidate 2. Supervisor’s declaration: I hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriate for the degree of Doctor of Philosophy in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree. Date Signature of supervisor 3. Permission for publication: In submitting this thesis to the University of St Andrews I understand that I am giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby.
    [Show full text]
  • Enzymatic Resolution of Α-Haloacids and Esters and Their Use in The
    Engineering of Candida antarctica lipase B for the kinetic resolution of α-halo esters A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Engineering and Physical Sciences 2010 Shu-Ling Tang School of Chemistry Contents Contents 2 Abstract 6 Declaration 7 Copyright statement 8 Acknowledgements 9 Abbreviations 10 1 Introduction 12 1.1 Chirality in nature 12 1.2 Enantiomers of drugs 12 1.2.1 Chiral pool asymmetric synthesis 14 1.2.2 Resolution 15 1.2.3 Auxiliary controlled stereoselectivity 17 1.2.4 Reagent controlled stereoselectivity 18 1.3 Pharmacologically active compounds 19 1.3.1 Retrosynthetic analyses of (S)-Keppra and (R)-Tiopronin 19 1.3.2 (S)-Keppra 19 1.3.3 (R)-Tiopronin 23 1.3.4 Synthesis of other chiral starting materials from 2-bromoalkanoic acid 24 1.3.5 2-Fluoropropionic acid 25 1.4 Chemical synthesis of α-haloacids 26 1.4.1 Chemical synthesis of 2-bromopropionic acid 26 1.4.2 Chemical synthesis of enantiopure 2-bromopropionic acid 27 1.4.3 Chemical synthesis of 2-fluoropropionic acid 28 1.4.4 Chemical synthesis of enantiopure 2-fluoropropionic acid 31 1.5 Biocatalysis 31 1.5.1 Biocatalysts 32 1.5.2 Enzyme classes 32 1.5.3 Hydrolases (EC 3.X.X.X) 33 1.6 Biosynthesis of halogenated compounds 34 2 1.6.1 Haloperoxidases and perhydrolases 34 1.6.2 Flavin dependent halogenases 36 1.6.3 Fluorinase 37 1.7 Kinetic resolution of α-haloacids and ester using lipases 38 1.7.1 Kinetic resolution of α-haloacids 39 1.7.2 Kinetic resolution of α-haloesters 40 1.8 Directed evolution
    [Show full text]
  • Characterization of a SAM-Dependent Fluorinase from a Latent Biosynthetic Pathway for Fluoroacetate and 4-Fluorothreonine Format
    F1000Research 2014, 3:61 Last updated: 16 MAY 2019 RESEARCH ARTICLE Characterization of a SAM-dependent fluorinase from a latent biosynthetic pathway for fluoroacetate and 4-fluorothreonine formation in Nocardia brasiliensis [version 1; peer review: 2 approved] Yaya Wang, Zixin Deng , Xudong Qu Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, China First published: 19 Feb 2014, 3:61 ( Open Peer Review v1 https://doi.org/10.12688/f1000research.3-61.v1) Latest published: 19 Feb 2014, 3:61 ( https://doi.org/10.12688/f1000research.3-61.v1) Reviewer Status Abstract Invited Reviewers Fluorination has been widely used in chemical synthesis, but is rare in 1 2 nature. The only known biological fluorination scope is represented by the fl pathway from Streptomyces cattleya that produces fluoroacetate (FAc) and version 1 4-fluorothreonine (4-FT). Here we report the identification of a novel published report report pathway for FAc and 4-FT biosynthesis from the actinomycetoma-causing 19 Feb 2014 pathogen Nocardia brasiliensis ATCC 700358. The new pathway shares overall conservation with the fl pathway in S. cattleya. Biochemical characterization of the conserved domains revealed a novel fluorinase 1 Changsheng Zhang, Chinese Academy of NobA that can biosynthesize 5’-fluoro-5’-deoxyadenosine (5’-FDA) from Sciences, Guangzhou, China inorganic fluoride and S-adenosyl-l-methionine (SAM). The NobA shows Cormac Murphy, University College Dublin, similar halide specificity and characteristics to the fluorination enzyme FlA 2 Dublin, Ireland of the fl pathway. Kinetic parameters for fluoride (Km 4153 μM, kcat 0.073 -1 -1 min ) and SAM (Km 416 μM, kcat 0.139 min ) have been determined, Any reports and responses or comments on the revealing that NobA is slightly (2.3 fold) slower than FlA.
    [Show full text]
  • Generated by SRI International Pathway Tools Version 25.0, Authors S
    Authors: Pallavi Subhraveti Ron Caspi Peter Midford Peter D Karp An online version of this diagram is available at BioCyc.org. Biosynthetic pathways are positioned in the left of the cytoplasm, degradative pathways on the right, and reactions not assigned to any pathway are in the far right of the cytoplasm. Transporters and membrane proteins are shown on the membrane. Ingrid Keseler Periplasmic (where appropriate) and extracellular reactions and proteins may also be shown. Pathways are colored according to their cellular function. Gcf_000190595Cyc: Cellulophaga lytica DSM 7489 Cellular Overview Connections between pathways are omitted for legibility. Anamika Kothari phosphate molybdate RS04350 RS08315 phosphate molybdate Macromolecule Modification tRNA-uridine 2-thiolation inosine 5'- Nucleoside and Nucleotide Degradation ditrans,octacis- a peptidoglycan with ditrans,octacis- a [bis(guanylyl an N-terminal- a [protein] and selenation (bacteria) phosphate guanosine ditrans,octacis- (4R)-4-hydroxy- an L-asparaginyl- an L-cysteinyl- Aminoacyl-tRNA Charging tRNA charging nucleotides undecaprenyldiphospho- (L-alanyl-γ-D-glutamyl- undecaprenyldiphospho- undecaprenyldiphospho- molybdopterin) cys L-methionyl-L- C-terminal glu degradation 2-oxoglutarate [tRNA Asn ] [tRNA Cys ] degradation III N-acetyl-(N-acetyl-β-D- L-lysyl-D-alanyl-D- N-acetyl-(N- N-acetyl-(N- cofactor cysteinyl-[protein] L-glutamate cys glucosaminyl)muramoyl- alanine) pentapeptide acetylglucosaminyl) acetylglucosaminyl) chaperone]- Phe IMP bifunctional phe a tRNA L-alanyl-γ-D-glutamyl-L-
    [Show full text]
  • Poster Abstracts
    ENGINEERING BACTERIAL NITROREDUCTASES FOR ANTICANCER GENE THERAPY AND TARGETED CELL ABLATION Abigail Sharrock, School of Biological Sciences, Victoria University of Wellington, NZ [email protected] Elsie Williams, Department of Chemistry, Emory University, USA Kelsi Hall, School of Biological Sciences, Victoria University of Wellington, NZ Jeff Mumm, Wilmer Eye Institute, Johns Hopkins University, USA David Ackerley, School of Biological Sciences, Victoria University of Wellington, NZ Key Words: nitroreductase, prodrug, directed evolution, gene therapy, cell ablation Bacterial nitroreductases are members of a diverse family of oxidoreductase enzymes that can catalyse the bioreductive activation of nitroaromatic compounds, including anti-cancer and antibiotic prodrugs. Nitroreductases have diverse applications in medicine and research, including anti-cancer gene therapy and targeted ablation of nitroreductase-expressing cells in transgenic zebrafish to model degenerative diseases. Research in these fields to date has focused almost exclusively on the canonical nitroreductase NfsB from Escherichia coli (NfsB_Ec), which is a relatively inefficient choice for most applications. By exploring alternative nitroreductase candidates from a variety of bacterial species, in concert with enzyme engineering to fine-tune specific activities, we have generated improved prodrug-activating enzymes. The nitroreductase NfsB from Vibrio vulnificus (NfsB_Vv) has been central to our efforts, and following solution of its crystal structure, was selected as a scaffold for directed evolution via site-saturation mutagenesis. By applying library screening strategies that involved rounds of both positive and negative selection, several mutants that displayed improved activity with a promising next-generation cancer prodrug were identified. In parallel work, an engineered NfsB_Vv variant from the same library was found to be substantially improved in activation of the antibiotic prodrug metronidazole, which is widely used for targeted cell ablation in transgenic zebrafish.
    [Show full text]
  • Enzymatic Defluorination of Fluorinated Compounds
    Seong et al. Appl Biol Chem (2019) 62:62 https://doi.org/10.1186/s13765-019-0469-6 INVITED REVIEW Open Access Enzymatic defuorination of fuorinated compounds Hyeon Jeong Seong†, Seong Woo Kwon†, Dong‑Cheol Seo, Jin‑Hyo Kim* and Yu‑Sin Jang* Abstract Fluorine‑containing compounds are widely used because they have properties required in textiles and coatings for electronic, automotive, and outdoor products. However, fuorinated compounds do not easily break down in nature, which has resulted in their accumulation in the environment as well as the human body. Recently, the enzymatic defuorination of fuorine‑containing compounds has gained increasing attention. Here, we review the enzymatic defuorination reactions of fuorinated compounds. Furthermore, we review the enzyme engineering strategies for cleaving C–F bonds, which have the highest dissociation energy found in organic compounds. Keywords: C–F bond, Defuorination, Fluorine, Perfuorinated compound Introduction and the corresponding enzymes, have not been well Because perfuorinated compounds (PFCs) repel both elucidated. water and oil, they are used as durable repellent treat- On the other hand, fuorinated compounds such as ments for textiles such as outdoor clothes and for home fuoroacetate and 5′-fuoro-5′-deoxyadenosine have been products such as carpets [1]. In addition, PFCs are widely identifed as natural products in nature [10, 11], dem- used in the production of fuoropolymers such as polyte- onstrating the existence of a biosynthesis pathway for trafuoroethylene (trade name, Tefon), which is widely fuorinated compounds in organisms [12]. A representa- used in coatings for electronic, automotive, and outdoor tive pathway of this type is the C–F bond forming reac- products [2].
    [Show full text]
  • Chemoenzymatic Enantioselective Synthesis
    TURUN YLIOPISTON JULKAISUJA ANNALES UNIVERSITATIS TURKUENSIS SARJA - SER. D OSA - TOM. 815 MEDICA - ODONTOLOGICA CHEMOENZYMATIC ENANTIOSELECTIVE SYNTHESIS OF b-AMINO ACID DERIVATIVES USING LIPASES by Xiang-Guo Li TURUN YLIOPISTO Turku 2008 Department of Pharmacology, Drug Development and Therapeutics/ Laboratory of Synthetic Drug Chemistry University of Turku Turku, Finland. Supervisor and custos: Professor Liisa T. Kanerva, Ph.D. Laboratory of Synthetic Drug Chemistry University of Turku Turku, Finland. Reviewers: Professor Ulf Hanefeld, Ph.D. Delft University of Technology Delft, The Netherlands and Professor Reko Leino, Ph.D. Åbo Academi University Turku, Finland Opponent: Professor Per Berglund, Ph.D. Royal Institute of Technology (KTH) Stockholm, Sweden ISBN 978-951-29-3671-7 (PRINT) ISBN 978-951-29-3672-4 (PDF) ISSN 0355-9483 Painosalama Oy – Turku, Finland 2008 CONTENTS Abstract Tiivistelmä (Abstract in Finnish) HAbbreviations HList of original papers H1 Introduction............................................................................................................................ H1 H2 Review of the literature .......................................................................................................... H3 H2.1 Kinetic resolution ........................................................................................................... H3 H2.2 Lipase catalysis .............................................................................................................. H5 H2.2.1 Lipases ...............................................................................................................
    [Show full text]
  • Organic & Biomolecular Chemistry
    Please do not adjust margins Organic & Biomolecular Chemistry Paper An enzymatic Finkelstein reaction: Fluorinase catalyses direct halogen exchange Received 00th January 20xx, Accepted 00th January 20xx Phillip T. Lowe,a Steven L. Cobbb and David O'Hagan*a DOI: 10.1039/x0xx00000x www.rsc.org/ The fluorinase enzyme from Streptomyces cattleya is shown to catalyse a direct displacement of bromide and iodide by fluoride ion from 5’-bromodeoxyadenosine (5’-BrDA) and 5’-iododeoxyadenosine (5’-IDA) respectively, to form 5’-fluorodeoxyadenosine (5’-FDA) in the absence of L-methionine (L-Met) or S-adenosyl- L-methionine (SAM). 5’-BrDA is the most efficient substrate for this enzyme catalysed Finkelstein reaction. Introduction 5’-chloro-5’-deoxy adenosine (5’-ClDA) 1 by L-Met to generate SAM Fluorine containing compounds have a remarkable record in tuning 2.15 When this reaction is conducted in the presence of fluoride ion, the properties of medicinal,1,2 agricultural3,4 and materials5 the resultant SAM 2 is then converted to 5’-FDA 3, to achieve a two- chemistry products. As such, in recent years the development of step transhalogenation from 5’-ClDA 1 to 5’-FDA 3. The substrate methodology to incorporate fluorine into biologically and flexibility of this ‘two-step’ fluorination process extends to 2’-deoxy commercially relevant compounds under mild conditions has analogues16 of 5’-ClDA 1 as well as C-2 decoration of the adenosine intensified. The naturally occurring fluorinase enzyme (5’-fluoro-5’- base of 5’-ClDA 1 with an acetylene, or terminally functionalised deoxy-adenosine synthase), originally isolated from Streptomyces acetylene moieties.17-23 In this manner, fluorinase-mediated trans- cattleya where it is involved in the C-F bond forming step in halogenation reactions have become established as a strategy for the fluoroacetate and 4-fluorothreonine biosynthesis, offers a rare synthesis of fluorinated bioactive compounds under experimentally opportunity to introduce fluorine enzymatically.
    [Show full text]
  • Generated by SRI International Pathway Tools Version 25.0, Authors S
    Authors: Pallavi Subhraveti Ron Caspi Peter Midford Peter D Karp An online version of this diagram is available at BioCyc.org. Biosynthetic pathways are positioned in the left of the cytoplasm, degradative pathways on the right, and reactions not assigned to any pathway are in the far right of the cytoplasm. Transporters and membrane proteins are shown on the membrane. Ingrid Keseler Periplasmic (where appropriate) and extracellular reactions and proteins may also be shown. Pathways are colored according to their cellular function. Gcf_000299235Cyc: Leptospirillum ferriphilum ML-04 Cellular Overview Connections between pathways are omitted for legibility. Anamika Kothari phosphate phosphate phosphate phosphate ammonium ammonium ammonium predicted ABC transporter RS12230 RS11960 RS10935 RS01775 RS01785 RS04585 of phosphate phosphate phosphate phosphate ammonium ammonium ammonium phosphate Macromolecule Modification Inorganic Nutrient Metabolism tRNA-uridine 2-thiolation UDP-N-acetyl- undecaprenyl- a carboxy- a nascent a peptidoglycan an [amino-group β 34 a nucleoside cob(II)yrinate a,c- Cofactor, Carrier, and Vitamin Biosynthesis selenate reduction α-D-glucosamine diphospho-N- adenylated- peptidoglycan a uracil in DNA D-glycero- -D- a cytidine lys dimer (meso- met carrier protein]- [2Fe-2S] iron-sulfur thioredoxin flavin biosynthesis I (bacteria and plants) lipoate biosynthesis lipoate salvage I benzimidazolyl adenosylcobamide lipoate biosynthesis pyridoxal 5'-phosphate biosynthesis I folate transformations III (E. coli) Fatty Acid and
    [Show full text]
  • Natural Production of Fluorinated Compounds and Biotechnological Prospects of the Fluorinase Enzyme
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repositório Científico do Instituto Politécnico do Porto Natural production of fluorinated compounds and biotechnological prospects of the fluorinase enzyme a b,c Maria F. Carvalho and Rui S. Oliveira aCIIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Porto, Portugal; bCentre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal; cDepartment of Environmental Health, Research Centre on Health and Environment, School of Allied Health Sciences, Polytechnic Institute of Porto, Porto, Portugal ABSTRACT Fluorinated compounds are finding increasing uses in several applications. They are employed in almost all areas of modern society. These compounds are all produced by chemical synthesis and their abundance highly contrasts with fluorinated molecules of natural origin. To date, only some plants and a handful of actinomycetes species are known to produce a small number of fluori- nated compounds that include fluoroacetate (FA), some x-fluorinated fatty acids, nucleocidin, 4-fluorothreonine (4-FT), and the more recently identified (2R3S4S)-5-fluoro-2,3,4-trihydroxypenta- noic acid. This largely differs from other naturally produced halogenated compounds, which totals more than 5000. The mechanisms underlying biological fluorination have been uncovered after discovering the first actinomycete species, Streptomyces cattleya, that is capable of produc- ing FA and 4-FT, and a fluorinase has been identified as the enzyme responsible for the forma- tion of the C–F bond. The discovery of this enzyme has opened new perspectives for the biotechnological production of fluorinated compounds and many advancements have been achieved in its application mainly as a biocatalyst for the synthesis of [18F]-labeled radiotracers for medical imaging.
    [Show full text]
  • A Localised Tolerance in the Substrate Specificity of the Fluorinase Enables ‘Last Step’ [18F]-Fluorination of a RGD Peptide Under Ambient Aqueous Conditions
    A localised tolerance in the substrate specificity of the fluorinase enables ‘last step’ [18F]-fluorination of a RGD peptide under ambient aqueous conditions Stephen Thompsona, Qingzhi Zhanga, Mayca Onegab, Stephen McMahona, Ian Flemingc, Sharon Ashworth,b James H. Naismitha, Jan Passchierb, David O’Hagana*. aSchool of Chemistry, University of St Andrews, North Haugh, St Andrews, KY16 9ST, UK. bImanova, Burlington Danes Building, Imperial College London, Hammersmith Hospital, Du Cane Road, London,W12 0NN, UK cAberdeen Biomedical Imaging Centre, School of Medicine and Dentistry, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK. dImperial College London, Departent of Medicine, Burlington Danes Building, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN (United Kingdom) ǂAuthors contributed equally. Abstract: A strategy for last step [18F]-fluorination of bioconjugated peptides is reported that exploits an ‘Achilles heel’ in the substrate specificity of the fluorinase enzyme. An acetylene functionality at C-2 of the adenosine substrate projects from the active site into the solvent. The fluorinase catalyses a transhalogenation of 5’-chlorodeoxy-2-ethynyladenosine (ClDEA) to 5’-fluorodeoxy-2-ethynyladenosine [18F]-FDEA. Extending a polyethelene glycol linker from the terminus of the acetylene allows bio- conjugation cargo to be presented to the enzyme for [18F]-labeling. The method benefits from using an 18 18 aqueous (H2 O) solution of [ F]-fluoride, generated by the cyclotron and has the modular capacity
    [Show full text]