- Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3-Hydroxyalkanoates
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Expanding the substrate scope of enzymes opens up new routes for synthesis of valuable chemicals. Ketone-functionalized fatty acid derivatives and corresponding chiral alcohols are valuable building blocks for the synthesis of a variety of chemicals including pharmaceuticals. The alcohol dehydrogenase from Candida parapsilosis (cpADH5) catalyzes the reversible oxidations of chiral alcohols and has a broad substrate range; a challenge for cpADH5 is to convert alcohols with small substituents (methyl or ethyl) next to the oxidized alcohol moiety. Molecular docking studies revealed that W286 is located in the small binding pocket and limits the access to substrates that contain aliphatic chains longer than ethyl substituent. In the current manuscript, we report that positions L119 and W286 are key residues to boost oxidation of medium chain methyl 3-hydroxy fatty acids; interestingly the enantiopreference toward methyl 3-hydroxybutyrate was inverted. Kinetic characterization of W286A showed a 5.5 fold increase of Vmax and a 9.6 fold decrease of Km values toward methyl 3-hydroxyhexanoate (Vmax: 2.48 U mg? and Km: 4.76 mm). Simultaneous saturation at positions 119 and 286 library yielded a double mutant (L119M/W286S) with more than 30-fold improved activity toward methyl 3-hydroxyoctanoate (WT: no conversion; L119M/W286S: 30 %) and inverted enantiopreference (S-enantiomer ≥99 % activity decrease and R-enantiomer >20-fold activity improvement) toward methyl 3-hydroxybutyrate.
- Ensari, Yunus,Dhoke, Gaurao V.,Davari, Mehdi D.,Bocola, Marco,Ruff, Anna Jo?lle,Schwaneberg, Ulrich
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p. 12636 - 12645
(2017/09/18)
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- Polyhydroxyalkanoate-based 3-hydroxyoctanoic acid and its derivatives as a platform of bioactive compounds
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A library of 18 different compounds was synthesized starting from (R)-3-hydroxyoctanoic acid which is derived from the bacterial polymer polyhydroxyalkanoate (PHA). Ten derivatives, including halo and unsaturated methyl and benzyl esters, were synthesized and characterized for the first time. Given that (R)-3-hydroxyalkanoic acids are known to have biological activity, the new compounds were evaluated for antimicrobial activity and in vitro antiproliferative effect with mammalian cell lines. The presence of the carboxylic group was essential for the antimicrobial activity, with minimal inhibitory concentrations against a panel of bacteria (Gram-positive and Gram-negative) and fungi (Candida albicans and Microsporum gypseum) in the range 2.87.0 mM and 0.16.3 mM, respectively. 3-Halogenated octanoic acids exhibited the ability to inhibit C. albicans hyphae formation. In addition, (R)-3-hydroxyoctanoic and (E)-oct-2-enoic acids inhibited quorum sensing-regulated pyocyanin production in the opportunistic pathogen Pseudomonas aeruginosa PAO1. Generally, derivatives did not inhibit mammalian cell proliferation even at 3-mM concentrations, while only (E)-oct-2-enoic and 3-oxooctanoic acid had IC50 values of 1.7 and 1.6 mM with the human lung fibroblast cell line.
- Radivojevic, Jelena,Skaro, Sanja,Senerovic, Lidija,Vasiljevic, Branka,Guzik, Maciej,Kenny, Shane T.,Maslak, Veselin,Nikodinovic-Runic, Jasmina,OConnor, Kevin E.
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p. 161 - 172
(2016/01/09)
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- Biochemical Studies of Mycobacterial Fatty Acid Methyltransferase: A Catalyst for the Enzymatic Production of Biodiesel
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Summary Transesterification of fatty acids yields the essential component of biodiesel, but current processes are cost-prohibitive and generate waste. Recent efforts make use of biocatalysts that are effective in diverting products from primary metabolism to yield fatty acid methyl esters in bacteria. These biotransformations require the fatty acid O-methyltransferase (FAMT) from Mycobacterium marinum (MmFAMT). Although this activity was first reported in the literature in 1970, the FAMTs have yet to be biochemically characterized. Here, we describe several crystal structures of MmFAMT, which highlight an unexpected structural conservation with methyltransferases that are involved in plant natural product metabolism. The determinants for ligand recognition are analyzed by kinetic analysis of structure-based active-site variants. These studies reveal how an architectural fold employed in plant natural product biosynthesis is used in bacterial fatty acid O-methylation. Mycobacterial fatty acid methyltransferases are employed as biocatalysts for the production of biodiesel. Petronikolou and Nair describe structural and biochemical characterization of a mycobacterial fatty acid methyltransferase, reveal an unexpected homology to enzymes involved in plant primary metabolism, and provide insights into substrate preference.
- Petronikolou, Nektaria,Nair, Satish K.
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p. 1480 - 1490
(2015/12/01)
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- Characterization of FabG and FabI of the Streptomyces coelicolor dissociated fatty acid synthase
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Streptomyces coelicolor produces fatty acids for both primary metabolism and for biosynthesis of the secondary metabolite undecylprodiginine. The first and last reductive steps during the chain elongation cycle of fatty acid biosynthesis are catalyzed by FabG and FabI. The S. coelicolor genome sequence has one fabI gene (SCO1814) and three likely fabG genes (SCO1815, SCO1345, and SCO1846). We report the expression, purification, and characterization of the corresponding gene products. Kinetic analyses revealed that all three FabGs and FabI are capable of utilizing both straight and branched-chain β-ketoacyl-NAC and enoyl-NAC substrates, respectively. Furthermore, only SCO1345 differentiates between ACPs from both biosynthetic pathways. The data presented provide the first experimental evidence that SCO1815, SCO1346, and SCO1814 have the catalytic capability to process intermediates in both fatty acid and undecylprodiginine biosynthesis.
- Singh, Renu,Reynolds, Kevin A.
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p. 631 - 640
(2015/03/31)
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- The synthesis of medium-chain-length β-hydroxy esters via the reformatsky reaction
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The synthesis of medium-chain-length β-hydroxy esters in good yield via the Reformatsky reaction is described. This work will be used as the basis for further investigation of hydroxyalkanoate polymers as potential feedstock for biofuel production.
- Sailer, Miloslav,Dubicki, Krystyn I.,Sorensen, John L.
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- Systematic investigation of the kinetic resolution of 3-hydroxy fatty acid esters using Candida antarctica lipase B (CALB) and the influence of competing oligomerization on the enantiomeric ratios
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The kinetic resolution of 3-hydroxy fatty acid esters C8:0 to C16:0 with Candida antarctica lipase B shows common plots of the enantiomeric excesses of the product and substrate, respectively, versus the conversion and an enantiomeric ratio E of 27 calculated from ee(p). Differences in E, either calculated from the products or the substrates, could be explained by competing oligomerization as a second substrate-consuming process. This reaction is slow compared to acylation, and the remaining enantiomer was oligomerized. Taylor & Francis Group, LLC.
- Braner, Markus,Zielonka, Stefan,Auras, Sylvia,Huettenhain, Stefan H.
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experimental part
p. 1019 - 1025
(2012/02/01)
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- Kinetic resolution of aliphatic acyclic β-hydroxyketones by recombinant whole-cell Baeyer-Villiger monooxygenases-Formation of enantiocomplementary regioisomeric esters
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A set of various linear aliphatic β-hydroxyketones was investigated as substrates in the enzymatic kinetic and regioselective Baeyer-Villiger oxidation catalyzed by 12 Baeyer-Villiger monooxygenases from different bacterial origin. Excellent enantioselect
- Rehdorf, Jessica,Lengar, Alenka,Bornscheuer, Uwe T.,Mihovilovic, Marko D.
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scheme or table
p. 3739 - 3743
(2010/02/28)
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- Gas chromatography/electron-capture negative ion mass spectrometry for the quantitative determination of 2- and 3-hydroxy fatty acids in bovine milk fat
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2- and 3-hydroxy fatty acids (2- and 3-OH-FAs) are bioactive substances reported in sphingolipids and bacteria. Little is known of their occurrence in food. For this reason, a method suitable for the determination of OH-FAs at trace levels in bovine milk fat was developed. OH-FAs (and conventional fatty acids in samples) were converted into methyl esters and the hydroxyl group was derivatized with pentafluorobenzoyl (PFBO) chloride to give PFBO-O-FA methyl esters. These derivatives with strong electron affinity were determined by gas chromatography interfaced to mass spectrometry using electron-capture negative ion in the selected ion monitoring mode (GC/ECNI-MS-SIM). This method proved to be highly sensitive and selective for PFBO-O-FA methyl esters. For the analysis of samples, two internal standards were used. For this purpose, 9,10-dideutero-2-OH-18:0 methyl ester (ISTD-1) from 2-OH-18:1(9c) methyl ester as well as the ethyl ester of 3-PFBO-O-12:0 (ISTD-2) was synthesized. ISTD-1 served as a recovery standard whereas ISTD-2 was used for GC/MS measurements. The whole-sample cleanup consisted of accelerated solvent extraction of dry bovine milk, addition of ISTD 1, saponification, conversion of fatty acids into methyl esters by use of boron trifluoride, separation of the methyl esters of OH-FAs from nonsubstituted FAs on activated silica, conversion of OH-FAs methyl esters into PFBO-O-FA methyl esters, addition of ISTD-2, and measurement by GC/ECNI-MS-SIM. By this method, ten OH-FAs were quantified in bovine milk fat with high precision in the range from 0.02 ± 0.00 to 4.49 ± 0.29 mg/100 g of milk fat.
- Jenske, Ramona,Vetter, Walter
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experimental part
p. 5500 - 5505
(2010/03/25)
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- Formation of an enolate intermediate is required for the reaction catalyzed by 3-hydroxyacyl-CoA dehydrogenase
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Fluorinated substrate analogs were synthesized and incubated with rat liver 3-hydroxyacyl-CoA dehydrogenase, which reveals that the formation of an enolate intermediate is required for the reaction catalyzed by the enzyme.
- Liu, Xiaojun,Deng, Guisheng,Chu, Xiusheng,Li, Nan,Wu, Long,Li, Ding
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p. 3187 - 3190
(2008/02/05)
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- Dibromomethane as one-carbon source in organic synthesis: Total synthesis of (±)- and (-)-methylenolactocin
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A general method was developed to construct monocyclic α-methylene- γ-butyrolactone moiety. The key step is to introduce the α-methylene group by the ozonolysis of mono-substituted alkenes followed by reacting with a preheated mixture of CH2Br2-Et2NH. Application of this key step in the total synthesis of the (±)- and (-)-methylenolactocin was described.
- Hon, Yung-Son,Hsieh, Cheng-Han,Liu, Yu-Wei
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p. 2713 - 2723
(2007/10/03)
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- Polyhydroxyalkanoate and manufacturing method thereof
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To provide a novel polyhydroxyalkanoate and a manufacturing method by a microorganism capable of substantially reducing unintended monomer units and obtaining the polyhydroxyalkanoate in a high yield. A microorganism capable of synthesizing a novel polyhydroxyalkanoate having 3-hydroxy-substituted benzoylalkanoic acid as a monomer unit using a substituted benzoylalkanoic acid as a material is cultured in the medium containing a substituted benzoylalkanoic acid, then the polyhydroxyalkanoate produced in the cultured bacteria is extracted and recovered.
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Page/Page column 35-36
(2008/06/13)
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- Copper-catalysed asymmetric 1,4-addition of organozinc compounds to linear aliphatic enones using 2,2′-dihydroxy 3,3′-dithioether derivatives of 1,1′-binaphthalene
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Directed ortho dilithiation of bis(diethylcarbamate) or bis(MOM)-protected (Sa)-1,1,′-bi(2-naphthol) followed by treatment with R2S2 [R = Me, Ph (X-ray structure)] or Me2Se2 cleanly affords the 3,3′ derivatives; the free naphthols are produced on deprotection. In the case of the bis(MOM) series, but not that of the bis(carbamates), some racemisation occurs. The ligand 2,2′-dihydroxy-3,3′-dimethylthio-1,1′-binaphthalene shows optimal performance in the addition of ZnEt2 to linear aliphatic enones (E)-R1C(O)CH=CHR2. Variation of the steric demands of R1 and R2 generates catalytic results consistent with binding of a zinc-based Lewis acid anti to the ene function and with the reactive conformation being s-cis. With enones containing the functions R2 = (CH2)nCH(OAlkyl)2 (n = 0-2), the ZnEt2 addition products undergo base-promoted cyclisation.
- Boerner, Christoph,Dennis, Michael R.,Sinn, Ekkehard,Woodward, Simon
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p. 2435 - 2446
(2007/10/03)
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- Chemistry of metapleural gland secretions of three attine ants, Atta sexdens rubropilosa, Atta cephalotes, and Acromyrmex octospinosus (Hymenoptera: Formicidae)
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The chemical composition of the secretions of the metapleural glands of workers and soldiers of two Atta species, atta sexdens rubropilosa and A. cephalotes, and workers of Acromyrmex oetospinoxus, has been studied. As indicated by infrared spectrometry and confirmed by the ninhydrin test, the secretions contain chiefly proteins. Of the volatile acidic portion, which is present as ionized salts, phenylacetic acid is the major component in workers and soldiers of A. s. rubropilosa and A. cephalotes. Both Atta species also contain 3 hydroxydecanoic acid and its homolog as minor components together with indoleacetic acid. While there are qualitative similarities in the acidic composition in the secretions of A. s. rubropilosa and A. cephalotes, they differ quantitatively. The secretion of Acromyrmex octospinosus contains 3-hydoxydecanoic and indoleacetic acids, but lacks phenylacetic acid The bactericidal and fungicidal actions of the three major substances have been confirmed.
- Do Nascimento, Ruth R.,Schoeters, Eric,Morgan, E. David,Billen, Johan,Stradling, David J.
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p. 987 - 1000
(2007/10/03)
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- Selective Transformations of threo-2,3-Dihydroxy Esters
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Two highly regio- and stereoselective transformations of threo-2,3-dihydroxy esters have been developed.In the first reaction, the α-hydroxy group is converted into a sulfonate group (tosylate or nosylate); the α-tosylates and α-nosylates are then subjected to basic conditions (K2CO3/ROH) to give erythro glycidic esters in high yield.The α-nosylates are also suitable electrophiles for azides, giving access to erythro-α-azido-β-hydroxy esters.The second reaction involves conversion of the diol esters to acetoxy bromo esters.The β-subsituent plays a key role in determining the regiochemistry since cases with β-alkyl substituents afford β-acetoxy-α-bromo esters exclusively, whereas a β-phenyl substitutent directs formation of the α-acetoxy-β-bromo ester.The acetoxy bromo esters can subsequently be converted to the threo glycidic esters (via the bromohydrin esters); selective hydrogenolysis of the bromine substituent can also be achieved.
- Fleming, Paul R.,Sharpless, K. Barry
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p. 2869 - 2875
(2007/10/02)
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- Studies on the Host-specific Pathotoxins Produced by Bipolaris (Helminthosporium) maydis, Race T, "Characterization of the Minor C35-, C47- and C49-Components of the Toxin Complex and Evidence Bearning on the Stereochemistry of Hydroxyl Groups"
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The host-specific pathotoxin complex isolated from the culture filtrate mycelial mat of bipolaris (Helminthosporium) maydis, race T, was reduced to hydrocarbons.The hydrocarbons were separated by molecular sieves and analyzed by GC-MS.The MS gave typical spectra of normal, odd carbon numbered hydrocarbons from C35H72 to C49H100.Compounds differing from hydrocarbons only in the presence of an internal tetrahydropyran ring were obtained as by-products from the toxin-complex reductions.These compounds were analyzed by CI (chemical ionization)-GC-MS and HR (high resoluton)-GC-Ms.The MS data supported the hypothesis that the C35- to C49-toxins are analogous compounds with many common partial structures.The configurations at both the C-6 and C-8 carbons of the major components appeared to be R.
- Kono, Yoshiki,Takeuchi, Setsuo,Kawarada, Akira,Daly, J. M.,Knoche, H. W.
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p. 2111 - 2116
(2007/10/02)
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