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David Ohagan - One of the best experts on this subject based on the ideXlab platform.

  • insights into fluorometabolite biosynthesis in Streptomyces Cattleya dsm46488 through genome sequence and knockout mutants
    Bioorganic Chemistry, 2012
    Co-Authors: Chunhua Zhao, Ryan P Mcglinchey, Zixin Deng, David Ohagan
    Abstract:

    Abstract Streptomyces Cattleya DSM 46488 is unusual in its ability to biosynthesise fluorine containing natural products, where it can produce fluoroacetate and 4-fluorothreonine. The individual enzymes involved in fluorometabolite biosynthesis have already been demonstrated in in vitro investigations. Candidate genes for the individual biosynthetic steps were located from recent genome sequences. In vivo inactivation of individual genes including those encoding the S -adenosyl- l -methionine:fluoride adenosyltransferase (fluorinase, SCATT_41540), 5′-fluoro-5′-deoxyadenosine phosphorylase (SCATT_41550), fluoroacetyl-CoA thioesterase (SCATT_41470), 5-fluoro-5-deoxyribose-1-phosphate isomerase (SCATT_20080) and a 4-fluorothreonine acetaldehyde transaldolase (SCATT_p11780) confirm that they are essential for fluorometabolite production. Notably gene disruption of the transaldolase (SCATT_p11780) resulted in a mutant which could produce fluoroacetate but was blocked in its ability to biosynthesise 4-fluorothreonine, revealing a branchpoint role for the PLP-transaldolase.

  • biological fluorination in Streptomyces Cattleya the fluorinase
    Fluorine and Health#R##N#Molecular Imaging Biomedical Materials and Pharmaceuticals, 2008
    Co-Authors: Hai Deng, David Ohagan, Fanglu Huang, Jonathan B. Spencer, James H Naismith, Xiaofeng Zhu
    Abstract:

    Publisher Summary This chapter reviews the recent developments on research into bacterial C-F bond forming enzyme. The fluorinase enzyme was isolated from Streptomyces Cattleya in 2002 and shown to catalyze the conversion of fluoride ion and S-adenosyl-L-methionine (SAM) to 5'-fluoro-5'-deoxyadenosine (5'-FDA) and L-methionine. Subsequently, the enzyme has been the subject of cloning, crystallization mechanism and substrate specificity studies. S. Cattleya is the producing organism for the b-lactam antibiotic, thienamycin 3, and during the efforts by Merck to improve the production titre of this antibiotic, they discovered a novel antibiotic activity when a particular soya protein was used in the fermentation nutrient. It subsequently transpired that the soya protein had significant levels of fluoride ion and that the novel antibiotic activity was due to 4-fluorothreonine (4-FT) 2 production. Positron emission tomography (PET) is an important non-invasive diagnostic method in the clinic for imaging tumors. The two most widely used positron emitting isotopes in PET are 11C (t1/2—11 min) and 18F (t1/2—110 min). Enzymes have not been used in PET ligand syntheses due to the virtual absence of appropriate enzymes to utilize the common sources of these isotopes. Fluoride ion is the preferred starting point because [18F]-fluoride is generated directly by the cyclotron in very high specific radioactivity (typically GBqs). Due to the relatively short half lives of the isotopes, syntheses methods for PET should ideally be rapid and incorporate isotope of high specific radioactivity.

  • mechanism of enzymatic fluorination in Streptomyces Cattleya
    Journal of the American Chemical Society, 2007
    Co-Authors: Xiaofeng Zhu, David Ohagan, Andrew R Mcewan, David A Robinson, James H Naismith
    Abstract:

    Recently a fluorination enzyme was identified and isolated from Streptomyces Cattleya, as the first committed step on the metabolic pathway to the fluorinated metabolites, fluoroacetate and 4-fluorothreonine. This enzyme, 5'-fluoro-5'-deoxy adenosine synthetase (FDAS), has been shown to catalyze C-F bond formation by nucleophilic attack of fluoride ion to S-adenosyl-l-methionine (SAM) with the concomitant displacement of l-methionine to generate 5'-fluoro-5'-deoxy adenosine (5'-FDA). Although the structures of FDAS bound to both SAM and products have been solved, the molecular mechanism remained to be elucidated. We now report site-directed mutagenesis studies, structural analyses, and isothermal calorimetry (ITC) experiments. The data establish the key residues required for catalysis and the order of substrate binding. Fluoride ion is not readily distinguished from water by protein X-ray crystallography; however, using chloride ion (also a substrate) with a mutant of low activity has enabled the halide ion to be located in nonproductive co-complexes with SAH and SAM. The kinetic data suggest the positively charged sulfur of SAM is a key requirement in stabilizing the transition state. We propose a molecular mechanism for FDAS in which fluoride weakly associates with the enzyme exchanging two water molecules for protein ligation. The binding of SAM expels remaining water associated with fluoride ion and traps the ion in a pocket positioned to react with SAM, generating l-methionine and 5'-FDA. l-methionine then dissociates from the enzyme followed by 5'-FDA.

  • recent developments on the fluorinase from Streptomyces Cattleya
    Journal of Fluorine Chemistry, 2006
    Co-Authors: David Ohagan
    Abstract:

    Abstract This is a summary of a lecture presented at the 100th Anniversary, Moissan Symposium in Paris on Friday, 10 November 2006. The lecture highlighted recent developments on the enzyme structure, mechanism and biotransformation prospects of the fluorinase enzyme identified in 2002 from the bacterium Streptomyces Cattleya . Over-expression of the enzyme has allowed it to be used as a catalyst for 18 F-incorporation during the synthesis of potential ligands for positron emission tomography (PET) applications.

  • the gene cluster for fluorometabolite biosynthesis in Streptomyces Cattleya a thioesterase confers resistance to fluoroacetyl coenzyme a
    Chemistry & Biology, 2006
    Co-Authors: Fanglu Huang, David Ohagan, Dieter Spiteller, Stephen F Haydock, Tatiana Mironenko, Peter F Leadlay, Jonathan B. Spencer
    Abstract:

    Summary A genomic library of Streptomyces Cattleya was screened to isolate a gene cluster encoding enzymes responsible for the production of fluorine-containing metabolites. In addition to the previously described fluorinase FlA which catalyzes the formation of 5′-fluoro-5′-deoxyadenosine from S -adenosylmethionine and fluoride, 11 other putative open reading frames have been identified. Three of the proteins encoded by these genes have been characterized. FlB was determined to be the second enzyme in the pathway, catalyzing the phosphorolytic cleavage of 5′-fluoro-5′-deoxyadenosine to produce 5-fluoro-5-deoxy- D -ribose-1-phosphate. The enzyme FlI was found to be an S -adenosylhomocysteine hydrolase, which may act to relieve S -adenosylhomocysteine inhibition of the fluorinase. Finally, flK encodes a thioesterase which catalyzes the selective breakdown of fluoroacetyl-CoA but not acetyl-CoA, suggesting that it provides the producing strain with a mechanism for resistance to fluoroacetate.

Hai Deng - One of the best experts on this subject based on the ideXlab platform.

  • mining complex bacteria media for all fluorinated compounds made possible by using hplc coupled parallel to fluorine specific and molecular specific detection
    Journal of Analytical Atomic Spectrometry, 2013
    Co-Authors: Andrea Raab, Hai Deng, Eva M Krupp, Jorg Feldmann
    Abstract:

    The lack of specific fluorine detection at trace levels hampers the screening of microorganisms which can form novel fluorometabolites. So far only target analysis by ESI-MS using accurate mass and certain fragmentation or preconcentration and clean-up procedures has been used to perform 19F and 13C/1H NMR for the identification of novel fluorocompounds. Here we demonstrate that the analysis of complex media from microorganism cultures can be screened using a set of HPLC separations which are coupled parallel to CS-MAS as a fluorine specific detector and the ESI-MS as the molecular detection. This makes it possible to identify fluorine containing species without prior knowledge of the compounds and the use of clean up procedures. Here specifically we identify traces of a fluorometabolite (5′-fluoro-5′-deoxy-adenosine, 5′-FDA) in complex media of Streptomyces Cattleya without preconcentration and target analysis.

  • biological fluorination in Streptomyces Cattleya the fluorinase
    Fluorine and Health#R##N#Molecular Imaging Biomedical Materials and Pharmaceuticals, 2008
    Co-Authors: Hai Deng, David Ohagan, Fanglu Huang, Jonathan B. Spencer, James H Naismith, Xiaofeng Zhu
    Abstract:

    Publisher Summary This chapter reviews the recent developments on research into bacterial C-F bond forming enzyme. The fluorinase enzyme was isolated from Streptomyces Cattleya in 2002 and shown to catalyze the conversion of fluoride ion and S-adenosyl-L-methionine (SAM) to 5'-fluoro-5'-deoxyadenosine (5'-FDA) and L-methionine. Subsequently, the enzyme has been the subject of cloning, crystallization mechanism and substrate specificity studies. S. Cattleya is the producing organism for the b-lactam antibiotic, thienamycin 3, and during the efforts by Merck to improve the production titre of this antibiotic, they discovered a novel antibiotic activity when a particular soya protein was used in the fermentation nutrient. It subsequently transpired that the soya protein had significant levels of fluoride ion and that the novel antibiotic activity was due to 4-fluorothreonine (4-FT) 2 production. Positron emission tomography (PET) is an important non-invasive diagnostic method in the clinic for imaging tumors. The two most widely used positron emitting isotopes in PET are 11C (t1/2—11 min) and 18F (t1/2—110 min). Enzymes have not been used in PET ligand syntheses due to the virtual absence of appropriate enzymes to utilize the common sources of these isotopes. Fluoride ion is the preferred starting point because [18F]-fluoride is generated directly by the cyclotron in very high specific radioactivity (typically GBqs). Due to the relatively short half lives of the isotopes, syntheses methods for PET should ideally be rapid and incorporate isotope of high specific radioactivity.

  • The identification of (3R,4S)-5-fluoro-5-deoxy-d-ribulose-1-phosphate as an intermediate in fluorometabolite biosynthesis in Streptomyces Cattleya
    Bioorganic chemistry, 2007
    Co-Authors: Mayca Onega, John T G Hamilton, Hai Deng, David O'hagan
    Abstract:

    (3R,4S)-5-Fluoro-5-deoxy-D-ribulose-1-phosphate (5-FDRulP) has been identified as the third fluorinated intermediate on the biosynthetic pathway to fluoroacetate and 4-fluorothreonine in Streptomyces Cattleya. 5-FDRulP is generated after formation of 5'-fluoro-5'-deoxyadenosine (5'-FDA) and then phosphorolysis of 5'-FDA to 5-fluoro-5-deoxy-D-ribose-1-phosphate (5-FDRP) by the action of a purine nucleoside phosphorylase. An isomerase mediates the conversion of 5-FDRP to 5-FDRulP. The identity of the (3R,4S) diastereoisomer of 5-FDRulP was established by comparative (19)F{(1)H} NMR studies whereby 5-FDRulP that accumulated in a cell free extract of S. Cattleya, was treated with a phytase to generate the non-phosphorylated sugar, 5-fluoro-5-deoxy-D-ribulose (5-FDRul). This S. Cattleya product was compared to the product of an in-vitro biotransformation where separately 5-fluoro-5-deoxy-D-ribose and 5-fluoro-5-deoxy-D-xylose were converted to 5-fluoro-5-deoxy-D-ribulose and 5-fluoro-5-deoxy-D-xylulose respectively by the action of glucose isomerase. It was demonstrated that 5-fluoro-5-deoxy-D-ribose gave the identical diastereoisomer to that observed from 5-FDRulP.

  • substrate specificity in enzymatic fluorination the fluorinase from Streptomyces Cattleya accepts 2 deoxyadenosine substrates
    Organic and Biomolecular Chemistry, 2006
    Co-Authors: Steven L Cobb, David Ohagan, Hai Deng, Andrew R Mcewan, James H Naismith, David A Robinson
    Abstract:

    The fluorinase enzyme from Streptomyces Cattleya displays an unusual ability in biocatalysis in that it forms a C–F bond. We now report that the enzyme will accept 2′-deoxyadenosine in place of adenosine substrates, and structural evidence reveals a reorganisation in hydrogen bonding to accommodate this substrate series. It emerges from this study that the enzyme does not require a planar ribose conformation of the substrate to catalyse C–F bond formation.

  • the fluorinase from Streptomyces Cattleya is also a chlorinase
    Angewandte Chemie, 2006
    Co-Authors: Hai Deng, Ryan P Mcglinchey, Steven L Cobb, David Ohagan, Andrew R Mcewan, James H Naismith, David A Robinson, Jonathan B. Spencer
    Abstract:

    Streptomyces Cattleya is unusual in that it elaborates organofluorine metabolites and in particular secretes fluoroacetate and 4-fluorothreonine as secondary metabolites when incubated in a medium supplemented with fluoride ions.[1,2] The first committed step on the biosynthetic pathway to these fluorometabolites involves the enzymatic mediated synthesis of 5′-fluoro-5′-deoxyadenosine (5′-FDA) from S-adenosyl-L-methionine (SAM) and fluoride ions.[3-5] After depurination, 5′-FDA is metabolized first to 5-fluoro-5-deoxyribose-1-phosphate (5′-FRP)[6] and then to fluorocetaldehyde.[7] Fluoroacetaldehyde is converted separately into fluoroacetate[8] and 4-fluorothreonine[9] (Scheme 1). Scheme 1 A summary of the biosynthesis of fluoroacetate and 4-fluorothreonine highlighting the fluorinase and the other characterized enzymes on the pathway. The fluorination enzyme, which has been the subject of a recent structural study,[10] has been isolated and fully characterized, the gene has been cloned, and the enzyme has been overexpressed. A stereochemical study that involved (5′S)-[5′-2H]-SAM has indicated that the C–F bond of 5′-FDA replaces the C–S bond of SAM with an inversion of configuration.[11,12] This observation, as well as a theoretical study[13] and structural analysis[10] all suggest an SN2 reaction mechanism for the fluorinase. Despite the high bond-dissociation energy of the C–F bond (the strongest bond in organic chemistry[14]), we herein report that the fluorinase operates in the reverse direction and that it utilizes a chloride ion. Incubation of 5′-FDA with L-(13C-methyl)methionine resulted in the formation of 13C-methyl-SAM, which was identified by HPLC–ES-MS. A similar experiment with L-selenomethionine (L-Se-met) and 5′-FDA resulted in a sixfold more-efficient reaction to generate L-Se-SAM, which was readily identified through the mass-spectrometry fragmentation product of methylselenoadenosine (MeSeAdo) by its signature isotope fingerprint in HPLC–ES-MS. Comparison with the Vmax values that were calculated at saturating kinetics (assays run in each direction) indicated that the equilibrium lies in favor of 5′-FDA by a factor of three (see Scheme 2). Scheme 2 The fluorinase operates in reverse with both 5′-FDA and 5′-ClDA. Efforts were made to replace the fluoride ion with a chloride ion as a substrate for the fluorinase by using standard assay protocols. Incubation with chloride ions in the presence of SAM has never resulted in the production of 5′-chloro-5′-deoxyadenosine (5′-ClDA) (by HPLC). It now emerges, however, that the failure to detect 5′-ClDA is due to the equilibrium of the chloride reaction lying significantly in favor of substrates over products rather than an inherent inability of the fluorinase to activate the chloride ions towards nucleophilic attack. This was revealed in two separate coupled-enzyme assays that were designed to shift the equilibrium of the reaction towards the organochlorine product. The first of these involved an assay in which the fluorinase was coupled to an L-amino acid oxidase (Bothrops atrox venom; purchased from Sigma Chem. Co. Ltd.). Removal of L-methionine, the coproduct of halide substitution by enzymatic oxidation, inhibits the reverse reaction. This assay therefore generated 5′-ClDA and allowed, for the first time, the detection of this product from SAM and chloride ions (see Scheme 3.) Scheme 3 Coupled enzyme assays with 5′-ClDA driving organochlorine synthesis. A related experiment involved a coupled fluorinase/adenyl acid deaminase (Aspergillus sp.)[15] assay. In this reaction the resultant 5′-ClDA was converted into 5′-chloro-5′-deoxyinosine (5′-ClDI). The production of 5′-ClDI was readily identified by HPLC–ES-MS against a reference compound.[16] These experiments indicated that the fluorinase can process chloride ions in a manner similar to that of fluoride ions. A set of fluorinase/L-methionine oxidase coupled assay experiments that were conducted at high concentrations of SAM (1 mM) and halide ion (25 mM) indicated a rate preference for F− over Cl− by a factor of 120. This preference was also demonstrated in a fluorinase-mediated trans-halogenation reaction as illustrated in Figure 1. Incubation of 5′-ClDA with fluoride ion, L-selenomethionine (catalyst), and the fluorinase resulted in an efficient and progressive conversion of 5′-ClDA to 5′-FDA, a reaction that proceeds through L-Se-SAM as a transient but detectable (by using HLPC) intermediate (see Figure 1). Figure 1 HPLC profile monitoring the fluorinase mediated transhalogenation reaction of 5′-ClDA to 5′-FDA with time and using L-Se-methionine as a co-catalyst. L-Se-SAM accumulates as a minor product. The increased size of the chloride ion over the fluoride ion raises the legitimate question of the ability of chloride ions or 5′-ClDA to coordinate to the fluoride- and 5′-FDA-binding sites of the protein. To explore this aspect further, 5′-ClDA was cocrystallised with overexpressed fluorinase (without added L-methionine). This resulted in a structure with 5′-ClDA bound to the active site of the enzyme as illustrated in Figure 2. There are three monomers in the asymmetric unit and all three have essentially the same structure although there are some small differences owing to crystal packing. Only one subunit of the trimer is discussed. Figure 2 Structure of the 5′-ClDA-fluorinase co-complex with contacts and distances between the halogens and Ser-156 N and O atoms shown. For 5′-ClDA, C=gray, N=blue, O=red, and Cl=purple. The protein is similarly colored except C=yellow. Shown ... The difference electron density shows that the C5′–Cl bond adopts two orientations that locate the organochlorine atom in one of two positions. One conformer (shown in Figure 2) is similar to that observed for 5′-FDA when bound to the fluorinase, however, the chlorine atom is displaced by 1.3 A relative to the location of the fluorine in the 5′-FDA complex. The chlorine atom is anchored to the main chain amide of Ser-156 by a hydrogen bond and a polar contact to the side chain hydroxy group of the same residue. Similar noncovalent contacts are also observed between the fluorine atom and Ser-156 although the distances are shorter for the 5′-FDA structure. We attribute this change in position to be a consequence of the larger van der Waals radius of the Cl atom relative to the F atom. The protein underwent no significant change in response to the larger atom, rather the substrate adjusts its position which perhaps suggests a rigid ligand binding site. The altered position of the halogen does not, however, prevent the enzyme from catalyzing the reaction. In the second conformation (not shown) the chlorine rotated out of the halogen binding site into the “empty” L-methionine sulfur binding site. Of course in the catalytic situation, L-methionine will occupy the space above the ribose ring and there will be no possibility of chlorine adopting this second orientation. This study reveals that the fluorinase will catalyze the combination of chloride ions and SAM to generate 5′-ClDA. The reaction is only observed in coupled-enzyme assays that prevent the reverse reaction from occurring. No organohalogen products were detected when Br− and I− were used in these reactions. This reaction extends the repertoire of the fluorinase and reveals a novel enzymatic chlorination reaction which operates by nucleophilic substitution rather than through the electrophilic hypochlorite[17] or flavin dependent enzymes[18] or by a recently reported enzymatic chlorine radical process.[19] A combination of nucleophilic chloride ion and SAM is responsible for the production of chloromethane in fungi.[20,21] This is the only other enzymatic reaction we are aware of in which Cl− acts as a nucleophile, interestingly with a different regiochemistry on the same substrate.

Michelle C Y Chang - One of the best experts on this subject based on the ideXlab platform.

  • discovery of a pathway for terminal alkyne amino acid biosynthesis
    Nature, 2019
    Co-Authors: Jorge A Marchand, M E Neugebauer, Jeffrey G Pelton, Michelle C Y Chang
    Abstract:

    Living systems can generate an enormous range of cellular functions, from mechanical infrastructure and signalling networks to enzymatic catalysis and information storage, using a notably limited set of chemical functional groups. This observation is especially notable when compared to the breadth of functional groups used as the basis for similar functions in synthetically derived small molecules and materials. The relatively small cross-section between biological and synthetic reactivity space forms the foundation for the development of bioorthogonal chemistry, in which the absence of a pair of reactive functional groups within the cell allows for a selective in situ reaction1–4. However, biologically ‘rare’ functional groups, such as the fluoro5, chloro6,7, bromo7,8, phosphonate9, enediyne10,11, cyano12, diazo13, alkene14 and alkyne15–17 groups, continue to be discovered in natural products made by plants, fungi and microorganisms, which offers a potential route to genetically encode the endogenous biosynthesis of bioorthogonal reagents within living organisms. In particular, the terminal alkyne has found broad utility via the Cu(i)-catalysed azide-alkyne cycloaddition ‘click’ reaction18. Here we report the discovery and characterization of a unique pathway to produce a terminal alkyne-containing amino acid in the bacterium Streptomyces Cattleya. We found that l-lysine undergoes an unexpected reaction sequence that includes halogenation, oxidative C–C bond cleavage and triple bond formation through a putative allene intermediate. This pathway offers the potential for de novo cellular production of halo-, alkene- and alkyne-labelled proteins and natural products from glucose for a variety of downstream applications. Microbial generation of a terminal-alkyne-containing amino acid can be encoded into E. coli and provides the potential for in vivo generation of proteins and natural products for click chemistry.

  • entropy drives selective fluorine recognition in the fluoroacetyl coa thioesterase from Streptomyces Cattleya
    Proceedings of the National Academy of Sciences of the United States of America, 2018
    Co-Authors: Amy M Weeks, Jeffrey G Pelton, Ningkun Wang, Michelle C Y Chang
    Abstract:

    Fluorinated small molecules play an important role in the design of bioactive compounds for a broad range of applications. As such, there is strong interest in developing a deeper understanding of how fluorine affects the interaction of these ligands with their targets. Given the small number of fluorinated metabolites identified to date, insights into fluorine recognition have been provided almost entirely by synthetic systems. The fluoroacetyl–CoA thioesterase (FlK) from Streptomyces Cattleya thus provides a unique opportunity to study an enzyme–ligand pair that has been evolutionarily optimized for a surprisingly high 106 selectivity for a single fluorine substituent. In these studies, we synthesize a series of analogs of fluoroacetyl–CoA and acetyl–CoA to generate nonhydrolyzable ester, amide, and ketone congeners of the thioester substrate to isolate the role of fluorine molecular recognition in FlK selectivity. Using a combination of thermodynamic, kinetic, and protein NMR experiments, we show that fluorine recognition is entropically driven by the interaction of the fluorine substituent with a key residue, Phe-36, on the lid structure that covers the active site, resulting in an ∼5- to 20-fold difference in binding (KD). Although the magnitude of discrimination is similar to that found in designed synthetic ligand–protein complexes where dipolar interactions control fluorine recognition, these studies show that hydrophobic and solvation effects serve as the major determinant of naturally evolved fluorine selectivity.

  • fluorothreonyl trna deacylase prevents mistranslation in the organofluorine producer Streptomyces Cattleya
    Proceedings of the National Academy of Sciences of the United States of America, 2017
    Co-Authors: Jonathan L Mcmurry, Michelle C Y Chang
    Abstract:

    Fluorine is an element with unusual properties that has found significant utility in the design of synthetic small molecules, ranging from therapeutics to materials. In contrast, only a few fluorinated compounds made by living organisms have been found to date, most of which derive from the fluoroacetate/fluorothreonine biosynthetic pathway first discovered in Streptomyces Cattleya. While fluoroacetate has long been known to act as an inhibitor of the tricarboxylic acid cycle, the fate of the amino acid fluorothreonine is still not well understood. Here, we show that fluorothreonine can be misincorporated into protein in place of the proteinogenic amino acid threonine. We have identified two conserved proteins from the organofluorine biosynthetic locus, FthB and FthC, that are involved in managing fluorothreonine toxicity. Using a combination of biochemical, genetic, physiological, and proteomic studies, we show that FthB is a trans-acting transfer RNA (tRNA) editing protein, which hydrolyzes fluorothreonyl-tRNA 670-fold more efficiently than threonyl-RNA, and assign a role to FthC in fluorothreonine transport. While trans-acting tRNA editing proteins have been found to counteract the misacylation of tRNA with commonly occurring near-cognate amino acids, their role has yet to be described in the context of secondary metabolism. In this regard, the recruitment of tRNA editing proteins to biosynthetic clusters may have enabled the evolution of pathways to produce specialized amino acids, thereby increasing the diversity of natural product structure while also attenuating the risk of mistranslation that would ensue.

  • Molecular Recognition of Fluorine Impacts Substrate Selectivity in the Fluoroacetyl-CoA Thioesterase FlK
    2015
    Co-Authors: Amy M Weeks, Neil S. Keddie, Rudy D. P. Wadoux, David O’hagan, Michelle C Y Chang
    Abstract:

    The fluoroacetate-producing bacterium Streptomyces Cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that exhibits a remarkably high level of discrimination for its cognate substrate compared to the cellularly abundant analogue acetyl-CoA, which differs only by the absence of the fluorine substitution. A major determinant of FlK specificity derives from its ability to take advantage of the unique properties of fluorine to enhance the reaction rate, allowing fluorine discrimination under physiological conditions where both substrates are likely to be present at saturating concentrations. Using a combination of pH–rate profiles, pre-steady-state kinetic experiments, and Taft analysis of wild-type and mutant FlKs with a set of substrate analogues, we explore the role of fluorine in controlling the enzyme acylation and deacylation steps. Further analysis of chiral (R)- and (S)-[2H1]­fluoroacetyl-CoA substrates demonstrates that a kinetic isotope effect (1.7 ± 0.2) is observed for only the (R)-2H1 isomer, indicating that deacylation requires recognition of the prochiral fluoromethyl group to position the α-carbon for proton abstraction. Taken together, the selectivity for the fluoroacetyl-CoA substrate appears to rely not only on the enhanced polarization provided by the electronegative fluorine substitution but also on molecular recognition of fluorine in both formation and breakdown of the acyl-enzyme intermediate to control active site reactivity. These studies provide insights into the basis of fluorine selectivity in a naturally occurring enzyme–substrate pair, with implications for drug design and the development of fluorine-selective biocatalysts

  • Temporal and Fluoride Control of Secondary Metabolism Regulates Cellular Organofluorine Biosynthesis
    2012
    Co-Authors: Mark C. Walker, Amy M Weeks, Miao Wen, Michelle C Y Chang
    Abstract:

    Elucidating mechanisms of natural organofluorine biosynthesis is essential for a basic understanding of fluorine biochemistry in living systems as well as for expanding biological methods for fluorine incorporation into small molecules of interest. To meet this goal we have combined massively parallel sequencing technologies, genetic knockout, and in vitro biochemical approaches to investigate the fluoride response of the only known genetic host of an organofluorine-producing pathway, Streptomyces Cattleya. Interestingly, we have discovered that the major mode of S. Cattleya’s resistance to the fluorinated toxin it produces, fluoroacetate, may be due to temporal control of production rather than the ability of the host’s metabolic machinery to discriminate between fluorinated and non-fluorinated molecules. Indeed, neither the acetate kinase/phosphotransacetylase acetate assimilation pathway nor the TCA cycle enzymes (citrate synthase and aconitase) exclude fluorinated substrates based on in vitro biochemical characterization. Furthermore, disruption of the fluoroacetate resistance gene encoding a fluoroacetyl-CoA thioesterase (FlK) does not appear to lead to an observable growth defect related to organofluorine production. By showing that a switch in central metabolism can mediate and control molecular fluorine incorporation, our findings reveal a new potential strategy toward diversifying simple fluorinated building blocks into more complex products

Gloria Blanco - One of the best experts on this subject based on the ideXlab platform.

  • activation and silencing of secondary metabolites in Streptomyces albus and Streptomyces lividans after transformation with cosmids containing the thienamycin gene cluster from Streptomyces Cattleya
    Archives of Microbiology, 2014
    Co-Authors: Alfredo F Brana, Miriam Rodriguez, Pallab Pahari, Jurgen Rohr, Luis A Garcia, Gloria Blanco
    Abstract:

    Activation and silencing of antibiotic production was achieved in Streptomyces albus J1074 and Streptomyces lividans TK21 after introduction of genes within the thienamycin cluster from S. Cattleya. Dramatic phenotypic and metabolic changes, involving activation of multiple silent secondary metabolites and silencing of others normally produced, were found in recombinant strains harbouring the thienamycin cluster in comparison to the parental strains. In S. albus, ultra-performance liquid chromatography purification and NMR structural elucidation revealed the identity of four structurally related activated compounds: the antibiotics paulomycins A, B and the paulomenols A and B. Four volatile compounds whose biosynthesis was switched off were identified by gas chromatography–mass spectrometry analyses and databases comparison as pyrazines; including tetramethylpyrazine, a compound with important clinical applications to our knowledge never reported to be produced by Streptomyces. In addition, this work revealed the potential of S. albus to produce many others secondary metabolites normally obtained from plants, including compounds of medical relevance as dihydro-β-agarofuran and of interest in perfume industry as β-patchoulene, suggesting that it might be an alternative model for their industrial production. In S. lividans, actinorhodins production was strongly activated in the recombinant strains whereas undecylprodigiosins were significantly reduced. Activation of cryptic metabolites in Streptomyces species might represent an alternative approach for pharmaceutical drug discovery.

  • mutational analysis of the thienamycin biosynthetic gene cluster from Streptomyces Cattleya
    Antimicrobial Agents and Chemotherapy, 2011
    Co-Authors: Miriam Rodriguez, Luz Elena Nunez, Alfredo F Brana, Carmen Mendez, Jose A Salas, Gloria Blanco
    Abstract:

    The generation of non-thienamycin-producing mutants with mutations in the thnL, thnN, thnO, and thnI genes within the thn gene cluster from Streptomyces Cattleya and their involvement in thienamycin biosynthesis and regulation were previously reported. Four additional mutations were independently generated in the thnP, thnG, thnR, and thnT genes by insertional inactivation. Only the first two genes were found to play a role in thienamycin biosynthesis, since these mutations negatively or positively affect antibiotic production. A mutation of thnP results in the absence of thienamycin production, whereas a 2- to 3-fold increase in thienamycin production was observed for the thnG mutant. On the other hand, mutations in thnR and thnT showed that although these genes were previously reported to participate in this pathway, they seem to be nonessential for thienamycin biosynthesis, as thienamycin production was not affected in these mutants. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of all available mutants revealed some putative intermediates in the thienamycin biosynthetic pathway. A compound with a mass corresponding to carbapenam-3-carboxylic acid was detected in some of the mutants, suggesting that the assembly of the bicyclic nucleus of thienamycin might proceed in a way analogous to that of the simplest natural carbapenem, 1-carbapen-2-em-3-carboxylic acid biosynthesis. The accumulation of a compound with a mass corresponding to 2,3-dihydrothienamycin in the thnG mutant suggests that it might be the last intermediate in the biosynthetic pathway. These data, together with the establishment of cross-feeding relationships by the cosynthesis analysis of the non-thienamycin-producing mutants, lead to a proposal for some enzymatic steps during thienamycin assembly.

  • transcriptional organization of thni regulated thienamycin biosynthetic genes in Streptomyces Cattleya
    The Journal of Antibiotics, 2010
    Co-Authors: Miriam Rodriguez, Carmen Mendez, Jose A Salas, Gloria Blanco
    Abstract:

    Transcriptional organization of ThnI-regulated thienamycin biosynthetic genes in Streptomyces Cattleya

  • identification of transcriptional activators for thienamycin and cephamycin c biosynthetic genes within the thienamycin gene cluster from Streptomyces Cattleya
    Molecular Microbiology, 2008
    Co-Authors: Miriam Rodriguez, Luz Elena Nunez, Alfredo F Brana, Carmen Mendez, Jose A Salas, Gloria Blanco
    Abstract:

    Summary Two regulatory genes, thnI and thnU, were identified in the thienamycin (thn) gene cluster from Streptomyces Cattleya. ThnI resembles LysR-type transcriptional activators and ThnU belongs to the SARP family of transcriptional activators. Their functional role was established after independent inactivation by gene replacement together with transcriptional analysis involving reverse transcription polymerase chain reaction (RT-PCR). Deletion of thnI abolished thienamycin production showing its involvement in thienamycin biosynthesis. Gene expression analysis applied to the thn gene cluster demonstrated that ThnI is a transcriptional activator essential for thienamycin biosynthesis that regulates the expression of nine genes involved in thienamycin assembly and export (thnH, thnJ, thnK, thnL, thnM, thnN, thnO, thnP and thnQ). Unexpectedly, the thnU disrupted mutant was not affected in thienamycin production but turned out to be essential for cephamycin C biosynthesis. Transcript analysis applied to early and late structural genes for cephamycin C biosynthesis (pcbAB and cmcI), revealed that ThnU is the transcriptional activator of these cephamycin C genes although they are not physically linked to the thn cluster. In addition, it was shown that deletion of thnI has an upregulatory effect on pcbAB and cmcI transcription consistent with a significant increase in cephamycin C biosynthesis in this mutant.

  • the biosynthetic gene cluster for the β lactam carbapenem thienamycin in Streptomyces Cattleya
    Chemistry & Biology, 2003
    Co-Authors: Luz Elena Nunez, Alfredo F Brana, Carmen Mendez, Gloria Blanco, Jose A Salas
    Abstract:

    Abstract β-lactam ring formation in carbapenem and clavam biosynthesis proceeds through an alternative mechanism to the biosynthetic pathway of classic β-lactam antibiotics. This involves the participation of a β-lactam synthetase. Using available information from β-lactam synthetases, we generated a probe for the isolation of the thienamycin cluster from Streptomyces Cattleya . Genes homologous to carbapenem and clavulanic acid biosynthetic genes have been identified. They would participate in early steps of thienamycin biosynthesis leading to the formation of the β-lactam ring. Other genes necessary for the biosynthesis of thienamycin have also been identified in the cluster (methyltransferases, cysteinyl transferases, oxidoreductases, hydroxylase, etc.) together with two regulatory genes, genes involved in exportation and/or resistance, and a quorum sensing system. Involvement of the cluster in thienamycin biosynthesis was demonstrated by insertional inactivation of several genes generating thienamycin nonproducing mutants.

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  • fluorometabolite biosynthesis and the fluorinase from Streptomyces Cattleya
    ChemInform, 2005
    Co-Authors: Hai Deng, David Ohagan, Christoph Schaffrath
    Abstract:

    This review outlines the recent developments in uncovering the enzymes and intermediates involved in fluorometabolite biosyntheses in the bacterium Streptomyces Cattleya. A particular emphasis is placed on the purification and characterisation of the fluorinase, the C-F bond forming enzyme which initiates the biosynthesis. Nature has hardly developed a biochemistry around fluorine, yet fluorinated organics are important commercial entities, therefore a biotransformation from inorganic to organic fluorine is novel and of contemporary interest.

  • The identification of 5'-fluoro-5-deoxyinosine as a shunt product in cell free extracts of Streptomyces Cattleya.
    Bioorganic chemistry, 2005
    Co-Authors: Steven L Cobb, Ryan P Mcglinchey, John T G Hamilton, Hai Deng, David O'hagan, Christoph Schaffrath
    Abstract:

    Abstract 5′-Fluoro-5′-deoxyinosine (5′-FDI) is identified as an adventitious side product that accumulates in cell free incubations of SAM and fluoride ion in Streptomyces Cattleya . 5′-FDI was identified by a combination of isotopic labelling studies and co-synthesis studies as well as enzymatic degradation. Although it is an efficiently generated end product of the cell free incubations, 5′-FDI is not a biosynthetic intermediate and it does not accumulate as a fluorometabolite with fluoroacetate and 4-fluorothreonine in whole cell incubations of S. Cattleya . Clearly the purine deaminase which converts 5′-fluoro-5′-deoxyadenosine (5′-FDA) to 5′-FDI in the cell free extract does not come into contact with 5′-FDA in whole cells, suggesting some level of compartmentalisation in cells of S. Cattleya . The biotransformation of 5′-FDI from fluoride ion extends the range of organofluorine products, beyond biosynthetic intermediates, that can be generated by this system, for applications such as enzymatic labelling with fluorine-18 for positron emission tomography applications.

  • crystal structure and mechanism of a bacterial fluorinating enzyme
    Nature, 2004
    Co-Authors: Changjiang Dong, David Ohagan, Hai Deng, Fanglu Huang, Jonathan B. Spencer, Christoph Schaffrath, James H Naismith
    Abstract:

    Fluorine is the thirteenth most abundant element in the earth's crust, but fluoride concentrations in surface water are low and fluorinated metabolites are extremely rare. The fluoride ion is a potent nucleophile in its desolvated state, but is tightly hydrated in water and effectively inert. Low availability and a lack of chemical reactivity have largely excluded fluoride from biochemistry: in particular, fluorine's high redox potential precludes the haloperoxidase-type mechanism used in the metabolic incorporation of chloride and bromide ions. But fluorinated chemicals are growing in industrial importance, with applications in pharmaceuticals, agrochemicals and materials products. Reactive fluorination reagents requiring specialist process technologies are needed in industry and, although biological catalysts for these processes are highly sought after, only one enzyme that can convert fluoride to organic fluorine has been described. Streptomyces Cattleya can form carbon-fluorine bonds and must therefore have evolved an enzyme able to overcome the chemical challenges of using aqueous fluoride. Here we report the sequence and three-dimensional structure of the first native fluorination enzyme, 5'-fluoro-5'-deoxyadenosine synthase, from this organism. Both substrate and products have been observed bound to the enzyme, enabling us to propose a nucleophilic substitution mechanism for this biological fluorination reaction.

  • crystallization and x ray diffraction of 5 fluoro 5 deoxyadenosine synthase a fluorination enzyme from Streptomyces Cattleya
    Acta Crystallographica Section D-biological Crystallography, 2003
    Co-Authors: Changjiang Dong, David Ohagan, Hai Deng, Christoph Schaffrath, Mark Dorward, James H Naismith
    Abstract:

    Organofluorine compounds are widely prepared throughout the chemicals industry, but their prepararion generally requires harsh fluorinating reagents and non-aqueous solvents. On the other hand, biology has hardly exploited organofluorine compounds. A very few organisms synthesize organofluorine metabolites, suggesting they have evolved a mechanism to overcome the kinetic desolvation barrier to utilizing F(-)(aq). Here, the purification and crystallization of an enzyme from Streptomyces Cattleya which is responsible for the synthesis of the C-F bond during fluoroacetate and 4-fluorothreonine biosynthesis is reported. The protein crystallizes in space group C222(1), with unit-cell parameters a = 75.9, b = 130.3, c = 183.4 A, alpha = beta = gamma = 90 degrees. Data were recorded to 1.9 A at the ESRF. The structure of the protein should provide important insights into the biochemical process of C-F bond formation.

  • isolation and characterisation of 5 fluorodeoxyadenosine synthase a fluorination enzyme from Streptomyces Cattleya
    FEBS Letters, 2003
    Co-Authors: Christoph Schaffrath, Hai Deng, David Ohagan
    Abstract:

    Abstract 5′-Fluorodeoxyadenosine synthase, a C–F bond-forming enzyme, has been purified from Streptomyces Cattleya. The enzyme mediates a reaction between inorganic fluoride and S-adenosyl- L -methionine (SAM) to generate 5′-fluoro-5′-deoxyadenosine. The molecular weight of the monomeric protein is shown to be 32.2 kDa by electrospray mass spectrometry. The kinetic parameters for SAM (Km 0.42 mM, Vmax 1.28 U/mg) and fluoride ion (Km 8.56 mM, Vmax 1.59 U/mg) have been evaluated. Both S-adenosyl- L -homocysteine (SAH) and sinefungin were explored as inhibitors of the enzyme. SAH emerged as a potent competitive inhibitor (Ki 29 μM) whereas sinefungin was only weakly inhibitory.