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Sample records for acyl hydrolase activity

  1. Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

    PubMed Central

    Stahl, U.; Banas, A.; Stymne, S.

    1995-01-01

    Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization. PMID:12228415

  2. Acyl hydrolases from trans-AT polyketide synthases target acetyl units on acyl carrier proteins.

    PubMed

    Jenner, Matthew; Afonso, Jose P; Kohlhaas, Christoph; Karbaum, Petra; Frank, Sarah; Piel, Jörn; Oldham, Neil J

    2016-04-18

    Acyl hydrolase (AH) domains are a common feature of trans-AT PKSs. They have been hypothesised to perform a proofreading function by removing acyl chains from stalled sites. This study determines the substrate tolerance of the AH PedC for a range of acyl-ACPs. Clear preference towards short, linear acyl-ACPs is shown, with acetyl-ACP the best substrate. These results imply a more targeted housekeeping role for PedC: namely the removal of unwanted acetyl groups from ACP domains caused by erroneous transfer of acetyl-CoA, or possibly by decarboxylation of malonyl-ACP.

  3. Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

    PubMed Central

    Kemp, Louise E.; Rusch, Marion; Adibekian, Alexander; Bullen, Hayley E.; Graindorge, Arnault; Freymond, Céline; Rottmann, Matthias; Braun-Breton, Catherine; Baumeister, Stefan; Porfetye, Arthur T.; Vetter, Ingrid R.; Hedberg, Christian; Soldati-Favre, Dominique

    2013-01-01

    In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein. PMID:23913689

  4. Novel Strategies for Upstream and Downstream Processing of Tannin Acyl Hydrolase

    PubMed Central

    Rodríguez-Durán, Luis V.; Valdivia-Urdiales, Blanca; Contreras-Esquivel, Juan C.; Rodríguez-Herrera, Raúl; Aguilar, Cristóbal N.

    2011-01-01

    Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme. PMID:21941633

  5. Unraveling the degradation of artificial amide bonds in nylon oligomer hydrolase: from induced-fit to acylation processes.

    PubMed

    Baba, Takeshi; Boero, Mauro; Kamiya, Katsumasa; Ando, Hiroyuki; Negoro, Seiji; Nakano, Masayoshi; Shigeta, Yasuteru

    2015-02-14

    To elucidate how the nylon oligomer hydrolase (NylB) acquires its peculiar degradation activity towards non-biological amide bonds, we inspected the underlying enzymatic processes going from the induced-fit upon substrate binding to acylation. Specifically we investigated the mutational effects of two mutants, Y170F and D181G, indicated in former experiments as crucial systems because of their specific amino acid residues. Therefore, by adopting first-principles molecular dynamics complemented with metadynamics we provide a detailed insight into the underlying acylation mechanism. Our results show that while in the wild type (WT) the Tyr170 residue points the NH group towards the proton-acceptor site of an artificial amide bond, hence ready to react, in the Y170F this does not occur. The reason is ascribed to the absence of Tyr170 in the mutant, which is replaced by phenylalanine, which is unable to form hydrogen bond with the amide bond; thus, resulting in an increase in the activation barrier of more than 10 kcal mol(-1). Nonetheless, despite the lack of hydrogen bonding between the Y170F and the substrate, the highest free energy barrier for the induced-fit is similar to that of WT. This seems to suggest that in the induced-fit process, kinetics is little affected by the mutation. On the basis of additional structural homology analyses on the enzymes of the same family, we suggest that natural selection is responsible for the development of the peculiar hydrolytic activity of Arthrobacter sp. KI72.

  6. Three-dimensional structure of the quorum-quenching N-acyl homoserine lactone hydrolase from Bacillus thuringiensis

    PubMed Central

    Liu, Dali; Lepore, Bryan W.; Petsko, Gregory A.; Thomas, Pei W.; Stone, Everett M.; Fast, Walter; Ringe, Dagmar

    2005-01-01

    The three-dimensional structure of the N-acyl-l-homoserine lactone hydrolase (AHL lactonase) from Bacillus thuringiensis has been determined, by using single-wavelength anomalous dispersion (SAD) phasing, to 1.6-Å resolution. AHLs are produced by many Gram-negative bacteria as signaling molecules used in quorum-sensing pathways that indirectly sense cell density and regulate communal behavior. Because of their importance in pathogenicity, quorum-sensing pathways have been suggested as potential targets for the development of novel therapeutics. Quorum-sensing can be disrupted by enzymes evolved to degrade these lactones, such as AHL lactonases. These enzymes are members of the metallo-β-lactamase superfamily and contain two zinc ions in their active sites. The zinc ions are coordinated to a number of ligands, including a single oxygen of a bridging carboxylate and a bridging water/hydroxide ion, thought to be the nucleophile that hydrolyzes the AHLs to ring-opened products, which can no longer act as quorum signals. PMID:16087890

  7. α-Ketoheterocycle Inhibitors of Fatty Acid Amide Hydrolase: Exploration of Conformational Constraints in the Acyl Side Chain

    PubMed Central

    Duncan, Katharine K.; Otrubova, Katerina; Boger, Dale L.

    2014-01-01

    A series of α-ketooxazoles containing heteroatoms embedded within conformational constraints in the C2 acyl side chain of 2 (OL-135) were synthesized and evaluated as inhibitors of fatty acid amide hydrolase (FAAH). The studies reveal that the installation of a heteroatom (O) in the conformational constraint is achievable, although the potency of these novel derivatives is reduced slightly relative to 2 and the analogous 1,2,3,4-tetrahydronaphthalene series. Interestingly, both enantiomers (R and S) of the candidate inhibitors bearing a chiral center adjacent to the electrophilic carbonyl were found to effectively inhibit FAAH. PMID:24690529

  8. Effects of hypo- and hyperthyroidism on rat liver microsomal long-chain fatty acyl-CoA synthetase and hydrolase

    SciTech Connect

    Dang, A.Q.; Faas, F.H.; Carter, W.J.

    1986-05-01

    The effects of hyperthyroidism (hyperT/sub 3/), (tri-iodothryonine (T/sub 3/) injected rats), and hypothyroidism (hypoT/sub 3/) (thyroidectomized rats) on the activation of fatty acids by a microsomal long-chain fatty acyl-CoA (LCA-CoA) synthetase and the degradation of LCA-CoA by a microsomal LCA-CoA hydrolase was determined. MAS was assayed by measuring the (1-/sup 14/C)-palmitate or -1-/sup 14/C) oleate incorporated into its water soluble CoA ester. MAH was assayed spectrophotomerically by following the reduction of 5',5'-dithiobis-(2-nitrobenzoic acid) by the CoA released from palmitoyl-CoA or oleoyl-CoA. Enzyme activities are given as mean (nmoles/mg/min) +/- SEM. MAS activities were decreased 36-44% (p < 0.01) in both hypoT/sub 3/ and hyperT/sub 3/ (controls = 101 +/- 4 (n = 11, (1-/sup 14/C)-palmitate) of 72 +/- 2 (n = 5,(1-/sup 14/C)oleate)). These decreases may contribute to the decreased triacelyglycerol (TG) and phospholipid contents in the hyperT/sub 3/ liver and the decreased clearance rate of plasma TG in the hypoT/sub 3/. MAH was decreased 27-42% (p<0.01) only in hypoT/sub 3/ (controls = 77 +/- 3 (n = 11, palmitoyl-CoA) or 45 +/- 1 (n = 5, oleoyl-CoA)). This decrease was corrected by T/sub 3/ treatment. Since the decreased MAH would increase the availability of LCA-CoA, it may contribute to the increased TG synthesis in hypoT/sub 3/.

  9. Activity of murein hydrolases in synchronized cultures of Escherichia coli.

    PubMed Central

    Hakenbeck, R; Messer, W

    1977-01-01

    Murein hydrolase activities were analyzed in synchronized cultures of Escherichia coli B/r. Cell wall-bound murein hydrolase activities, including the penicillin-sensitive endopeptidase, increased discontinuously during the cell cycle and showed maximum activity at a cell age of 30 to 35 min (generation time, 43 min). Maximum activity was observed at the same time that the rate of cell wall synthesis reached its maximum. These oscillations depended on the termination of replication: no increase in hydrolase activity was found if deoxyribonucleic acid synthesis was inhibited at an early time in the life cycle. In contrast, the activity of another murein hydrolase that was not tightly bound to the membrane (transglycosylase) increased exponentially with time, even when deoxyribonucleic acid synthesis was inhibited. PMID:321419

  10. Acyl silicates and acyl aluminates as activated intermediates in peptide formation on clays

    NASA Technical Reports Server (NTRS)

    White, D. H.; Kennedy, R. M.; Macklin, J.

    1984-01-01

    Glycine reacts with heating on dried clays and other minerals to give peptides in much better yield than in the absence of mineral. This reaction was proposed to occur by way of an activated intermediate such as an acyl silicate or acyl aluminate analogous to acyl phosphates involved in several biochemical reactions including peptide bond synthesis. The proposed mechanism has been confirmed by trapping the intermediate, as well as by direct spectroscopic observation of a related intermediate. The reaction of amino acids on periodically dried mineral surfaces represents a widespead, geologically realistic setting for prebiotic peptide formation via in situ activation.

  11. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    SciTech Connect

    John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

    2016-10-25

    A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacylethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings. The subject matter disclosed herein relates to enhancers of amidohydrolase activity.

  12. Identification of an Acyl-Enzyme Intermediate in a meta-Cleavage Product Hydrolase Reveals the Versatility of the Catalytic Triad

    SciTech Connect

    Ruzzini, Antonio C.; Ghosh, Subhangi; Horsman, Geoff P.; Foster, Leonard J.; Bolin, Jeffrey T.; Eltis, Lindsay D.

    2012-03-14

    Meta-cleavage product (MCP) hydrolases are members of the {alpha}/{beta}-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C-C bond. BphD, the MCP hydrolase from the biphenyl degradation pathway, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. A 1.6 {angstrom} resolution crystal structure of BphD H265Q incubated with HOPDA revealed that the enzyme's catalytic serine was benzoylated. The acyl-enzyme is stabilized by hydrogen bonding from the amide backbone of 'oxyanion hole' residues, consistent with formation of a tetrahedral oxyanion during nucleophilic attack by Ser112. Chemical quench and mass spectrometry studies substantiated the formation and decay of a Ser112-benzoyl species in wild-type BphD on a time scale consistent with turnover and incorporation of a single equivalent of {sup 18}O into the benzoate produced during hydrolysis in H{sub 2}{sup 18}O. Rapid-scanning kinetic studies indicated that the catalytic histidine contributes to the rate of acylation by only an order of magnitude, but affects the rate of deacylation by over 5 orders of magnitude. The orange-colored catalytic intermediate, ES{sup red}, previously detected in the wild-type enzyme and proposed herein to be a carbanion, was not observed during hydrolysis by H265Q. In the newly proposed mechanism, the carbanion abstracts a proton from Ser112, thereby completing tautomerization and generating a serinate for nucleophilic attack on the C6-carbonyl. Finally, quantification of an observed pre-steady-state kinetic burst suggests that BphD is a half-site reactive enzyme. While the updated catalytic mechanism shares features with the serine proteases, MCP hydrolase-specific chemistry highlights the versatility of the Ser-His-Asp triad.

  13. Oxidative activation of dihydropyridine amides to reactive acyl donors.

    PubMed

    Funder, Erik Daa; Trads, Julie B; Gothelf, Kurt V

    2015-01-07

    Amides of 1,4-dihydropyridine (DHP) are activated by oxidation for acyl transfer to amines, alcohols and thiols. In the reduced form the DHP amide is stable towards reaction with amines at room temperature. However, upon oxidation with DDQ the acyl donor is activated via a proposed pyridinium intermediate. The activated intermediate reacts with various nucleophiles to give amides, esters, and thio-esters in moderate to high yields.

  14. Influence of the degree of unsaturation of the acyl side chain upon the interaction of analogues of 1-arachidonoylglycerol with monoacylglycerol lipase and fatty acid amide hydrolase.

    PubMed

    Vandevoorde, Séverine; Saha, Bijali; Mahadevan, Anu; Razdan, Raj K; Pertwee, Roger G; Martin, Billy R; Fowler, Christopher J

    2005-11-11

    Little is known as to the structural requirements of the acyl side chain for interaction of acylglycerols with monoacylglycerol lipase (MAGL), the enzyme chiefly responsible for the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain. In the present study, a series of twelve analogues of 1-AG (the more stable regioisomer of 2-AG) were investigated with respect to their ability to inhibit the metabolism of 2-oleoylglycerol by cytosolic and membrane-bound MAGL. In addition, the ability of the compounds to inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) was investigated. For cytosolic MAGL, compounds with 20 carbon atoms in the acyl chain and 2-5 unsaturated bonds inhibited the hydrolysis of 2-oleoylglycerol with similar potencies (IC50 values in the range 5.1-8.2 microM), whereas the two compounds with a single unsaturated bond were less potent (IC50 values 19 and 21 microM). The fully saturated analogue 1-monoarachidin did not inhibit the enzyme, whereas the lower side chain analogues 1-monopalmitin and 1-monomyristin inhibited the enzyme with IC50 values of 12 and 32 microM, respectively. The 22-carbon chain analogue of 1-AG was also potent (IC50 value 4.5 microM). Introduction of an alpha-methyl group for the C20:4, C20:3, and C22:4 compounds did not affect potency in a consistent manner. For the FAAH and the membrane-bound MAGL, there was no obvious relationship between the degree of unsaturation of the acyl side chain and the ability to inhibit the enzymes. It is concluded that increasing the number of unsaturated bonds on the acyl side chain of 1-AG from 1 to 5 has little effect on the affinity of acylglycerols for cytosolic MAGL.

  15. An inducible hydrolase from Aspergillus niger, acting on carbon–carbon bonds, for phlorrhizin and other C-acylated phenols

    PubMed Central

    Minamikawa, T.; Jayasankar, N. P.; Bohm, B. A.; Taylor, I. E. P.; Towers, G. H. N.

    1970-01-01

    1. An inducible enzyme catalysing the hydrolysis of phloretin to form phloroglucinol and phloretic acid has been extracted from the acetone-dried powders of the mycelial felts of an Aspergillus niger strain grown in the presence of phlorrhizin. The enzyme was partially purified by treatment with protamine sulphate, ammonium sulphate fractionation, negative adsorption on tricalcium phosphate gel, and DEAE-cellulose column chromatography. 2. The hydrolytic activity on phloretin appeared to be maximal at about pH9.6. However, the characteristics of the enzyme were studied at pH7.2, because of the lability of the product, phloroglucinol, under alkaline conditions. 3. The apparent Km value at pH7.2 was about 0.3–0.4mm for phloretin and 0.15mm for 3′-methylphloracetophenone. 4. Maximum activity of the enzyme was obtained without the addition of any cofactor or metal ion. The involvement of thiol groups in the reaction was demonstrated by the potent inhibitory action of both heavy-metal ions and p-chloromercuribenzoate. 5. The enzyme showed a rather broad substrate specificity, and some other C-acylated phenols related to phloretin were hydrolysed. It was found that 3′-methylphloracetophenone, phloracetophenone and 2′,4,4′-trihydroxydihydrochalcone were attacked more efficiently than phloretin. We propose the systematic name C-acylphenol acylhydrolase for the enzyme. This enzyme belongs to EC group 3.7.1. PMID:5441377

  16. The activity of Rhizomuchor miehei lipase as a biocatalyst in enzymatic acylation of cyclic alcohol

    NASA Astrophysics Data System (ADS)

    Iftitah, Elvina Dhiaul; Srihardyastuti, Arie; Ariefin, Mokhamat

    2017-03-01

    We report the activity of Rhizomuchor miehei lipase (RML) as a biocatalyst, in particular the investigations concerning the effort of substrate-structure reactivity on the enzymatic acylation. The acylation was studied using acetic anhydride as an acyl donor and performed in n-hexane as a solvent. The selectivity of the enzymatic acylation was revealed by Gas Chromatography-Mass Spectra. We observed that, RML has shown different behavior when catalyzing the acylation of isopulegol and mixture of isopulegol and citronellal (ratio 1:1). The chemoselectivity for the O-acylation was improved when the acyl acceptor included mixture of isopulegol and citronellal

  17. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; Udell, Hannah S.; Yik, Eric J.; Bergeman, Lai F.; Fox, Brian G.

    2015-01-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  18. [S-Acyl derivatives of thiosalicylamides having antifungal activity. II].

    PubMed

    Mazza, M; Modena, T; Montanari, L; Pavanetto, F

    1978-07-01

    Some S-acyl derivatives of N-alkylthiosalicylamides [Table I: substances (I leads to XXXI)] were prepared and tested for antifungal activity. The substances, most of which had not been previously reported, were prepared by condensation of 2-mercapto-N-alkylbenzamides with suitable acylating agents. The antifungal activity of the compounds was tested in vitro against Candida albicans and Trichophyton mentagrophytes. For some compounds the was tested activity against the above strains fungicidal, Candida tropicalis and Saccharomyces cerevisiae. Many of the compounds proved to have high antifungal activity comparable with that of Clotrimazol. The results extended knowledge on the structure-antifungal activity relationships of this class of compounds. The compounds with the highest antifungal activity were: 2-acetylmercapto-N,n-heptylbenzamide (XXVIII); 2-acetylmercapto-5-Cl-N,n-propylbenzamide (XIV); 2-acetylmercapto-N,n-octylbenzamide (XXXI); 2-acetylmercapto-N,n-pentylbenzamide (XXV); 2-acetylmercapto-N,n-hexylbenzamide (XXVII).

  19. Acetylcarnitine hydrolase activity in bovine caudal epididymal spermatozoa

    SciTech Connect

    Bruns, K.; Foster, R.A.; Casillas, E.R.

    1986-05-01

    Recently, the authors identified mM concentrations of acetylcarnitine in epidiymal fluids and have investigated the metabolism of acetylcarnitine by bovine and hamster caudal epididymal spermatozoa. (1-/sup 14/C)acetyl-L-carnitine is oxidized to /sup 14/CO/sub 2/ by washed, intact hamster and bovine sperm at maximal rates of 8.4 and 15.2 nmol/hr/10/sup 7/ cells respectively. Conversely, the carnitine moiety of acetyl-L-(/sup 3/H-methyl)carnitine is not accumulated by sperm under similar conditions. Hydrolysis of (/sup 3/H)acetyl-L-carnitine and competition of uptake of (/sup 3/H)acetate by unlabeled acetate was demonstrated in incubations of intact cells of both species. The amount of (/sup 3/H)acetate accumulated in the incubation medium is time-dependent and also depends on the concentration of unlabeled acetate. A partial solubilization of acetylcarnitine hydrolase activity from washed, intact bovine caudal epididymal spermatozoa in buffer or 0.01% Triton X-100 is observed. There is an enrichment of acetylcarnitine hydrolase activity in purified plasma membranes from bovine caudal epididymal spermatozoa when compared to the activity present in broken cell preparations or other cellular fractions. The results suggest that acetylcarnitine is a substrate for spermatozoa as they traverse the epididymis.

  20. Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria.

    PubMed

    Pinnell, Lee J; Dunford, Eric; Ronan, Patrick; Hausner, Martina; Neufeld, Josh D

    2014-07-01

    Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [(13)C]glucose and [(13)C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.

  1. A Substrate-Assisted Mechanism of Nucleophile Activation in a Ser-His-Asp Containing C-C Bond Hydrolase

    SciTech Connect

    Ruzzini, Antonio C.; Bhowmik, Shiva; Ghosh, Subhangi; Yam, Katherine C.; Bolin, Jeffrey T.; Eltis, Lindsay D.

    2013-11-12

    The meta-cleavage product (MCP) hydrolases utilize a Ser–His–Asp triad to hydrolyze a carbon–carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ESred, which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ESred decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2nuc ~ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His–Asp pair does not play an essential role. The data further suggest that ESred represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.

  2. The Responses of Rat Intestinal Brush Border and Cytosol Peptide Hydrolase Activities to Variation in Dietary Protein Content DIETARY REGULATION OF INTESTINAL PEPTIDE HYDROLASES

    PubMed Central

    Nicholson, J. Alex; McCarthy, Denis M.; Kim, Young S.

    1974-01-01

    The effects of variation in dietary protein content on small intestinal brush border and cytosol peptide hydrolase activities have been investigated. One group of rats was fed a high protein diet (55% casein) and another group was fed a low protein diet (10% casein). After 1 wk, brush border peptide hydrolase activity (L-leucyl-β-naphthylamide as substrate) and cytosol peptide hydrolase activity (L-prolyl-L-leucine as substrate) were determined in mucosae taken from the proximal, middle, and distal small intestine. As judged by several parameters, brush border peptide hydrolase activity was significantly greater in rats fed the high protein diet when data for corresponding segments were compared. In contrast, no significant difference was seen in cytosol peptide hydrolase activity. In a second study, brush border and cytosol peptide hydrolase activities were determined in the proximal intestine by utilizing an additional three peptide substrates: L-leucyl-L-alanine, L-phenylalanylglycine, and glycyl-L-phenylalanine. Sucrase, maltase, and alkaline phosphatase activities were also determined. As before, brush border peptide hydrolase activities were significantly greater in rats fed the high protein diet. However, activities of the nonproteolytic brush border enzymes did not vary significantly with diet. In contrast to the results obtained with L-prolyl-L-leucine as substrate for the cytosol enzymes, cytosol activity against the three additional peptide substrates was greater in rats fed the high protein diet. It is suggested that the brush border peptide hydrolase response to variation in dietary protein content represents a functional adaptation analogous to the regulation of intestinal disaccharidases by dietary carbohydrates. The implication of the differential responses of the cytosol peptide hydrolases is uncertain, since little is known of the functional role of these nonorgan-specific enzymes. PMID:4430719

  3. The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase.

    PubMed

    Kim, Myung Hee; Choi, Won-Chan; Kang, Hye Ok; Lee, Jong Suk; Kang, Beom Sik; Kim, Kyung-Jin; Derewenda, Zygmunt S; Oh, Tae-Kwang; Lee, Choong Hwan; Lee, Jung-Kee

    2005-12-06

    In many Gram-negative bacteria, including a number of pathogens such as Pseudomonas aeruginosa and Erwinia carotovora, virulence factor production and biofilm formation are linked to the quorum-sensing systems that use diffusible N-acyl-L-homoserine lactones (AHLs) as intercellular messenger molecules. A number of organisms also contain genes coding for lactonases that hydrolyze AHLs into inactive products, thereby blocking the quorum-sensing systems. Consequently, these enzymes attract intense interest for the development of antiinfection therapies. However, the catalytic mechanism of AHL-lactonase is poorly understood and subject to controversy. We here report a 2.0-angstroms resolution structure of the AHL-lactonase from Bacillus thuringiensis and a 1.7-angstroms crystal structure of its complex with L-homoserine lactone. Despite limited sequence similarity, the enzyme shows remarkable structural similarities to glyoxalase II and RNase Z proteins, members of the metallo-beta-lactamase superfamily. We present experimental evidence that AHL-lactonase is a metalloenzyme containing two zinc ions involved in catalysis, and we propose a catalytic mechanism for bacterial metallo-AHL-lactonases.

  4. Mapping human brain fatty acid amide hydrolase activity with PET

    PubMed Central

    Rusjan, Pablo M; Wilson, Alan A; Mizrahi, Romina; Boileau, Isabelle; Chavez, Sofia E; Lobaugh, Nancy J; Kish, Stephen J; Houle, Sylvain; Tong, Junchao

    2013-01-01

    Endocannabinoid tone has recently been implicated in a number of prevalent neuropsychiatric conditions. [11C]CURB is the first available positron emission tomography (PET) radiotracer for imaging fatty acid amide hydrolase (FAAH), the enzyme which metabolizes the prominent endocannabinoid anandamide. Here, we sought to determine the most suitable kinetic modeling approach for quantifying [11C]CURB that binds selectively to FAAH. Six healthy volunteers were scanned with arterial blood sampling for 90 minutes. Kinetic parameters were estimated regionally using a one-tissue compartment model (TCM), a 2-TCM with and without irreversible trapping, and an irreversible 3-TCM. The 2-TCM with irreversible trapping provided the best identifiability of PET outcome measures among the approaches studied (coefficient of variation (COV) of the net influx constant Ki and the composite parameter λk3 (λ=K1/k2) <5%, and COV(k3)<10%). Reducing scan time to 60 minutes did not compromise the identifiability of rate constants. Arterial spin labeling measures of regional cerebral blood flow were only slightly correlated with Ki, but not with k3 or λk3. Our data suggest that λk3 is sensitive to changes in FAAH activity, therefore, optimal for PET quantification of FAAH activities with [11C]CURB. Simulations showed that [11C]CURB binding in healthy subjects is far from a flow-limited uptake. PMID:23211960

  5. Acylated quercetagetin glycosides with antioxidant activity from Tagetes maxima.

    PubMed

    Parejo, Irene; Bastida, Jaume; Viladomat, Francesc; Codina, Carles

    2005-10-01

    The fractionation of a methanolic extract of Tagetes maxima guided for antioxidant activity resulted in the isolation of three acylated quercetagetin glycosides, quercetagetin-7-O-(6-O-caffeoyl-beta-D-glucopyranoside), quercetagetin-7-O-(6-O-p-coumaroyl-beta-D-glucopyranoside) and quercetagetin-7-O-(6-O-galloyl-beta-D-glucopyranoside), as well as four known flavonoid glycosides. The structural elucidation was accomplished by spectroscopic methods (ESI-MS/MS and NMR). The antioxidant activity of fractions and isolated compounds was determined by checking the scavenging activity against three different radicals: 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH*), hydroxyl (*OH), and superoxide (O2*-). The three isolated compounds exhibited a high radical scavenging activity in comparison with reference compounds.

  6. Molecular Basis of Arabinobio-hydrolase Activity in Phytopathogenic Fungi

    PubMed Central

    Carapito, Raphaël; Imberty, Anne; Jeltsch, Jean-Marc; Byrns, Simon C.; Tam, Pui-Hang; Lowary, Todd L.; Varrot, Annabelle; Phalip, Vincent

    2009-01-01

    The phytopathogenic fungus Fusarium graminearum secretes a very diverse pool of glycoside hydrolases (GHs) aimed at degrading plant cell walls. α-l-Arabinanases are essential GHs participating in the complete hydrolysis of hemicellulose, a natural resource for various industrial processes, such as bioethanol or pharmaceuticals production. Arb93A, the exo-1,5-α-l-arabinanase of F. graminearum encoded by the gene fg03054.1, belongs to the GH93 family, for which no structural data exists. The enzyme is highly active (1065 units/mg) and displays a strict substrate specificity for linear α-1,5-l-arabinan. Biochemical assays and NMR experiments demonstrated that the enzyme releases α-1,5-l-arabinobiose from the nonreducing end of the polysaccharide. We determined the crystal structure of the native enzyme and its complex with α-1,5-l-arabinobiose, a degradation product of α-Me-1,5-l-arabinotetraose, at 1.85 and 2.05Å resolution, respectively. Arb93A is a monomeric enzyme, which presents the six-bladed β-propeller fold characteristic of sialidases of clan GHE. The configuration of the bound arabinobiose is consistent with the retaining mechanism proposed for the GH93 family. Catalytic residues were proposed from the structural analysis, and site-directed mutagenesis was used to validate their role. They are significantly different from those observed for GHE sialidases. PMID:19269961

  7. Soluble epoxide hydrolase inhibitory activity of anthraquinone components from Aloe.

    PubMed

    Sun, Ya Nan; Kim, Jang Hoon; Li, Wei; Jo, A Reum; Yan, Xi Tao; Yang, Seo Young; Kim, Young Ho

    2015-10-15

    Aloe is a short-stemmed succulent herb widely used in traditional medicine to treat various diseases and as raw material in cosmetics and heath foods. In this study, we isolated and identified two new anthraquinone derivatives, aloinoside C (6) and aloinoside D (7), together with six known compounds from an aqueous dissolved Aloe exudate. Their structures were identified by spectroscopic analysis. The inhibitory effects of the isolated compounds on soluble epoxide hydrolase (sEH) were evaluated. Compounds 1-8 inhibited sEH activity potently, with IC50 values ranging from 4.1±0.6 to 41.1±4.2 μM. A kinetic analysis of compounds 1-8 revealed that the inhibitory actions of compounds 1, 6 and 8 were non-competitive, whereas those of compounds 2-5 and 7 were the mixed-type. Molecular docking increases our understanding of receptor-ligand binding of all compounds. These results demonstrate that compounds 1-8 from Aloe are potential sEH inhibitors.

  8. A molecular model for the active site of S-adenosyl- l-homocysteine hydrolase

    NASA Astrophysics Data System (ADS)

    Yeh, Jerry C.; Borchardt, Ronald T.; Vedani, Angelo

    1991-06-01

    S-adenosyl- l-homocysteine hydrolase (AdoHcy hydrolase, EC 3.3.1.1.), a specific target for antiviral drug design, catalyzes the hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy) as well as the synthesis of AdoHcy from Ado and Hcy. The enzyme isolated from different sources has been shown to contain tightly bound NAD+. Based on the 2.0 Å-resolution X-ray crystal structure of dogfish lactate dehydrogenase (LDH), which is functionally homologous to AdoHcy hydrolase, and the primary sequence of rat liver AdoHcy hydrolase, we have derived a molecular model of an extended active site for AdoHcy hydrolase. The computational mutation was performed using the software MUTAR (Yeh et al., University of Kansas, Lawrence), followed by molecular mechanics optimizations using the programs AMBER (Singh et al., University of California, San Francisco) and YETI (Vedani, University of Kansas). Solvation of the model structure was achieved by use of the program SOLVGEN (Jacober, University of Kansas); 56 water molecules were explicitly included in all refinements. Some of these may be involved in the catalytic reaction. We also studied a model of the complex of AdoHcy hydrolase with NAD+, as well as the ternary complexes of the redox reaction catalyzed by AdoHcy hydrolase and has been used to differentiate the relative binding strength of inhibitors.

  9. Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria*

    PubMed Central

    Yao, Jiangwei; Bruhn, David F.; Frank, Matthew W.; Lee, Richard E.; Rock, Charles O.

    2016-01-01

    Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria. PMID:26567338

  10. Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

    PubMed Central

    2011-01-01

    Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316

  11. Development of a differential medium for bile salt hydrolase-active Lactobacillus spp.

    PubMed Central

    Dashkevicz, M P; Feighner, S D

    1989-01-01

    An agar plate assay was developed to detect bile salt hydrolase activity in lactobacilli. On Lactobacillus-selective MRS or Rogosa SL medium supplemented with taurodeoxycholic, taurocholic, or taurochenodeoxycholic acids, bile salt hydrolysis was manifested at two intensities: (i) the formation of precipitate halos around colonies or (ii) the formation of opaque granular white colonies. Sixty-six lactobacilli were tested for bile salt hydrolase activity by both the plate assay and a sensitive radiochemical assay. No false-positive or false-negative results were detected by the plate assay. Based on results of experiments with Eubacterium lentum and Bacteroides species, the plate assay was dependent on two factors: (i) the presence of bile salt hydrolytic activity and (ii) the ability of the organism to sufficiently acidify the medium to protonate free bile acids. The availability of a differential medium for determination of bile salt hydrolase activity will provide a rapid method for determining shifts in a specific functional activity of intestinal Lactobacillus species and provide a rapid screening capability for identifying bile salt hydrolase-deficient mutants. The latter application should allow bile salt hydrolase activity to be used as a marker enzyme in genetic experiments. Images PMID:2705765

  12. A specific acyl-ACP thioesterase implicated in medium-chain fatty acid production in immature cotyledons of Umbellularia californica.

    PubMed

    Pollard, M R; Anderson, L; Fan, C; Hawkins, D J; Davies, H M

    1991-02-01

    Umbellularia californica (California Bay) seeds accumulate 10:0 and 12:0 as principal reserve fatty acyl groups. An in vitro fatty acid synthesis system from the developing cotyledons produces chiefly 10:0 and 12:0, in approximately the same proportions as the intact tissue. The kinetics of acyl thioester and free fatty acid formation in this system suggest that a medium-chain specific acyl-acyl-carrier protein (ACP) hydrolysis mechanism is responsible for the preponderance of medium-chain products. A crude extract of the developing cotyledons exhibits hydrolytic activity toward acyl-ACPs, with marked preference for 12:0-ACP and 18:1-ACP in the test series 6:0, 8:0, 10:0, 11:0, 12:0, 14:0, 16:0, and 18:1-ACPs. Partial purification of the 12:0-ACP hydrolytic activity has resulted in its separation from the 18:1-ACP hydrolase(s) and the 12:0-coenzyme A hydrolase(s) that are also present, thereby demonstrating its specificity for the 12-carbon acyl chain length and the ACP derivative. During cotyledon development, as the proportion of medium-chain to other fatty acyl groups increases, the extractable yield of this activity also increases substantially. Collectively these results suggest a role for this 12-ACP thioesterase in medium-chain production in vivo.

  13. Murein hydrolase activity of surface layer proteins from Lactobacillus acidophilus against Escherichia coli.

    PubMed

    Meng, Jun; Gao, Shu-Ming; Zhang, Qiu-Xiang; Lu, Rong-Rong

    2015-08-01

    The aim of this study was to investigate the murein hydrolase activities of the surface layer proteins (SLPs) from two strains of Lactobacillus acidophilus using zymography. The influence of these hydrolase activities on Escherichia coli ATCC 43893 was also evaluated by analysing their growth curve, cell morphology and physiological state. After the incubation of E. coli with SLPs, growth was inhibited, the number of viable cells was significantly reduced, examination by transmission electron microscopy showed that the cell wall was damaged and flow cytometry results indicated that the majority of the cells were sublethally injured. All of these results suggested that the SLPs of both L. acidophilus strains possessed murein hydrolase activities that were sublethal to E. coli cells.

  14. Acyl-CoA synthetase activity links wild-type but not mutant α-synuclein to brain arachidonate metabolism

    PubMed Central

    Golovko, Mikhail Y.; Rosenberger, Thad A.; Faergeman, Nils J.; Feddersen, Søren; Cole, Nelson B.; Pribill, Ingrid; Berger, Johannes; Nussbaum, Robert L.; Murphy, Eric J.

    2008-01-01

    Because α-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that the loss of α-synuclein had on brain arachidonic acid (20:4n-6) metabolism in vivo using Snca-/- mice. We measured [1-14C]20:4n-6 incorporation and turnover kinetics in brain phospholipids using an established steady-state kinetic model. Liver was used as a negative control and no changes were observed between groups. In Snca-/- brains, there was a marked reduction in 20:4n-6-CoA mass and in microsomal acyl-CoA synthetases (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was completely restored after the addition of exogenous wt mouse or human α-synuclein, but not by A30P, E46K, and A53T forms of α-synuclein. Acsl and acyl-CoA hydrolase expression was not different between groups. The incorporation and turnover of 20:4n-6 into brain phospholipid pools was markedly reduced. The dilution coefficient lambda, which indicates 20:4n-6 recycling between the acyl-CoA pool and brain phospholipids, was increased 3.3-fold, indicating more 20:4n-6 was entering the 20:4n-6-CoA pool from the plasma relative to that being recycled from the phospholipids. This is consistent with the reduction in Acsl activity observed in the Snca-/- mice. Using titration microcalorimetry, we determined that α-synuclein bound free 20:4n-6 (Kd of 3.7 μM), but did not bind 20:4n-6-CoA. These data suggest α-synuclein is involved in substrate presentation to Acsl rather than product removal. In summary, our data demonstrate that α-synuclein has a major role in brain 20:4n-6 metabolism through its modulation of endoplasmic reticulum localized acyl-CoA synthetase activity, although mutants forms of α-synuclein fail to restore this activity. PMID:16734431

  15. Data set of optimal parameters for colorimetric red assay of epoxide hydrolase activity.

    PubMed

    de Oliveira, Gabriel Stephani; Adriani, Patricia Pereira; Borges, Flavia Garcia; Lopes, Adriana Rios; Campana, Patricia T; Chambergo, Felipe S

    2016-09-01

    The data presented in this article are related to the research article entitled "Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization" [1]. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. This article describes the optimal parameters for the colorimetric red assay to determine the enzymatic activity, with an emphasis on the characterization of the kinetic parameters, pH optimum and thermal stability of this enzyme. The effects of reagents that are not resistant to oxidation by sodium periodate on the reactions can generate false positives and interfere with the final results of the red assay.

  16. Epoxide hydrolase activities and epoxy fatty acids in the mosquito Culex quinquefasciatus

    PubMed Central

    Xu, Jiawen; Morisseau, Christophe; Yang, Jun; Mamatha, Dadala M.

    2015-01-01

    Culex mosquitoes have emerged as important model organisms for mosquito biology, and are disease vectors for multiple mosquito-borne pathogens, including West Nile virus. We characterized epoxide hydrolase activities in the mosquito Culex quinquefasciatus, which suggested multiple forms of epoxide hydrolases were present. We found EH activities on epoxy eicosatrienoic acids (EETs). EETs and other eicosanoids are well-established lipid signaling molecules in vertebrates. We showed EETs can be synthesized in vitro from arachidonic acids by mosquito lysate, and EETs were also detected in vivo both in larvae and adult mosquitoes by LC-MS/MS. The EH activities on EETs can be induced by blood feeding, and the highest activity was observed in the midgut of female mosquitoes. The enzyme activities on EETs can be inhibited by urea-based inhibitors designed for mammalian soluble epoxide hydrolases (sEH). The sEH inhibitors have been shown to play diverse biological roles in mammalian systems, and they can be useful tools to study the function of EETs in mosquitoes. Besides juvenile hormone metabolism and detoxification, insect epoxide hydrolases may also play a role in regulating lipid signaling molecules, such as EETs and other epoxy fatty acids, synthesized in vivo or obtained from blood feeding by female mosquitoes. PMID:25686802

  17. Enhanced Activity of Nanocrystalline Beta Zeolite for Acylation of Veratrole with Acetic Anhydride.

    PubMed

    Aisha Mahmood Abdulkareem, Al-Turkustani; Selvin, Rosilda

    2016-04-01

    Friedel-Craft acylation of veratrole using homogeneous acid catalysts such as AlCl3, FeCl3, ZnCl2, and HF etc. produces acetoveratrone, (3',4'-dimethoxyacetophenone), which is the intermediate for synthesis of papavarine alkaloids. The problems associated with these homogeneous catalysts can be overcome by using heterogeneous solid catalysts. Since acetoveratrone is a larger molecule, large pore Beta zeolites with smaller particle sizes are beneficial for the liquid-phase acylation of veratrole, for easy diffusion of reactants and products. The present study aims in the acylation of veratrole with acetic anhydride using nanocrystalline Beta Zeolite catalyst. A systematic investigation of the effects of various reaction parameters was done. The catalysts were characterized for their structural features by using XRD, TEM and DLS analyses. The catalytic activity of nanocrystalline Beta zeolite was compared with commercial Beta zeolite for the acylation and was found that nanocrystalline Beta zeolite possessed superior activity.

  18. A single amino acid substitution in a chitinase of the legume Medicago truncatula is sufficient to gain Nod-factor hydrolase activity

    PubMed Central

    Zhang, Lan-Yue; Cai, Jie; Li, Ru-Jie; Liu, Wei; Wagner, Christian; Wong, Kam-Bo; Xie, Zhi-Ping; Staehelin, Christian

    2016-01-01

    The symbiotic interaction between nitrogen-fixing rhizobia and legumes depends on lipo-chitooligosaccharidic Nod-factors (NFs). The NF hydrolase MtNFH1 of Medicago truncatula is a symbiotic enzyme that hydrolytically inactivates NFs with a C16 : 2 acyl chain produced by the microsymbiont Sinorhizobium meliloti 1021. MtNFH1 is related to class V chitinases (glycoside hydrolase family 18) but lacks chitinase activity. Here, we investigated the substrate specificity of MtNFH1-related proteins. MtCHIT5a and MtCHIT5b of M. truncatula as well as LjCHIT5 of Lotus japonicus showed chitinase activity, suggesting a role in plant defence. The enzymes failed to hydrolyse NFs from S. meliloti. NFs from Rhizobium leguminosarum with a C18 : 4 acyl moiety were neither hydrolysed by these chitinases nor by MtNFH1. Construction of chimeric proteins and further amino acid replacements in MtCHIT5b were performed to identify chitinase variants that gained the ability to hydrolyse NFs. A single serine-to-proline substitution was sufficient to convert MtCHIT5b into an NF-cleaving enzyme. MtNFH1 with the corresponding proline-to-serine substitution failed to hydrolyse NFs. These results are in agreement with a substrate-enzyme model that predicts NF cleavage when the C16 : 2 moiety is placed into a distinct fatty acid-binding cleft. Our findings support the view that MtNFH1 evolved from the ancestral MtCHIT5b by gene duplication and subsequent symbiosis-related neofunctionalization. PMID:27383628

  19. Hydrolase activity in the venom of the pupal endoparasitic wasp, Pimpla hypochondriaca.

    PubMed

    Dani, M P; Edwards, J P; Richards, E H

    2005-07-01

    Venom from the pupal endoparasitoid, Pimpla hypochondriaca has previously been shown to contain a mixture of biologically active molecules. Currently, P. hypochondriaca venom was examined for the presence of hydrolase activity. Six hydrolases were consistently detected using the API ZYM semiquantitative colourimetric kit. The main hydrolases detected were; acid phosphatase, beta-glucosidase, esterase, beta-galactosidase, esterase lipase, and lipase. The most rapid and intense colour reaction was detected for acid phosphatase. The pH optimum and the specific activity of venom acid phosphatase was determined using p-nitrophenol phosphate as a substrate and were 4.8 and 0.47 nmol p-nitrophenol/min/microg of venom protein, respectively. The acid phosphatase activity was inhibited in a dose dependent manner by sodium fluoride (IC(50) 4.2 x 10(-4) M), and by cocktail inhibitor 2 (CI 2). P. hypochondriaca venom has previously been shown to display potent cytotoxic activity towards Lacanobia oleracea haemocytes maintained in vitro. The contribution of acid phosphatase in venom to this cytotoxic activity was investigated by titrating venom against CI 2 prior to the addition of L. oleracea haemocytes. The results suggest that, despite the relatively high levels of acid phosphatase activity in venom, venom acid phosphatase plays no role in the antihaemocytic activity of P. hypochondriaca venom in vitro.

  20. Effects of proteins and polynucleotides on the activity of various hydrolases

    PubMed Central

    Palmieri, M. J.; Koldovský, O.

    1972-01-01

    The effect of various macromolecules on the activity of several hydrolases was studied. Dilution of partially purified acid β-galactosidase from ileal mucosa of suckling rats resulted in a decrease of specific activity. The relationship between specific activity and dilution of the enzyme suggests a dissociation of the enzyme. This could be prevented by addition of several proteins tested. However, addition of DNA to the assay mixture for acid β-galactosidase caused an inhibition. This inhibition could be prevented by addition of proteins. Other polynucleotides and tRNA also exert an inhibitory effect that is prevented by albumin, but nucleotides have no effect. This inhibition occurs maximally at a low pH (3.0–4.0); no inhibition is observed at pH5.5. A similar pH-dependent inhibition by DNA was also found with various other acid hydrolases. PMID:5076227

  1. A fluorescent assay to quantitatively measure in vitro acyl CoA:diacylglycerol acyltransferase activity.

    PubMed

    McFie, Pamela J; Stone, Scot J

    2011-09-01

    Triacylglycerols (TG) are the major storage form of energy in eukaryotic organisms and are synthesized primarily by acyl CoA:1,2-diacylglycerol acyltransferase (DGAT) enzymes. In vitro DGAT activity has previously been quantified by measuring the incorporation of either radiolabeled fatty acyl CoA or diacylglycerol (DG) into TG. We developed a modified acyltransferase assay using a fluorescent fatty acyl CoA substrate to accurately quantify in vitro DGAT activity. In the modified assay, radioactive fatty acyl CoA is replaced with fluorescent NBD-palmitoyl CoA, which is used as a substrate by DGAT with DG to produce NBD-TG. After extraction with organic solvents and separation by thin layer chromatography, NBD-TG formation can be detected and accurately quantified using a fluorescent imaging system. We demonstrate that this method can be adapted to detect other acyltransferase activities. Because NBD-palmitoyl CoA is commercially available at a much lower cost compared with radioactive acyl CoA substrates, it is a more economical alternative to radioactive tracers. In addition, the exposure of laboratory personnel to radioactivity is greatly reduced.

  2. Synthesis, Surface Active Properties and Cytotoxicity of Sodium N-Acyl Prolines.

    PubMed

    Sreenu, Madhumanchi; Narayana Prasad, Rachapudi Badari; Sujitha, Pombala; Kumar, Chityal Ganesh

    2015-01-01

    Sodium N-acyl prolines (NaNAPro) were synthesized using mixture of fatty acids obtained from coconut, palm, karanja, Sterculia foetida and high oleic sunflower oils via Schotten-Baumann reaction in 58-75% yields to study the synergetic effect of mixture of hydrophobic fatty acyl functionalities like saturation, unsaturation and cyclopropene fatty acids with different chain lengths and aliphatic hetero cyclic proline head group on their surface and cytotoxicity activities. The products were characterized by chromatographic and spectral techniques. The synthesized products were evaluated for their surface active properties such as surface tension, wetting power, foaming characteristics, emulsion stability, calcium tolerance, critical micelle concentration (CMC) and thermodynamic properties. The results revealed that all the products exhibited superior surface active properties like CMC, calcium tolerance and emulsion stability as compared to the standard surfactant, sodium lauryl sulphate (SLS). In addition, palm, Sterculia foetida and high oleic sunflower fatty N-acyl prolines exhibited promising cytotoxicity against different tumor cell lines.

  3. Long chain acyl-CoA synthetases and other acyl activating enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proper synthesis and breakdown of molecules containing carboxylic acids is a vital part of metabolism in all living organisms. Given the relatively inert chemical nature of many carboxylic acids, activation is a necessary step prior to use in the various anabolic and catabolic pathways that utilize...

  4. Diadenosine tetraphosphate hydrolase is part of the transcriptional regulation network in immunologically activated mast cells.

    PubMed

    Carmi-Levy, Irit; Yannay-Cohen, Nurit; Kay, Gillian; Razin, Ehud; Nechushtan, Hovav

    2008-09-01

    We previously discovered that microphthalmia transcription factor (MITF) and upstream stimulatory factor 2 (USF2) each forms a complex with its inhibitor histidine triad nucleotide-binding 1 (Hint-1) and with lysyl-tRNA synthetase (LysRS). Moreover, we showed that the dinucleotide diadenosine tetraphosphate (Ap(4)A), previously shown to be synthesized by LysRS, binds to Hint-1, and as a result the transcription factors are released from their suppression. Thus, transcriptional activity is regulated by Ap(4)A, suggesting that Ap(4)A is a second messenger in this context. For Ap(4)A to be unambiguously established as a second messenger, several criteria have to be fulfilled, including the presence of a metabolizing enzyme. Since several enzymes are able to hydrolyze Ap(4)A, we provided here evidence that the "Nudix" type 2 gene product, Ap(4)A hydrolase, is responsible for Ap(4)A degradation following the immunological activation of mast cells. The knockdown of Ap(4)A hydrolase modulated Ap(4)A accumulation, resulting in changes in the expression of MITF and USF2 target genes. Moreover, our observations demonstrated that the involvement of Ap(4)A hydrolase in gene regulation is not a phenomenon exclusive to mast cells but can also be found in cardiac cells activated with the beta-agonist isoproterenol. Thus, we have provided concrete evidence establishing Ap(4)A as a second messenger in the regulation of gene expression.

  5. Ubiquitin dimers control the hydrolase activity of UCH-L3.

    PubMed

    Setsuie, Rieko; Sakurai, Mikako; Sakaguchi, Yuriko; Wada, Keiji

    2009-01-01

    Ubiquitin (Ub) carboxy terminal hydrolase (UCH)-L1 and UCH-L3 are two of the deubiquitinating enzymes expressed in the brain. Both gad mice, which lack UCH-L1 expression and Uchl3 knockout mice exhibit neurodegeneration, although at distinct areas. These phenotypes indicate the importance of UCH-L1 and UCH-L3 in the regulation of the central nervous system. However, molecular substrates and the molecular regulators of UCH-L1 and UCH-L3 remain poorly identified. Here we show that Ub dimers interact non-covalently with UCH-L3 in vitro and in cells. These interactions were not observed with UCH-L1 in cells. In vitro, K48-linked Ub dimers pronouncedly inhibited the hydrolase activity of UCH-L3, while mono-Ub, a previously identified interacting protein, inhibited the hydrolase activity of UCH-L1. These results indicate that mono-Ub and Ub dimers may regulate the enzymatic functions of UCH-L1 and UCH-L3, respectively, in vivo.

  6. Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

    NASA Astrophysics Data System (ADS)

    Legler, Patricia; Boisvert, Susanne; Compton, Jaimee; Millard, Charles

    2014-07-01

    We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine O?. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. pNBE was tested as a surrogate scaffold for mammalian esterases. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1. We discuss the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases.

  7. Unacylated ghrelin promotes adipogenesis in rodent bone marrow via ghrelin O-acyl transferase and GHS-R1a activity: evidence for target cell-induced acylation

    PubMed Central

    Hopkins, Anna L.; Nelson, Timothy A. S.; Guschina, Irina A.; Parsons, Lydia C.; Lewis, Charlotte L.; Brown, Richard C.; Christian, Helen C.; Davies, Jeffrey S.; Wells, Timothy

    2017-01-01

    Despite being unable to activate the cognate ghrelin receptor (GHS-R), unacylated ghrelin (UAG) possesses a unique activity spectrum that includes promoting bone marrow adipogenesis. Since a receptor mediating this action has not been identified, we re-appraised the potential interaction of UAG with GHS-R in the regulation of bone marrow adiposity. Surprisingly, the adipogenic effects of intra-bone marrow (ibm)-infused acylated ghrelin (AG) and UAG were abolished in male GHS-R-null mice. Gas chromatography showed that isolated tibial marrow adipocytes contain the medium-chain fatty acids utilised in the acylation of UAG, including octanoic acid. Additionally, immunohistochemistry and immunogold electron microscopy revealed that tibial marrow adipocytes show prominent expression of the UAG-activating enzyme ghrelin O-acyl transferase (GOAT), which is located in the membranes of lipid trafficking vesicles and in the plasma membrane. Finally, the adipogenic effect of ibm-infused UAG was completely abolished in GOAT-KO mice. Thus, the adipogenic action of exogenous UAG in tibial marrow is dependent upon acylation by GOAT and activation of GHS-R. This suggests that UAG is subject to target cell-mediated activation – a novel mechanism for manipulating hormone activity. PMID:28361877

  8. Fatty acyl-CoA inhibition of beta-hydroxy-beta-methylglutaryl-CoA reductase activity.

    PubMed

    Faas, F H; Carter, W J; Wynn, J O

    1978-11-22

    The influence of the fatty acyl-CoA thioesters on rat liver microsomal hydroxymethylglutaryl-CoA reductase activity was tested in vitro to determine if the previously demonstrated inhibition of [14C]acetate incorporation into cholesterol is due to inhibition of this rate limiting step in cholesterol synthesis. The polyunsaturated fatty acyl-CoA thioesters caused the greatest inhibition of enzyme activity, 50 micron arachidonoyl-CoA inhibiting 67% and 5 micron inhibiting 22%. 50 micron linoleoyl-CoA inhibited 56% with the more saturated thioesters causing less inhibition. 50--100 micron free fatty acids, free CoA, cholesterol esters, phospholipids, carnitine derivatives, prostaglandins and non-specific detergents caused little or no inhibition of enzyme activity. Kinetic studies revealed the inhibition to be noncompetitive with respect to hydroxymethylglutaryl-CoA with a Ki for arachidonoyl CoA of 3.10 micron. Fatty acyl-CoA inhibition of in vitro cholesterol synthesis is due to inhibition of hydroxymethylglutaryl-CoA reductase activity. Variation in intracellular concentrations of fatty acyl-CoA thioesters may signficantly alter cholesterol synthesis.

  9. Multicomponent synthesis of acylated short peptoids with antifungal activity against plant pathogens.

    PubMed

    Galetti, Matías D; Cirigliano, Adriana M; Cabrera, Gabriela M; Ramírez, Javier A

    2012-02-01

    In this article, we describe the synthesis of a small library of short peptoids composed of four glycine residues and acylated with a fatty acid that showed a remarkable in vitro activity against two fungal plant pathogens. Their straightforward synthesis implied two consecutive Ugi reactions and can be efficiently extended to the construction of highly diverse libraries.

  10. Genome-level and biochemical diversity of the acyl-activating enzyme superfamily in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In higher plants, the superfamily of carboxyl-CoA ligases and related proteins, collectively called acyl activating enzymes (AAEs), has evolved to provide enzymes for many pathways of primary and secondary metabolism and for the conjugation of hormones to amino acids. Across the superfamily there is...

  11. Remodeling Natural Products: Chemistry and Serine Hydrolase Activity of a Rocaglate-Derived β-Lactone

    PubMed Central

    2015-01-01

    Flavaglines are a class of natural products with potent insecticidal and anticancer activities. β-Lactones are a privileged structural motif found in both therapeutic agents and chemical probes. Herein, we report the synthesis, unexpected light-driven di-epimerization, and activity-based protein profiling of a novel rocaglate-derived β-lactone. In addition to in vitro inhibition of the serine hydrolases ABHD10 and ACOT1/2, the most potent β-lactone enantiomer was also found to inhibit these enzymes, as well as the serine peptidases CTSA and SCPEP1, in PC3 cells. PMID:24447064

  12. Structural Milestones in the Reaction Pathway of an Amide Hydrolase: Substrate, Acyl, and Product Complexes of Cephalothin with AmpC [beta]-Lactamase

    SciTech Connect

    Beadle, Beth M.; Trehan, Indi; Focia, Pamela J.; Shoichet, Brian K.

    2010-03-05

    {beta}-lactamases hydrolyze {beta}-lactam antibiotics and are the leading cause of bacterial resistance to these drugs. Although {beta}-lactamases have been extensively studied, structures of the substrate-enzyme and product-enzyme complexes have proven elusive. Here, the structure of a mutant AmpC in complex with the {beta}-lactam cephalothin in its substrate and product forms was determined by X-ray crystallography to 1.53 {angstrom} resolution. The acyl-enzyme intermediate between AmpC and cephalothin was determined to 2.06 {angstrom} resolution. The ligand undergoes a dramatic conformational change as the reaction progresses, with the characteristic six-membered dihydrothiazine ring of cephalothin rotating by 109{sup o}. These structures correspond to all three intermediates along the reaction path and provide insight into substrate recognition, catalysis, and product expulsion.

  13. Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

    SciTech Connect

    Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew; Sadler, Natalie C.; Brown, Robert W.; Smith, Richard D.; Wright, Aaron T.; Grundner, Christoph

    2016-02-01

    The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

  14. A real-time fluorogenic assay for the visualization of glycoside hydrolase activity in planta.

    PubMed

    Ibatullin, Farid M; Banasiak, Alicja; Baumann, Martin J; Greffe, Lionel; Takahashi, Junko; Mellerowicz, Ewa J; Brumer, Harry

    2009-12-01

    There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M(-1) cm(-1)), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-beta-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-beta-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

  15. Organophosphate and Pyrethroid Hydrolase Activities of Mutant Esterases from the Cotton Bollworm Helicoverpa armigera

    PubMed Central

    Li, Yongqiang; Farnsworth, Claire A.; Coppin, Chris W.; Teese, Mark G.; Liu, Jian-Wei; Scott, Colin; Zhang, Xing; Russell, Robyn J.; Oakeshott, John G.

    2013-01-01

    Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes’ active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4–6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively. PMID:24204917

  16. Acylated anthocyanins from sprouts of Raphanus sativus cv. Sango: isolation, structure elucidation and antioxidant activity.

    PubMed

    Matera, Riccardo; Gabbanini, Simone; Berretti, Serena; Amorati, Riccardo; De Nicola, Gina Rosalinda; Iori, Renato; Valgimigli, Luca

    2015-01-01

    Little is known on structure-activity relationships of antioxidant anthocyanins. Raphanus sativus cv Sango sprouts are among the richest sources (270 mg/100 g fresh weight). We isolated from sprouts' juice 9 acylated anthocyanins, including 4 new compounds. All comprise a cyanidin core bearing 3-4 glucose units, multiply acylated with malonic and phenolic acids (ferulic and sinapic). All compounds were equally effective in inhibiting the autoxidation of linoleic acid in aqueous micelles, with rate constant for trapping peroxyl radicals kinh=(3.8 ± 0.7) × 10(4)M(-1)s(-1) at 37 °C. In acetonitrile solution kinh varied with acylation: (0.9-2.1) × 10(5)M(-1)s(-1) at 30 °C. Each molecule trapped a number n of peroxyl radicals ranging from 4 to 7. Anthocyanins bearing sinapic acid were more effective than those bearing the ferulic moiety. Under identical settings, deacylated cyanin, ferulic and sinapic acids had kinh of 0.4 × 10(5), 0.3 × 10(5) and 1.6 × 10(5)M(-1)s(-1) respectively, with n ranging 2-3. Results show the major role of acylation on antioxidant performance.

  17. Nostoc commune UTEX 584 gene expressing indole phosphate hydrolase activity in Escherichia coli.

    PubMed Central

    Xie, W Q; Whitton, B A; Simon, J W; Jäger, K; Reed, D; Potts, M

    1989-01-01

    A gene encoding an enzyme capable of hydrolyzing indole phosphate was isolated from a recombinant gene library of Nostoc commune UTEX 584 DNA in lambda gt10. The gene (designated iph) is located on a 2.9-kilobase EcoRI restriction fragment and is present in a single copy in the genome of N. commune UTEX 584. The iph gene was expressed when the purified 2.9-kilobase DNA fragment, free of any vector sequences, was added to a cell-free coupled transcription-translation system. A polypeptide with an Mr of 74,000 was synthesized when the iph gene or different iph-vector DNA templates were expressed in vitro. When carried by different multicopy plasmids and phagemids (pMP005, pBH6, pB8) the cyanobacterial iph gene conferred an Iph+ phenotype upon various strains of Escherichia coli, including a phoA mutant. Hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate was detected in recombinant E. coli strains grown in phosphate-rich medium, and the activity persisted in assay buffers that contained phosphate. In contrast, indole phosphate hydrolase activity only developed in cells of N. commune UTEX 584, when they were partially depleted of phosphorus, and the activity associated with these cells was suppressed partially by the addition of phosphate to assay buffers. Indole phosphate hydrolase activity was detected in periplasmic extracts from E. coli (Iph+) transformants. Images PMID:2536677

  18. Alteration of the mutagenicity 3,3'-dichlorobenzidine by modifiers of rat hepatic epoxide hydrolase activity

    SciTech Connect

    Iba, M.M.

    1986-03-05

    The involvement of arene oxides in the activation of benzidines was assessed by examining the effect of (I) the epoxide hydrolase inhibitor trichloropropylene oxide (TCPO), (II) purified rat liver microsomal (P) epoxide hydrolase (EH), and (III) pretreatment of rats with phenobarbital (PB) on hepatic Sg- or P-catalyzed mutagenicity of benzidine (BZ) and 3,3'-dichlorobenzidine (DCB) to Salmonella TA 98. When catalyzed by Sg from untreated rats, the mutagenicity of DCB and BZ was 601 +/- 101 and 79 +/- 25 (His/sup +/ revertants/plate) respectively, but was 345 +/- 55 and 226 +/- 30 respectively, when catalyzed by microsomes (P) from untreated rats. PB-pretreatment enhanced the Sg-catalyzed mutagenicity of DCB and BZ (2.3-fold and 1.7-fold, respectively) and the P-catalyzed mutagenicity of DCB (1.7-fold), but totally inhibited the P-catalyzed mutagenicity of BZ. In TCPO-supplemented activating systems from PB-pretreated rats, the mutagenicity of DCB was enhanced in both Sg and P (1.9-fold and 1.6-fold, respectively), whereas that of BZ was unchanged. Added EH enhanced the P-catalyzed mutagenicity of DCB (1.4-fold) but had no effect on that of BZ, suggesting that the activity of the enzyme on DCB metabolites may not be entirely detoxifying. The data suggest that epoxidation may contribute to the activation of DCB but not BZ.

  19. Revised molecular basis of the promiscuous carboxylic acid perhydrolase activity in serine hydrolases.

    PubMed

    Yin, DeLu Tyler; Kazlauskas, Romas J

    2012-06-25

    Several serine hydrolases catalyze a promiscuous reaction: perhydrolysis of carboxylic acids to form peroxycarboxylic acids. The working hypothesis is that perhydrolases are more selective than esterases for hydrogen peroxide over water. In this study, we tested this hypothesis, and focused on L29P-PFE (Pseudomonas fluorescens esterase), which catalyzes perhydrolysis of acetic acid 43-fold faster than wild-type PFE. This hypothesis predicts that L29P-PFE should be approximately 43-fold more selective for hydrogen peroxide than wild-type PFE, but experiments show that L29P-PFE is less selective. The ratio of hydrolysis to perhydrolysis of methyl acetate at different concentrations of hydrogen peroxide fit a kinetic model for nucleophile selectivity. L29P-PFE (β(0)=170  M(-1)) is approximately half as selective for hydrogen peroxide over water than wild-type PFE (β(0)=330  M(-1)), which contradicts the working hypothesis. An alternative hypothesis is that carboxylic acid perhydrolases increase perhydrolysis by forming the acyl-enzyme intermediate faster. Consistent with this hypothesis, the rate of acetyl-enzyme formation, measured by (18)O-water exchange into acetic acid, was 25-fold faster with L29P-PFE than with wild-type PFE, which is similar to the 43-fold faster perhydrolysis with L29P-PFE. Molecular modeling of the first tetrahedral intermediate (T(d)1) suggests that a closer carbonyl group found in perhydrolases accepts a hydrogen bond from the leaving group water. This revised understanding can help design more efficient enzymes for perhydrolysis and shows how subtle changes can create new, unnatural functions in enzymes.

  20. Syntheses and Antibacterial Activity of N-Acylated Ciprofloxacin Derivatives Based on the Trimethyl Lock

    PubMed Central

    2015-01-01

    Several N-acyl ciprofloxacin quinone derivatives based on a trimethyl lock structure were synthesized, and their in vitro antibacterial activity against a panel of clinically relevant bacteria was evaluated. A few new analogues displayed enhanced activity against Gram-positive species compared to the parent drug. Additionally, studies of 8-Cip, which was the most potent compound tested, indicate that it may act through a dual-action mechanism. PMID:26101578

  1. Cold-active hydrolases producing bacteria from two different sub-glacial Himalayan lakes.

    PubMed

    Sahay, Harmesh; Babu, Bandamaravuri Kishore; Singh, Surendra; Kaushik, Rajeev; Saxena, Anil K; Arora, Dilip K

    2013-08-01

    Microorganisms, native to the cold environments have successfully acclimatized their physiological, metabolic, and biological features, exhibiting uniqueness in their enzymes, proteins, and membrane structures. These cold-active enzymes have immense biotechnological potential. The diversity of culturable bacteria in two different water lakes (the sub-glacial freshwater and the brackish) of Himalayas was analyzed using SYBR green staining and cultural methods. A total of 140 bacteria were isolated and were grouped as psychrophiles, psychrotrophs, and psychrotolerant organisms, based on their optimal temperature for growth. The amplified ribosomal DNA restriction analysis using three restriction enzymes facilitated the grouping of these isolates into 96 genotypes at ≥85% polymorphism. Phylogenetic analysis using 16S rRNA gene sequences revealed that the bacterial strains from both lakes belonged to Firmicutes, Proteobacteria (α, β, and γ) or Actinobacteria. Screening of the germplasm for the activity of different cold-active hydrolases such as protease, amylase, xylanase, and cellulase, revealed that about 16 isolates were positive, and exhibiting a wide range of stability at various temperature and pH. Our results suggest that the distinctly different ecosystems of sub-glacial freshwater and brackish water lakes have diverse groups of bacteria, which can be an excellent source of extracellular hydrolases with a wide range of thermal stability.

  2. Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

    PubMed Central

    Legler, Patricia M.; Boisvert, Susanne M.; Compton, Jaimee R.; Millard, Charles B.

    2014-01-01

    We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine Oγ. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1 (hCE1). We discuss the use of pNBE as a surrogate scaffold for the mammalian esterases, and the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases. PMID:25077141

  3. Mfge8 regulates enterocyte lipid storage by promoting enterocyte triglyceride hydrolase activity

    PubMed Central

    Khalifeh-Soltani, Amin; Gupta, Deepti; Ha, Arnold; Iqbal, Jahangir; Hussain, Mahmood; Podolsky, Michael J.

    2016-01-01

    The small intestine has an underappreciated role as a lipid storage organ. Under conditions of high dietary fat intake, enterocytes can minimize the extent of postprandial lipemia by storing newly absorbed dietary fat in cytoplasmic lipid droplets. Lipid droplets can be subsequently mobilized for the production of chylomicrons. The mechanisms that regulate this process are poorly understood. We report here that the milk protein Mfge8 regulates hydrolysis of cytoplasmic lipid droplets in enterocytes after interacting with the αvβ3 and αvβ5 integrins. Mice deficient in Mfge8 or the αvβ3 and αvβ5 integrins accumulate excess cytoplasmic lipid droplets after a fat challenge. Mechanistically, interruption of the Mfge8-integrin axis leads to impaired enterocyte intracellular triglyceride hydrolase activity in vitro and in vivo. Furthermore, Mfge8 increases triglyceride hydrolase activity through a PI3 kinase/mTORC2–dependent signaling pathway. These data identify a key role for Mfge8 and the αvβ3 and αvβ5 integrins in regulating enterocyte lipid processing. PMID:27812539

  4. [Antibacterial Activity of Alkylated and Acylated Derivatives of Low-Molecular Weight Chitosan].

    PubMed

    Shagdarova, B Ts; Il'ina, A V; Varlamov, V P

    2016-01-01

    A number of alkylated (quaternized) and acylated derivatives of low-molecular weight chitosan were obtained. The structure and composition of the compounds were confirmed by the results of IR and PMR spectroscopy, as well as conductometric titration. The effect of the acyl substituent and the degree of substitution of N-(2-hydroxy-3-trimethylammonium) with the propyl fragment appended to amino groups of the C2 atom of polymer chains on antibacterial activity against typical representatives of gram-positive and gram-negative microorganisms (Staphylococcus epidermidis and Escherichia coli) was studied. The highest activity was in the case of N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride with the maximal substitution (98%). The minimal inhibitory concentration of the derivative was 0.48 µg/mL and 3.90 µg/mL for S. epidermis and E. coli, respectively.

  5. Role of phosphatase activity of soluble epoxide hydrolase in regulating simvastatin-activated endothelial nitric oxide synthase.

    PubMed

    Hou, Hsin-Han; Liao, Yi-Jen; Hsiao, Sheng-Huang; Shyue, Song-Kun; Lee, Tzong-Shyuan

    2015-08-25

    Soluble epoxide hydrolase (sEH) has C-terminal epoxide hydrolase and N-terminal lipid phosphatase activity. Its hydrolase activity is associated with endothelial nitric oxide synthase (eNOS) dysfunction. However, little is known about the role of sEH phosphatase in regulating eNOS activity. Simvastatin, a clinical lipid-lowering drug, also has a pleiotropic effect on eNOS activation. However, whether sEH phosphatase is involved in simvastatin-activated eNOS activity remains elusive. We investigated the role of sEH phosphatase activity in simvastatin-mediated activation of eNOS in endothelial cells (ECs). Simvastain increased the phosphatase activity of sEH, which was diminished by pharmacological inhibitors of sEH phosphatase. In addition, pharmacological inhibition of sEH phosphatase or overexpressing the inactive phosphatase domain of sEH enhanced simvastatin-induced NO bioavailability, tube formation and phosphorylation of eNOS, Akt, and AMP-activated protein kinase (AMPK). In contrast, overexpressing the phosphatase domain of sEH limited the simvastatin-increased NO biosynthesis and eNOS phosphorylation at Ser1179. Simvastatin evoked epidermal growth factor receptor-c-Src-increased Tyr phosphorylation of sEH and formation of an sEH-Akt-AMPK-eNOS complex, which was abolished by the c-Src kinase inhibitor PP1 or c-Src dominant-negative mutant K298M. These findings suggest that sEH phosphatase activity negatively regulates simvastatin-activated eNOS by impeding the Akt-AMPK-eNOS signaling cascade.

  6. Role of phosphatase activity of soluble epoxide hydrolase in regulating simvastatin-activated endothelial nitric oxide synthase

    PubMed Central

    Hou, Hsin-Han; Liao, Yi-Jen; Hsiao, Sheng-Huang; Shyue, Song-Kun; Lee, Tzong-Shyuan

    2015-01-01

    Soluble epoxide hydrolase (sEH) has C-terminal epoxide hydrolase and N-terminal lipid phosphatase activity. Its hydrolase activity is associated with endothelial nitric oxide synthase (eNOS) dysfunction. However, little is known about the role of sEH phosphatase in regulating eNOS activity. Simvastatin, a clinical lipid-lowering drug, also has a pleiotropic effect on eNOS activation. However, whether sEH phosphatase is involved in simvastatin-activated eNOS activity remains elusive. We investigated the role of sEH phosphatase activity in simvastatin-mediated activation of eNOS in endothelial cells (ECs). Simvastain increased the phosphatase activity of sEH, which was diminished by pharmacological inhibitors of sEH phosphatase. In addition, pharmacological inhibition of sEH phosphatase or overexpressing the inactive phosphatase domain of sEH enhanced simvastatin-induced NO bioavailability, tube formation and phosphorylation of eNOS, Akt, and AMP-activated protein kinase (AMPK). In contrast, overexpressing the phosphatase domain of sEH limited the simvastatin-increased NO biosynthesis and eNOS phosphorylation at Ser1179. Simvastatin evoked epidermal growth factor receptor–c-Src–increased Tyr phosphorylation of sEH and formation of an sEH–Akt–AMPK–eNOS complex, which was abolished by the c-Src kinase inhibitor PP1 or c-Src dominant-negative mutant K298M. These findings suggest that sEH phosphatase activity negatively regulates simvastatin-activated eNOS by impeding the Akt–AMPK–eNOS signaling cascade. PMID:26304753

  7. Aminoalcohol-Induced Activation of Organophosphorus Hydrolase (OPH) towards Diisopropylfluorophosphate (DFP)

    PubMed Central

    Li, Dandan; Zhang, Yunze; Song, Haitao; Lu, Liangqiu; Liu, Deli; Yuan, Yongze

    2017-01-01

    Aminoalcohols have been addressed as activating buffers for alkaline phosphatase. However, there is no record on the buffer activation regarding organophosphorus hydrolase (OPH). Here we reported the activating effects of aminoalcohols on OPH-catalyzed hydrolysis of diisopropylfluorophosphate (DFP), an analog molecule of G-type warfare agents. The kinetic parametors kcat, Vmax and kcat/Km in the OPH reaction were remarkably increased in the buffers (pH 8.0, 25°C) containing aminoalcohols with C2 between nitrogen (N) and oxygen (O) in their structures, including triethanolamine (TEA), diethanolamine, monoethanolamine, 1-amino-2-propanol, 2-amino-2-methyl-1-propanol, and triisopropanolamine. In contrast, much lower or no rate-enhancing effects were observed in the adding of amines, alcohols, amine/alcohol mixtures, or 3-amino-1-propanol (C3 between N and O). The 300 mM TEA further increased DFP-degrading activities of OPH mutants F132Y and L140Y, the previously reported OPH mutants with desirable activities towards DFP. However, the treatment of ethylenediaminetetraacetate (EDTA) markedly abolished the TEA-induced activation of OPH. The product fluoride effectively inhibited OPH-catalyzed hydrolysis of DFP by a linear mixed inhibition (inhibition constant Ki ~ 3.21 mM), which was partially released by TEA adding at initial or later reaction stage. The obtained results indicate the activation of OPH by aminoalcohol buffers could be attributed to the reduction of fluoride inhibition, which would be beneficial to the hydrolase-based detoxification of organophosphofluoridate. PMID:28085964

  8. Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC

    PubMed Central

    Mavrici, Daniela; Marakalala, Mohlopheni J.; Holton, James M.; Prigozhin, Daniil M.; Gee, Christine L.; Zhang, Yanjia J.; Rubin, Eric J.; Alber, Tom

    2014-01-01

    Bacterial growth and cell division are coordinated with hydrolysis of the peptidoglycan (PG) layer of the cell wall, but the mechanisms of regulation of extracellular PG hydrolases are not well understood. Here we report the biochemical, structural, and genetic analysis of the Mycobacterium tuberculosis homolog of the transmembrane PG-hydrolase regulator, FtsX. The purified FtsX extracellular domain binds the PG peptidase Rv2190c/RipC N-terminal segment, causing a conformational change that activates the enzyme. Deletion of ftsEX and ripC caused similar phenotypes in Mycobacterium smegmatis, as expected for genes in a single pathway. The crystal structure of the FtsX extracellular domain reveals an unprecedented fold containing two lobes connected by a flexible hinge. Mutations in the hydrophobic cleft between the lobes reduce RipC binding in vitro and inhibit FtsX function in M. smegmatis. These studies suggest how FtsX recognizes RipC and support a model in which a conformational change in FtsX links the cell division apparatus with PG hydrolysis. PMID:24843173

  9. Signature motifs identify an Acinetobacter Cif virulence factor with epoxide hydrolase activity.

    PubMed

    Bahl, Christopher D; Hvorecny, Kelli L; Bridges, Andrew A; Ballok, Alicia E; Bomberger, Jennifer M; Cady, Kyle C; O'Toole, George A; Madden, Dean R

    2014-03-14

    Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

  10. Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling.

    PubMed Central

    Faergeman, N J; Knudsen, J

    1997-01-01

    The intracellular concentration of free unbound acyl-CoA esters is tightly controlled by feedback inhibition of the acyl-CoA synthetase and is buffered by specific acyl-CoA binding proteins. Excessive increases in the concentration are expected to be prevented by conversion into acylcarnitines or by hydrolysis by acyl-CoA hydrolases. Under normal physiological conditions the free cytosolic concentration of acyl-CoA esters will be in the low nanomolar range, and it is unlikely to exceed 200 nM under the most extreme conditions. The fact that acetyl-CoA carboxylase is active during fatty acid synthesis (Ki for acyl-CoA is 5 nM) indicates strongly that the free cytosolic acyl-CoA concentration is below 5 nM under these conditions. Only a limited number of the reported experiments on the effects of acyl-CoA on cellular functions and enzymes have been carried out at low physiological concentrations in the presence of the appropriate acyl-CoA-buffering binding proteins. Re-evaluation of many of the reported effects is therefore urgently required. However, the observations that the ryanodine-senstitive Ca2+-release channel is regulated by long-chain acyl-CoA esters in the presence of a molar excess of acyl-CoA binding protein and that acetyl-CoA carboxylase, the AMP kinase kinase and the Escherichia coli transcription factor FadR are affected by low nanomolar concentrations of acyl-CoA indicate that long-chain acyl-CoA esters can act as regulatory molecules in vivo. This view is further supported by the observation that fatty acids do not repress expression of acetyl-CoA carboxylase or Delta9-desaturase in yeast deficient in acyl-CoA synthetase. PMID:9173866

  11. Expression and characterization of an epoxide hydrolase from Anopheles gambiae with high activity on epoxy fatty acids

    PubMed Central

    Xu, Jiawen; Morisseau, Christophe; Hammock, Bruce D.

    2014-01-01

    In insects, epoxide hydrolases (EHs) play critical roles in the metabolism of xenobiotic epoxides from the food resources and in the regulation of endogenous chemical mediators, such as juvenile hormones. Using the baculovirus expression system, we expressed and characterized an epoxide hydrolase from Anopheles gambiae (AgEH) that is distinct in evolutionary history from insect juvenile hormone epoxide hydrolases (JHEHs). We partially purified the enzyme by ion exchange chromatography and isoelectric focusing. The experimentally determined molecular weight and pI were estimated to be 35kD and 6.3 respectively, different than the theoretical ones. The AgEH had the greatest activity on long chain epoxy fatty acids such as 14,15-epoxyeicosatrienoic acids (14,15-EET) and 9,10-epoxy-12Z-octadecenoic acids (9,10-EpOME or leukotoxin) among the substrates evaluated. Juvenile hormone III, a terpenoid insect growth regulator, was the next best substrate tested. The AgEH showed kinetics comparable to the mammalian soluble epoxide hydrolases, and the activity could be inhibited by AUDA [12-(3-adamantan-1-yl-ureido) dodecanoic acid], a urea-based inhibitor designed to inhibit the mammalian soluble epoxide hydrolases. The rabbit serum generated against the soluble epoxide hydrolase of Mus musculus can both cross-react with natural and denatured forms of the AgEH, suggesting immunologically they are similar. The study suggests there are mammalian sEH homologs in insects, and epoxy fatty acids may be important chemical mediators in insects. PMID:25173592

  12. A calcium-dependent acyltransferase that produces N-acyl phosphatidylethanolamines

    PubMed Central

    Ogura, Yuji; Parsons, William H.; Kamat, Siddhesh S.; Cravatt, Benjamin F.

    2016-01-01

    More than 30 years ago, a calcium-dependent enzyme activity was described that generates N-acyl phosphatidylethanolamines (NAPEs), which are precursors for N-acyl ethanolamine (NAE) lipid transmitters, including the endocannabinoid anandamide. The identity of this calcium-dependent N-acyltransferase (Ca-NAT) has remained mysterious. Here, we use activity-based protein profiling to identify the poorly characterized serine hydrolase PLA2G4E as a mouse brain Ca-NAT and show that this enzyme generates NAPEs and NAEs in mammalian cells. PMID:27399000

  13. Acylated pregnane glycosides from Caralluma tuberculata and their antiparasitic activity.

    PubMed

    Abdel-Sattar, Essam; Harraz, Fathalla M; Al-ansari, Soliman Mohammed Abdullah; El-Mekkawy, Sahar; Ichino, Chikara; Kiyohara, Hiroaki; Ishiyama, Aki; Otoguro, Kazuhiko; Omura, Satoshi; Yamada, Haruki

    2008-08-01

    Five pregnane glycosides were isolated from Caralluma tuberculata (1-5), in addition to a known one (russelioside E, 6). The structures of the isolated compounds were elucidated by the analysis of NMR data and FAB-MS experiments. All the isolated compounds were tested for their antimalarial and antitrypanosomal activities as well as their cytotoxicity against human diploid embryonic cell line (MRC5).

  14. Acylated flavonol glycosides from Tagetes minuta with antibacterial activity.

    PubMed

    Shahzadi, Irum; Shah, Mohammad M

    2015-01-01

    Wild marigold (Tagetes minuta), a flowering plant of the family Asteraceae contains compounds of pharmaceutical and nutritional importance especially essential oils and flavonols. Identification, characterization of flavonols and determination of their antibacterial activity were major objectives of the current study. The isolation and purification of flavonols was accomplished using chromatographic techniques while structural elucidation was completed by LC-MS and NMR spectroscopy. The extracts and purified compounds were tested against various bacterial strains for antibacterial activity. A total of 19 flavonols were isolated from this species. Of these, 17 were of butanol and two of ethyl acetate extracts. Based on the concentration and purity, eight potential flavonols were selected and structurally elucidated. Four flavonols, 6-hydroxyquercetin 7-O-β-(6''-galloylglucopyranoside; 2), 6-hydroxykaempferol 7-O-β-glucopyranoside (5), 6-hydroxykaempferol 7-O-β-(6''-galloylglucopyranoside; 7), 6-hydroxyquercetin 7-O-β-(6''-caffeoylglucopyranoside; 9), were identified for the first time from T. minuta. Butanol and ethyl acetate extracts of flowers and seeds showed significant antibacterial activity against Micrococcus leteus, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas pikettii. Among the isolated flavonols only 1, 2, and 18 were found to possess significant antibacterial activity against M. luteus. The extracts and purified flavonols from T. minuta can be potential candidates for antibacterial drug discovery and support to ethnopharmacological use.

  15. Acylated flavone glycosides from the roots of Saussurea lappa and their antifungal activity.

    PubMed

    Rao, Kolisetty Sambasiva; Babu, Goriparthi Venu; Ramnareddy, Yemireddy Venkata

    2007-03-07

    The isolation of four novel acylated flavonoid glycosides from the roots of Saussurea lappa and their identification using a combination of 1D and 2D NMR and mass spectrometry is described. The in vitro antifungal and antibacterial activities of the isolated compounds and their mixture were tested on nine fungal and four bacterial strains, using the microdilution method. The compounds and mixture showed moderate to high antifungal activity against most of the fungi tested, compared to a miconazole standard, while only one compound and the mixture showed antibacterial activity against all strains tested.

  16. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    ERIC Educational Resources Information Center

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  17. Soluble Epoxide Hydrolase Inhibitory Activity of Selaginellin Derivatives from Selaginella tamariscina.

    PubMed

    Kim, Jang Hoon; Cho, Chong Woon; Tai, Bui Huu; Yang, Seo Young; Choi, Gug-Seoun; Kang, Jong Seong; Kim, Young Ho

    2015-12-02

    Selaginellin derivatives 1-3 isolated from Selaginella tamariscina were evaluated for their inhibition of soluble epoxide hydrolase (sEH) to demonstrate their potential for the treatment of cardiovascular disease. All selaginellin derivatives (1-3) inhibited sEH enzymatic activity and PHOME hydrolysis, in a dose-dependent manner, with IC50 values of 3.1 ± 0.1, 8.2 ± 2.2, and 4.2 ± 0.2 μM, respectively. We further determined that the derivatives function as non-competitive inhibitors. Moreover, the predicted that binding sites and interaction between 1-3 and sEH were solved by docking simulations. According to quantitative analysis, 1-3 were confirmed to have high content in the roots of S. tamariscina; among them, selaginellin 3 exhibited the highest content of 189.3 ± 0.0 μg/g.

  18. A glycoside hydrolase family 31 dextranase with high transglucosylation activity from Flavobacterium johnsoniae.

    PubMed

    Gozu, Yoshifumi; Ishizaki, Yuichi; Hosoyama, Yuhei; Miyazaki, Takatsugu; Nishikawa, Atsushi; Tonozuka, Takashi

    2016-08-01

    Glycoside hydrolase family (GH) 31 enzymes exhibit various substrate specificities, although the majority of members are α-glucosidases. Here, we constructed a heterologous expression system of a GH31 enzyme, Fjoh_4430, from Flavobacterium johnsoniae NBRC 14942, using Escherichia coli, and characterized its enzymatic properties. The enzyme hydrolyzed dextran and pullulan to produce isomaltooligosaccharides and isopanose, respectively. When isomaltose was used as a substrate, the enzyme catalyzed disproportionation to form isomaltooligosaccharides. The enzyme also acted, albeit inefficiently, on p-nitrophenyl α-D-glucopyranoside, and p-nitrophenyl α-isomaltoside was the main product of the reaction. In contrast, Fjoh_4430 did not act on trehalose, kojibiose, nigerose, maltose, maltotriose, or soluble starch. The optimal pH and temperature were pH 6.0 and 60 °C, respectively. Our results indicate that Fjoh_4430 is a novel GH31 dextranase with high transglucosylation activity.

  19. The effects of modulation of microsomal epoxide hydrolase activity on microsome-catalyzed activation of benzo[alpha]pyrene and its covalent binding to DNA.

    PubMed

    Guenthner, T M; Oesch, F

    1981-01-01

    The effects of modulation of microsomal epoxide hydrolase activity on the binding of calf thymus DNA of benzo[alpha]pyrene metabolically activated by rat liver microsomes were investigated. In systems where microsomal epoxide hydrolase levels were not manipulated, 2 major bound species, one derived from 9-hydroxybenzo[alpha]pyrene and the other derived from benzo[alpha]pyrene 7,8-dihydrodiol, were found in approximately equivalent amounts. When epoxide hydrolase levels were increased, either by addition in vitro of purified enzyme or by induction in vivo by trans-stilbene oxide, the binding of the benzo[alpha]pyrene 7,8-dihydrodiol product was increased, while the binding of the 9-hydroxybenzo[alpha]pyrene product was practically eliminated. When microsomal epoxide hydrolase activity was decreased by selective inhibition with low concentrations of 1,1,1-trichloropropene 2,3-oxide, the binding of the species derived from 9-hydroxybenzo[alpha]pyrene was increased several-fold, while that of the species derived from benzo[alpha]pyrene 7,8-dihydrodiol was greatly decreased. The results indicate that the binding species derived from 9-hydroxybenzo[alpha]pyrene is formed through a metabolic pathway leading to an epoxide which is a substrate of microsomal epoxide hydrolase and that microsomal epoxide hydrolase is important in regulating the pattern of binding of individual microsomally-formed benzo[alpha]pyrene metabolites to DNA.

  20. Formation of acylated growth hormone-releasing peptide-6 by poly(lactide-co-glycolide) and its biological activity.

    PubMed

    Na, Dong Hee; Lee, Jeong Eun; Jang, Sun Woo; Lee, Kang Choon

    2007-06-08

    The purpose of this study was to investigate the formation of acylated impurity resulting from a chemical reaction between the growth hormone-releasing peptide-6 (GHRP-6) and poly(lactide-co-glycolide) (PLGA) and the effect of peptide acylation on the in vivo biological activity of GHRP-6. The peptide acylation pattern of GHRP-6 by hydrophilic PLGA polymers with different molecular weights was characterized by reversed-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Higher levels of acylated GHRP-6 were produced with the higher molecular weight PLGA, which might be due to the slower degradation rate of the polymer. The evaluation of the biological activity in rats showed that the acylated GHRP-6 had a much lower activity than the intact GHRP-6. This finding suggests that the acylation reaction would decrease the effectiveness of the GHRP-6 formulation such as PLGA microspheres. Therefore, a strategy for stabilizing the GHRP-6 will be necessary for the development of a successful formulation of PLGA microspheres.

  1. Generation of fatty acids by an acyl esterase in the bioluminescent system of Photobacterium phosphoreum

    SciTech Connect

    Carey, L.M.; Rodriguez, A.; Meighen, E.

    1984-08-25

    The fatty acid reductase complex from Photobacterium phosphoreum has been discovered to have a long chain ester hydrolase activity associated with the 34K protein component of the complex. This protein has been resolved from the other components (50K and 58K) of the fatty acid reductase complex with a purity of > 95% and found to catalyze the transfer of acyl groups from acyl-CoA primarily to thiol acceptors with a low level of transfer to glycerol and water. Addition of the 50K protein of the complex caused a dramatic change in specificity increasing the transfer to oxygen acceptors. The acyl-CoA hydrolase activity increased almost 10-fold, and hence free fatty acids can be generated by the 34K protein when it is present in the fatty acid reductase complex. Hydrolysis of acyl-S-mercaptoethanol and acyl-1-glycerol and the ATP-dependent reduction of the released fatty acids to aldehyde for the luminescent reaction were also demonstrated for the reconstituted fatty acid reductase complex, raising the possibility that the immediate source of fatty acids for this reaction in vivo could be the membrane lipids and/or the fatty acid synthetase system.

  2. Synthesis of Phenoxyacyl-Ethanolamides and Their Effects on Fatty Acid Amide Hydrolase Activity*

    PubMed Central

    Faure, Lionel; Nagarajan, Subbiah; Hwang, Hyeondo; Montgomery, Christa L.; Khan, Bibi Rafeiza; John, George; Koulen, Peter; Blancaflor, Elison B.; Chapman, Kent D.

    2014-01-01

    N-Acylethanolamines (NAEs) are involved in numerous biological activities in plant and animal systems. The metabolism of these lipids by fatty acid amide hydrolase (FAAH) is a key regulatory point in NAE signaling activity. Several active site-directed inhibitors of FAAH have been identified, but few compounds have been described that enhance FAAH activity. Here we synthesized two sets of phenoxyacyl-ethanolamides from natural products, 3-n-pentadecylphenolethanolamide and cardanolethanolamide, with structural similarity to NAEs and characterized their effects on the hydrolytic activity of FAAH. Both compounds increased the apparent Vmax of recombinant FAAH proteins from both plant (Arabidopsis) and mammalian (Rattus) sources. These NAE-like compounds appeared to act by reducing the negative feedback regulation of FAAH activity by free ethanolamine. Both compounds added to seedlings relieved, in part, the negative growth effects of exogenous NAE12:0. Cardanolethanolamide reduced neuronal viability and exacerbated oxidative stress-mediated cell death in primary cultured neurons at nanomolar concentrations. This was reversed by FAAH inhibitors or exogenous NAE substrate. Collectively, our data suggest that these phenoxyacyl-ethanolamides act to enhance the activity of FAAH and may stimulate the turnover of NAEs in vivo. Hence, these compounds might be useful pharmacological tools for manipulating FAAH-mediated regulation of NAE signaling in plants or animals. PMID:24558037

  3. Isolation and characterization of an extracellular glycosylated protein complex from Clostridium thermosaccharolyticum with pectin methylesterase and polygalacturonate hydrolase activity.

    PubMed Central

    Van Rijssel, M; Gerwig, G J; Hansen, T A

    1993-01-01

    An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous culture of Clostridium thermosaccharolyticum Haren. The complex possessed both pectin methylesterase (EC 3.1.1.11) and exo-poly-alpha-galacturonate hydrolase (EC 3.2.1.82) activity and produced digalacturonate from the nonreducing end of the pectin chain. The protein consisted of 230- and 25-kDa subunits. The large subunit contained 10% (wt/wt) sugars (N-acetylgalactosamine and galactose). Under physiological conditions both activities acted in a coordinated manner: the ratio between methanol and digalacturonate released during degradation was constant and equal to the degree of esterification of the pectin used. Prolonged incubation of the enzyme with pectin led to a nondialyzable fraction that was enriched in neutral sugars, such as arabinose, rhamnose, and galactose; the high rhamnose/galacturonic acid ratio was indicative of hairy region-like structures. The smallest substrate utilized by the hydrolase was a tetragalacturonate. Vmax with oligogalacturonates increased with increasing chain length. The Km and Vmax for the polygalacturonate hydrolase with citrus pectate as a substrate were 0.8 g liter-1 and 180 mumol min-1 mg of protein-1, respectively. The Km and Vmax for the esterase with citrus pectin as a substrate were 1.2 g liter-1 and 440 mumol min-1 mg of protein-1, respectively. The temperature optima for the hydrolase and esterase were 70 and 60 degrees C, respectively. Both enzyme activities were stable for more than 1 h at 70 degrees C. The exo-polygalacturonate hydrolase of Clostridium thermosulfurogenes was partially purified while the methylesterase was also copurified. Images PMID:8481009

  4. Novel endogenous N-acyl amides activate TRPV1-4 receptors, BV-2 microglia, and are regulated in brain in an acute model of inflammation

    PubMed Central

    Raboune, Siham; Stuart, Jordyn M.; Leishman, Emma; Takacs, Sara M.; Rhodes, Brandon; Basnet, Arjun; Jameyfield, Evan; McHugh, Douglas; Widlanski, Theodore; Bradshaw, Heather B.

    2014-01-01

    A family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (Anandamide), and called N-acyl amides have emerged as a family of biologically active compounds at TRP receptors. N-acyl amides are constructed from an acyl group and an amine via an amide bond. This same structure can be modified by changing either the fatty acid or the amide to form potentially hundreds of lipids. More than 70 N-acyl amides have been identified in nature. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, using a unique in-house library of over 70 N-acyl amides we tested the following three hypotheses: (1) Additional N-acyl amides will have activity at TRPV1-4, (2) Acute peripheral injury will drive changes in CNS levels of N-acyl amides, and (3) N-acyl amides will regulate calcium in CNS-derived microglia. Through these studies, we have identified 20 novel N-acyl amides that collectively activate (stimulating or inhibiting) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we showed that levels of at least 10 of these N-acyl amides that activate TRPVs are regulated in brain after intraplantar carrageenan injection. We then screened the BV2 microglial cell line for activity with this N-acyl amide library and found overlap with TRPV receptor activity as well as additional activators of calcium mobilization from these lipids. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at ion channels, in microglia, and in the brain in the context of inflammation. PMID:25136293

  5. Characterization of multiple epoxide hydrolase activities in mouse liver nuclear envelope.

    PubMed

    Guenthner, T M

    1986-10-01

    A nuclear envelope-associated epoxide hydrolase in mouse liver that hydrates trans-stilbene oxide has been identified and characterized. This epoxide hydrolase is distinct from the enzyme in nuclear envelopes that hydrates benzo[a]pyrene 4,5-oxide and other arene oxides. This distinction was demonstrated by the criteria of pH optima, response to specific inhibitors in vitro, and precipitation by specific antibodies. The new epoxide hydrolase had a pH optimum of 6.8, was poorly inhibited by trichloropropene oxide, was potently inhibited by 4-phenylchalcone oxide, and did not bind to antiserum against benzo[a]pyrene 4,5-oxide hydrolase. This nuclear enzyme is similar in many of its properties to cytosolic and microsomal trans-stilbene oxide hydrolases and may be nuclear envelope-bound form of these other epoxide hydrolases. It differed from these other trans-stilbene oxide hydrolases in that its affinities for both trans-stilbene oxide (measured as apparent Km) and 4-phenylchalcone oxide (measured as I50) were 4- to 20-fold lower than those of either the cytosolic or microsomal forms.

  6. A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation.

    PubMed

    Pucher, Marion E; Steiger, Matthias G; Mach, Robert L; Mach-Aigner, Astrid R

    2011-06-10

    Hypocrea jecorina (anamorph Trichoderma reesei) is a saprophytic fungus that produces hydrolases, which are applied in different types of industries and used for the production of biofuel. A recombinant Hypocrea strain, which constantly expresses the main transcription activator of hydrolases (Xylanase regulator 1), was found to grow faster on xylan and its monomeric backbone molecule d-xylose. This strain also showed improved ability of clearing xylan medium on plates. Furthermore, this strain has a changed transcription profile concerning genes encoding for hydrolases and enzymes associated with degradation of (hemi)celluloses. We demonstrated that enzymes of this strain from a xylan cultivation favoured break down of hemicelluloses to the monomer d-xylose compared to the parental strain, while the enzymes of the latter one formed more xylobiose. Applying supernatants from cultivation on carboxymethylcellulose in enzymatic conversion of hemicelluloses, the enzymes of the recombinant strain were clearly producing more of both, d-xylose and xylobiose, compared to the parental strain. Altogether, these results point to a changed hydrolase expression profile, an enhanced capability to form the xylan-monomer d-xylose and the assumption that there is a disordered induction pattern if the Xylanase regulator 1 is de-regulated in Hypocrea.

  7. Balancing the stability and the catalytic specificities of OP hydrolases with enhanced V-agent activities.

    PubMed

    Reeves, T E; Wales, M E; Grimsley, J K; Li, P; Cerasoli, D M; Wild, J R

    2008-06-01

    Rational site-directed mutagenesis and biophysical analyses have been used to explore the thermodynamic stability and catalytic capabilities of organophosphorus hydrolase (OPH) and its genetically modified variants. There are clear trade-offs in the stability of modifications that enhance catalytic activities. For example, the H254R/H257L variant has higher turnover numbers for the chemical warfare agents VX (144 versus 14 s(-1) for the native enzyme (wild type) and VR (Russian VX, 465 versus 12 s(-1) for wild type). These increases are accompanied by a loss in stability in which the total Gibb's free energy for unfolding is 19.6 kcal/mol, which is 5.7 kcal/mol less than that of the wild-type enzyme. X-ray crystallographic studies support biophysical data that suggest amino acid residues near the active site contribute to the chemical and thermal stability through hydrophobic and cation-pi interactions. The cation-pi interactions appear to contribute an additional 7 kcal/mol to the overall global stability of the enzyme. Using rational design, it has been possible to make amino acid changes in this region that restored the stability, yet maintained effective V-agent activities, with turnover numbers of 68 and 36 s(-1) for VX and VR, respectively. This study describes the first rationally designed, stability/activity balance for an OPH enzyme with a legitimate V-agent activity, and its crystal structure.

  8. The soluble epoxide hydrolase determines cholesterol homeostasis by regulating AMPK and SREBP activity.

    PubMed

    Mangels, Nicole; Awwad, Khader; Wettenmann, Annika; Dos Santos, Laila Romagueira Bichara; Frömel, Timo; Fleming, Ingrid

    2016-09-01

    Inhibition or deletion of the soluble epoxide hydrolase (sEH) has been linked to reduced cholesterol and protection against atherosclerosis. This study set out to identify sEH substrate(s) or product(s), altered in livers from sEH(-/-) mice that contribute to these beneficial effects. In livers and isolated hepatocytes, deletion of sEH decreased expression of HMG CoA reductase, fatty acid synthase and low density lipoprotein receptor. Sterol regulatory element binding proteins (SREBPs) regulate the expression of all three enzymes and SREBP activation was attenuated in the absence of sEH. The effect was attributed to the AMPK-activated protein kinase (AMPK) which was activated in the absence of sEH. Livers from wild-type versus sEH(-/-) littermates contained significantly higher levels of the sEH substrate 12,13-epoxyoctadecenoic acid, which elicited AMPK activation, while the corresponding sEH product was inactive. Thus, AMPK activation and subsequent inhibition of SREBP can account for the altered expression of lipid metabolizing enzymes in sEH(-/-) mice.

  9. Characterisation of the organophosphate hydrolase catalytic activity of SsoPox

    PubMed Central

    Hiblot, Julien; Gotthard, Guillaume; Chabriere, Eric; Elias, Mikael

    2012-01-01

    SsoPox is a lactonase endowed with promiscuous phosphotriesterase activity isolated from Sulfolobus solfataricus that belongs to the Phosphotriesterase-Like Lactonase family. Because of its intrinsic thermal stability, SsoPox is seen as an appealing candidate as a bioscavenger for organophosphorus compounds. A comprehensive kinetic characterisation of SsoPox has been performed with various phosphotriesters (insecticides) and phosphodiesters (nerve agent analogues) as substrates. We show that SsoPox is active for a broad range of OPs and remains active under denaturing conditions. In addition, its OP hydrolase activity is highly stimulated by anionic detergent at ambient temperature and exhibits catalytic efficiencies as high as kcat/KM of 105 M−1s−1 against a nerve agent analogue. The structure of SsoPox bound to the phosphotriester fensulfothion reveals an unexpected and non-productive binding mode. This feature suggests that SsoPox's active site is sub-optimal for phosphotriester binding, which depends not only upon shape but also on localised charge of the ligand. PMID:23139857

  10. Colloid-based multiplexed method for screening plant biomass-degrading glycoside hydrolase activities in microbial communities

    SciTech Connect

    Reindl, W.; Deng, K.; Gladden, J.M.; Cheng, G.; Wong, A.; Singer, S.W.; Singh, S.; Lee, J.-C.; Yao, J.-S.; Hazen, T.C.; Singh, A.K; Simmons, B.A.; Adams, P.D.; Northen, T.R.

    2011-05-01

    The enzymatic hydrolysis of long-chain polysaccharides is a crucial step in the conversion of biomass to lignocellulosic biofuels. The identification and characterization of optimal glycoside hydrolases is dependent on enzyme activity assays, however existing methods are limited in terms of compatibility with a broad range of reaction conditions, sample complexity, and especially multiplexity. The method we present is a multiplexed approach based on Nanostructure-Initiator Mass Spectrometry (NIMS) that allowed studying several glycolytic activities in parallel under diverse assay conditions. Although the substrate analogs carried a highly hydrophobic perfluorinated tag, assays could be performed in aqueous solutions due colloid formation of the substrate molecules. We first validated our method by analyzing known {beta}-glucosidase and {beta}-xylosidase activities in single and parallel assay setups, followed by the identification and characterization of yet unknown glycoside hydrolase activities in microbial communities.

  11. The Structural Basis of Exopolygalacturonase Activity in a Family 28 Glycoside Hydrolase

    SciTech Connect

    Abbott,D.; Boraston, A.

    2007-01-01

    Family 28 glycoside hydrolases (polygalacturonases) are found in organisms across the plant, fungal and bacterial kingdoms, where they are central to diverse biological functions such as fruit ripening, biomass recycling and plant pathogenesis. The structures of several polygalacturonases have been reported; however, all of these enzymes utilize an endo-mode of digestion, which generates a spectrum of oligosaccharide products with varying degrees of polymerization. The structure of a complementary exo-acting polygalacturonase and an accompanying explanation of the molecular determinants for its specialized activity have been noticeably lacking. We present the structure of an exopolygalacturonase from Yersinia enterocolitica, YeGH28 in a native form (solved to 2.19 {angstrom} resolution) and a digalacturonic acid product complex (solved to 2.10 {angstrom} resolution). The activity of YeGH28 is due to inserted stretches of amino acid residues that transform the active site from the open-ended channel observed in the endopolygalacturonases to a closed pocket that restricts the enzyme to the exclusive attack of the non-reducing end of oligogalacturonide substrates. In addition, YeGH28 possesses a fused FN3 domain with unknown function, the first such structure described in pectin active enzymes.

  12. N-terminal domain of soluble epoxide hydrolase negatively regulates the VEGF-mediated activation of endothelial nitric oxide synthase

    PubMed Central

    Hou, Hsin-Han; Hammock, Bruce D.; Su, Kou-Hui; Morisseau, Christophe; Kou, Yu Ru; Imaoka, Susumu; Oguro, Ami; Shyue, Song-Kun; Zhao, Jin-Feng; Lee, Tzong-Shyuan

    2012-01-01

    Aims The mammalian soluble epoxide hydrolase (sEH) has both an epoxide hydrolase and a phosphatase domain. The role of sEH hydrolase activity in the metabolism of epoxyeicosatrienoic acids (EETs) and the activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) has been well defined. However, far less is known about the role of sEH phosphatase activity in eNOS activation. In the present study, we investigated whether the phosphatase domain of sEH was involved in the eNOS activation in ECs. Methods and results The level of eNOS phosphorylation in aortas is higher in the sEH knockout (sEH−/−) mice than in wild-type mice. In ECs, pharmacological inhibition of sEH phosphatase or overexpressing sEH with an inactive phosphatase domain enhanced vascular endothelial growth factor (VEGF)-induced NO production and eNOS phosphorylation. In contrast, overexpressing the phosphatase domain of sEH prevented the VEGF-mediated NO production and eNOS phosphorylation at Ser617, Ser635, and Ser1179. Additionally, treatment with VEGF induced a c-Src kinase-dependent increase in transient tyrosine phosphorylation of sEH and the formation of a sEH–eNOS complex, which was abolished by treatment with a c-Src kinase inhibitor, PP1, or the c-Src dominant-negative mutant K298M. We also demonstrated that the phosphatase domain of sEH played a key role in VEGF-induced angiogenesis by detecting the tube formation in ECs and neovascularization in Matrigel plugs in mice. Conclusion In addition to epoxide hydrolase activity, phosphatase activity of sEH plays a pivotal role in the regulation of eNOS activity and NO-mediated EC functions. PMID:22072631

  13. Targeted acylation for all the hydroxyls of (+)-catechin and evaluation of their individual contribution to radical scavenging activity.

    PubMed

    Hong, Shan; Liu, Songbai

    2016-04-15

    The reactivity profile of all the hydroxyl groups in (+)-catechin towards acylation and their respective contribution to radical scavenging activity were systematically explored in this work. Selective acylation of the hydroxyls on different rings was carried out employing either a basic or acidic activation strategy. Monoacylation of B ring was achieved effectively with the aid of dimethyltin dichloride. Monoacylation of A ring was accomplished by sequential protection and deprotection of B and C rings. Based on specific acylation of all the individual hydroxyls of (+)-catechins, the structure radical scavenging activity relationship of each hydroxyl of (+)-catechin was established. It was demonstrated that the vicinal phenolic hydroxyls on B ring played the most important role in the ABTS radical scavenging activity and those on A and C rings made a much smaller contribution. This study has laid solid groundwork for further modification of the catechins and improvement of their properties.

  14. Oxidative acylation using thioacids

    NASA Technical Reports Server (NTRS)

    Liu, R.; Orgel, L. E.

    1997-01-01

    Several important prebiotic reactions, including the coupling of amino acids into polypeptides by the formation of amide linkages, involve acylation. Theae reactions present a challenge to the understanding of prebiotic synthesis. Condensation reactions relying on dehydrating agents are either inefficient in aqueous solution or require strongly acidic conditions and high temperatures. Activated amino acids such as thioester derivatives have therefore been suggested as likely substrates for prebiotic peptide synthesis. Here we propose a closely related route to amide bond formation involving oxidative acylation by thioacids. We find that phenylalanine, leucine and phenylphosphate are acylated efficiently in aqueous solution by thioacetic acid and an oxidizing agent. From a prebiotic point of view, oxidative acylation has the advantage of proceeding efficiently in solution and under mild conditions. We anticipate that oxidative acylation should prove to be a general method for activating carboxylic acids, including amino acids.

  15. A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.

    PubMed

    Kage, Karen L; Richardson, Paul L; Traphagen, Linda; Severin, Jean; Pereda-Lopez, Ana; Lubben, Thomas; Davis-Taber, Rachel; Vos, Melissa H; Bartley, Diane; Walter, Karl; Harlan, John; Solomon, Larry; Warrior, Usha; Holzman, Thomas F; Faltynek, Connie; Surowy, Carol S; Scott, Victoria E

    2007-03-30

    Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC). This assay gives an excellent signal window for measuring FAAH activity and, as a continuous assay, inherently offers improved sensitivity and accuracy over previously reported endpoint assays. The assay was validated using a panel of known FAAH inhibitors and purified recombinant human FAAH, then converted to a 384 well format and used to screen a large library of compounds (>600,000 compounds) to identify FAAH inhibitors. This screen identified numerous novel FAAH inhibitors of diverse chemotypes. These hits confirmed using a native FAAH substrate, anandamide, and had very similar rank order potency to that obtained using the D-AMC substrate. Collectively these data demonstrate that D-AMC can be successfully used to rapidly and effectively identify novel FAAH inhibitors for potential therapeutic use.

  16. Identification, quantification and antioxidant activity of acylated flavonol glycosides from sea buckthorn (Hippophae rhamnoides ssp. sinensis).

    PubMed

    Chen, Chu; Xu, Xue-Min; Chen, Yang; Yu, Meng-Yao; Wen, Fei-Yan; Zhang, Hao

    2013-12-01

    A novel acylated flavonol glycoside: isorhamnetin (3-O-[(6-O-E-sinapoyl)-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside) (1), together with two known acylated flavonol glycosides: quercetin (3-O-[(6-O-E-sinapoyl)-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside) (2) and kaempferol (3-O-[(6-O-E-sinapoyl)-β-D-glucopyranosyl-(1→2)]-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside) (3) were isolated from the n-butanol fraction of sea buckthorn (Hippophae rhamnoides ssp. sinensis) berries for the first time by chromatographic methods, and their structures were elucidated using UV, MS, (1)H and (13)C NMR, and 2D NMR. Compounds 1-3 showed good scavenging activities, with respective IC50 values of 8.91, 4.26 and 30.90 μM toward the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical; respective Trolox equivalent antioxidant capacities of 2.89, 4.04 and 2.44 μM μM(-1) toward 2,2'-azino-bis-3-ethyl-benzothiazoline-6-sulphonate (ABTS) radical. The quantitative analysis of the isolated acylated flavonol glycosides was performed by HPLC-DAD method. The contents of compounds 1-3 were in the range of 12.2-31.4, 4.0-25.3, 7.5-59.7 mg/100 g dried berries and 9.1-34.5, 75.1-182.1, 29.2-113.4 mg/100 g dried leaves, respectively.

  17. First Glycoside Hydrolase Family 2 Enzymes from Thermus antranikianii and Thermus brockianus with β-Glucosidase Activity

    PubMed Central

    Schröder, Carola; Blank, Saskia; Antranikian, Garabed

    2015-01-01

    Two glycoside hydrolase encoding genes (tagh2 and tbgh2) were identified from different Thermus species using functional screening. Based on amino acid similarities, the enzymes were predicted to belong to glycoside hydrolase (GH) family 2. Surprisingly, both enzymes (TaGH2 and TbGH2) showed twofold higher activities for the hydrolysis of nitrophenol-linked β-D-glucopyranoside than of -galactopyranoside. Specific activities of 3,966 U/mg for TaGH2 and 660 U/mg for TbGH2 were observed. In accordance, Km values for both enzymes were significantly lower when β-D-glucopyranoside was used as substrate. Furthermore, TaGH2 was able to hydrolyze cellobiose. TaGH2 and TbGH2 exhibited highest activity at 95 and 90°C at pH 6.5. Both enzymes were extremely thermostable and showed thermal activation up to 250% relative activity at temperatures of 50 and 60°C. Especially, TaGH2 displayed high tolerance toward numerous metal ions (Cu2+, Co2+, Zn2+), which are known as glycoside hydrolase inhibitors. In this study, the first thermoactive GH family 2 enzymes with β-glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to other enzymes of GH family 2. Our work contributes to a broader knowledge of substrate specificities in GH family 2. PMID:26090361

  18. Erythrocyte L-aspartyl-L-phenylalanine hydrolase activity and plasma phenylalanine and aspartate concentrations in children consuming diets high in aspartame.

    PubMed

    Stegink, L D; Lindgren, S D; Brummel, M C; Stumbo, P J; Wolraich, M L

    1995-12-01

    A deficit of alpha-aspartyl-phenylalanine (alpha-Asp-Phe) hydrolase activity has been suggested as a cause of possible adverse effects of aspartame ingestion. Twenty-five normal preschool children and 23 school-age children described by their parents as sensitive to sugar were fed diets high in sucrose, aspartame, or saccharin for three successive 3-wk periods. Blood samples were obtained at baseline (fasting) and within the last 3 d of each dietary period (postprandial). alpha-Asp-Phe concentrations were below detection limits (0.5 mumol/L) in all plasma samples and Phe and Asp concentrations remained within normal limits, alpha-Asp-Phe hydrolase activities in baseline hemolysate samples did not differ between groups. One subject had a plasma alpha-Asp-Phe hydrolase activity > 2 SD below the mean. Despite this low activity, this subject did not show consistent cognitive or behavioral anomalies that could be linked to low hydrolase activity.

  19. Colorimetric assay for S-adenosylhomocysteine hydrolase activity and inhibition using fluorosurfactant-capped gold nanoparticles.

    PubMed

    Lin, Jia-Hui; Chang, Chung-Wei; Wu, Zong-Han; Tseng, Wei-Lung

    2010-11-01

    This study reports a simple colorimetric method for the sensitive detection of S-adenosylhomocysteine hydrolase (SAHH) activity and inhibition using fluorosurfactant-capped gold nanoparticles (FSN-AuNPs). FSN stabilizes the AuNPs against conditions of high ionic strength, and FSN-AuNPs are merely aggregated in the presence of homocysteine (HCys) and cysteine. Because of this feature, FSN-AuNPs were found to be dispersed in the presence of S-adenosylhomocysteine (SAH) that lacks a free thiol group. After SAHH catalyzed the hydrolysis of SAH, the produced HCys molecules were bound to the surface of AuNPs through the formation of Au-S bonds. As a result, the nanoparticle (NP) aggregation occurred through electrostatic attraction between each HCys-attached AuNP. This approach had a minimum detectable concentration of 100 units/L (~6 nM). Additionally, because adenosine analogs are capable of inhibiting SAHH activity, the addition of adenosine analogs to a solution containing SAH and SAHH resulted in the suppression of hydrolyzed SAH-induced NP aggregation. Adenosine analogs exhibited the following trend in the half-maximal inhibitory concentrations: adenosine > adenosine monophosphate > adenosine diphosphate ~ adenosine triphosphate. We have demonstrated that the combination of SAHH inhibition and FSN-AuNPs can be utilized for the selective detection of adenosine.

  20. Synthesis and Biological Activity of Mono- and Di-N-acylated Aminoglycosides

    PubMed Central

    2015-01-01

    Despite issues with oto/nephrotoxicity and bacterial resistance, aminoglycosides (AGs) remain an effective and widely used class of antibacterial agents. For decades now, efforts toward the development of novel AGs with potential to overcome some of these problems have been major research focuses. 1-N-Acylation, especially γ-amino-β-hydroxybutyrate (AHB) derivatization, has proven to be one of the most successful strategies for improving the overall properties of AGs, including their ability to avoid certain resistance mechanisms. More recently, 6′-N-acylation arose as another possible strategy to improve the properties of these drugs. In this study, we report on the glycinyl, carboxybenzyl, and AHB mono- and diderivatization at the 1-, 6′-, and/or 4‴-amines of the AGs amikacin, kanamycin A, netilmicin, sisomicin, and tobramycin. We also present the antibacterial activities and the reduced reactivity of AG-modifying enzymes (AMEs) toward these new AG derivatives, and identify the AMEs present in the bacterial strains tested. PMID:26617967

  1. A quantitation of the factors which affect the hydrolase and transgalactosylase activities of beta-galactosidase (E. coli) on lactose.

    PubMed

    Huber, R E; Kurz, G; Wallenfels, K

    1976-05-04

    A study was implemented to quantitate the hydrolase and transgalactosylase activities of beta-galactosidase (E. coli) with lactose as the substrate and to investigate various factors which affect these activities. At low lactose concentrations the rate of galactose production was equal to the rate of glucose production. The rate of galactose production relative to glucose, however, dropped dramatically at lactose concentrations higher than 0.05 M and production of trisaccharides and tetrasaccharides began (galactose/glucose ratios of about 2:1 and 3:1, respectively, were found for these two types of oligosaccharides). At least five different trissacharides were formed and their patterns of formation showed that they probably utilized both lactose and allolactose as galactosyl acceptors. Allolactose was produced in amounts proportional to glucose at all lactose concentrations (ratios of allolactose/glucose were about 0.88). Analyses of various data, including a reaction analyzed at very early times, showed that the major means of production of allolactose (and the only means initially) was the direct enzymatic transfer of galactose from the 4 position to the 6 position of the glucose moiety of lactose without prior release of glucose from the enzyme. It was shown, however, that allolactose could also be formed in significant quantities by the transfer of galactose to the 6 position of free glucose, and also by hydrolysis of preformed trisaccharide. A mechanism which fits the initial velocity data was proposed in which the steps involving the formation of an enzyme-gallactose-glucose complex, the formation and breakage of allolactose on the enzyme, and the release of glucose all seem to be of roughly equal magnitude and rate determining. Various factors affected the amounts of transgalactosylase and hydrolase activities occurring. At high pH values (greater than 7.8) the transgalactosylase/hydrolyase activity ratio increased dramatically while it decreased at low p

  2. Probing Mechanisms for Enzymatic Activity Enhancement of Organophosphorus Hydrolase in Functionalized Mesoporous Silica

    SciTech Connect

    Chen, Baowei; Lei, Chenghong; Shin, Yongsoon; Liu, Jun

    2009-12-25

    We have previously reported that organophosphorus hydrolase (OPH) can be spontaneously entrapped in functionalized mesoporous silica (FMS) with HOOC - as the functional groups and the entrapped OPH in HOOC-FMS showed enhanced enzyme specific activity. This work is to study the mechanisms that why OPH entrapped in FMS displayed the enhanced activity in views of OPH-FMS interactions using spectroscopic methods. The circular dichroism (CD) spectra show that, comparing to the secondary structure of OPH free in solution, OPH in HOOC-FMS displayed increased a-helix/b-strand transition of OPH with increased OPH loading density. The fluorescence emission spectra of Trp residues were used to assess the tertiary structural changes of the enzyme. There was a 42% increase in fluorescence. This is in agreement with the fact that the fluorescence intensity of OPH was increased accompanying with the increased OPH activity when decreasing urea concentrations in solution. The steady-state anisotropy was increased after OPH entrapping in HOOC-FMS comparing to the free OPH in solution, indicating that protein mobility was reduced upon entrapment. The solvent accessibility of Trp residues of OPH was probed by using acrylamide as a collisional quencher. Trp residues of OPH-FMS had less solvent exposure comparing with free OPH in solution due to its electrostatical binding to HOOC-FMS thereby displaying the increased fluorescence intensity. These results suggest the interactions of OPH with HOOC-FMS resulted in the protein immobilization and a favorable conformational change for OPH in the crowded confinement space and accordingly the enhanced activity.

  3. Insights into Exo- and Endoglucanase Activities of Family 6 Glycoside Hydrolases from Podospora anserina

    PubMed Central

    Poidevin, Laetitia; Feliu, Julia; Doan, Annick; Berrin, Jean-Guy; Bey, Mathieu; Coutinho, Pedro M.; Henrissat, Bernard; Record, Eric

    2013-01-01

    The ascomycete Podospora anserina is a coprophilous fungus that grows at late stages on droppings of herbivores. Its genome encodes a large diversity of carbohydrate-active enzymes. Among them, four genes encode glycoside hydrolases from family 6 (GH6), the members of which comprise putative endoglucanases and exoglucanases, some of them exerting important functions for biomass degradation in fungi. Therefore, this family was selected for functional analysis. Three of the enzymes, P. anserina Cel6A (PaCel6A), PaCel6B, and PaCel6C, were functionally expressed in the yeast Pichia pastoris. All three GH6 enzymes hydrolyzed crystalline and amorphous cellulose but were inactive on hydroxyethyl cellulose, mannan, galactomannan, xyloglucan, arabinoxylan, arabinan, xylan, and pectin. PaCel6A had a catalytic efficiency on cellotetraose comparable to that of Trichoderma reesei Cel6A (TrCel6A), but PaCel6B and PaCel6C were clearly less efficient. PaCel6A was the enzyme with the highest stability at 45°C, while PaCel6C was the least stable enzyme, losing more than 50% of its activity after incubation at temperatures above 30°C for 24 h. In contrast to TrCel6A, all three studied P. anserina GH6 cellulases were stable over a wide range of pHs and conserved high activity at pH values of up to 9. Each enzyme displayed a distinct substrate and product profile, highlighting different modes of action, with PaCel6A being the enzyme most similar to TrCel6A. PaCel6B was the only enzyme with higher specific activity on carboxymethylcellulose (CMC) than on Avicel and showed lower processivity than the others. Structural modeling predicts an open catalytic cleft, suggesting that PaCel6B is an endoglucanase. PMID:23645193

  4. Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates.

    PubMed Central

    Fernández-Valverde, M; Reglero, A; Martinez-Blanco, H; Luengo, J M

    1993-01-01

    Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase [ACoAS]) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source. The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol. Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively. Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids). The broad substrate specificity of ACoAS from P. putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins. Images PMID:8476289

  5. Continuous recording of long-chain acyl-coenzyme a synthetase activity using fluorescently labeled bovine serum albumin.

    PubMed

    Demant, E J; Nystrøm, B T

    2001-08-01

    The fluorescence-based long-chain fatty acid probe BSA-HCA (bovine serum albumin labeled with 7-hydroxycoumarin-4-acetic acid) is shown to respond to binding of long-chain acyl-CoA thioesters by quenching of the 450 nm fluorescence emission. As determined by spectrofluorometric titration, binding affinities for palmitoyl-, stearoyl-, and oleoyl-CoA (Kd = 0.2-0.4 microM) are 5-10 times lower than those for the corresponding nonesterified fatty acids. In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) above the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles. This allows BSA-HCA to be used as a fluorescent probe for continuous recording of fatty acid concentrations in detergent solution with little interference from acyl-CoA. Using a calibration of the fluorescence signal with fatty acids in the C14 to C20 chain-length range, fatty acid consumption by Pseudomonas fragi and rat liver microsomal acyl-CoA synthetase activities are measured down to 0.05 microM/min with a data sampling rate of 10 points per second. This new method provides a very promising spectrofluorometric approach to the study of acyl-CoA synthetase reaction kinetics at physiologically relevant (nM) aqueous phase concentrations of fatty acid substrates and at a time resolution that cannot be obtained in isotopic sampling or enzyme-coupled assays.

  6. Highly active β-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates.

    PubMed

    Jordan, Douglas B; Wagschal, Kurt; Grigorescu, Arabela A; Braker, Jay D

    2013-05-01

    The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. β-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 is recognized as a source of highly active β-xylosidases, some of which could have practical applications. The biochemical details of four GH43 β-xylosidases (those from Alkaliphilus metalliredigens QYMF, Bacillus pumilus, Bacillus subtilis subsp. subtilis str. 168, and Lactobacillus brevis ATCC 367) are examined here. Sedimentation equilibrium experiments indicate that the quaternary states of three of the enzymes are mixtures of monomers and homodimers (B. pumilus) or mixtures of homodimers and homotetramers (B. subtilis and L. brevis). k cat and k cat/K m values of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of other GH43 β-xylosidases. The K i values for D-glucose (83.3-357 mM) and D-xylose (15.6-70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature (K t (0.5) ∼ 45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from L. brevis ATCC 367 displays the highest reported k cat and k cat/K m on natural substrates xylobiose (407 s(-1), 138 s(-1) mM(-1)), xylotriose (235 s(-1), 80.8 s(-1) mM(-1)), and xylotetraose (146 s(-1), 32.6 s(-1) mM(-1)).

  7. Contribution of active-site glutamine to rate enhancement in ubiquitin carboxy terminal hydrolases

    PubMed Central

    Boudreaux, David; Chaney, Joseph; Maiti, Tushar K.; Das, Chittaranjan

    2012-01-01

    Ubiquitin carboxy terminal hydrolases (UCHs) are cysteine proteases featuring a classical cysteine-histidine-aspartate catalytic triad, also a highly conserved glutamine thought to be a part of the oxyanion hole. However, the contribution of this side chain to the catalysis by UCH enzymes is not known. Herein, we demonstrate that the glutamine side chain contributes to rate enhancement in UCHL1, UCHL3 and UCHL5. Mutation of the glutamine to alanine in these enzymes impairs the catalytic efficiency mainly due to a 16 to 30-fold reduction in kcat, which is consistent with a loss of approximately 2 kcal/mol in transition-state stabilization. However, the contribution to transition-state stabilization observed here is rather modest for the side chain’s role in oxyanion stabilization. Interestingly, we discovered that the carbonyl oxygen of this side chain is engaged in a C—H•••O hydrogen-bonding contact with the CεH group of the catalytic histidine. Upon further analysis, we found that this interaction is a common active-site structural feature in most cysteine proteases, including papain, belonging to families with the QCH(N/D) type of active-site configuration. It is possible that removal of the glutamine side chain might have abolished the C—H•••O interaction, which typically accounts for 2 kcal/mol of stabilization, leading to the effect on catalysis observed here. Additional studies performed on UCHL3 by mutating the glutamine to glutamate (strong C—H•••O acceptor but oxyanion destabilizer) and to lysine (strong oxyanion stabilizer but lacking C—H•••O hydrogen-bonding property) suggest that the C—H•••O hydrogen bond could contribute to catalysis. PMID:22284438

  8. Rational design of a thermostable glycoside hydrolase from family 3 introduces β-glycosynthase activity.

    PubMed

    Pozzo, Tania; Romero-García, Javier; Faijes, Magda; Planas, Antoni; Nordberg Karlsson, Eva

    2017-01-01

    The thermostable β-glucosidase from Thermotoga neapolitana, TnBgl3B, is a monomeric three-domain representative from glycoside hydrolase family 3. By using chemical reactivation with exogenous nucleophiles in previous studies with TnBg13B, the catalytic nucleophile (D242) and corresponding acid/base residue (E458) were determined. Identifying these residues led to the attempt of converting TnBgl3B into a β-glucosynthase, where three nucleophilic variants were created (TnBgl3B_D242G, TnBgl3B_D242A, TnBgl3B_D242S) and all of them failed to exhibit glucosynthase activity. A deeper analysis of the TnBgl3B active site led to the generation of three additional variants, each of which received a single-point mutation. Two of these variants were altered at the -1 subsite (Y210F, W243F) and the third received a substitution near the binding site's aglycone region (N248R). Kinetic evaluation of these three variants revealed that W243F substitution reduced hydrolytic turnover while maintaining KM This key W243F mutation was then introduced into the original nucleophile variants and the resulting double mutants were successfully converted into β-glucosynthases that were assayed using two separate biosynthetic methods. The first reaction used an α-glucosyl fluoride donor with a 4-nitrophenyl-β-d-glucopyranoside (4NPGlc) acceptor, and the second used 4NPGlc as both the donor and acceptor in the presence of the exogenous nucleophile formate. The primary specificity observed was a β-1,3-linked disaccharide product, while a secondary β-1,4-linked disaccharide product was observed with increased incubation times. Additional analysis revealed that substituting quercetin-3-glycoside for the second reaction's acceptor molecule resulted in the successful production of quercetin-3,4'-diglycosides with yields up to 40%.

  9. Iron Acyl Thiolato Carbonyls: Structural Models for the Active Site of the [Fe]-Hydrogenase (Hmd)

    PubMed Central

    Royer, Aaron M.; Salomone-Stagni, Marco

    2012-01-01

    Phosphine-modified thioester derivatives are shown to serve as efficient precursors to phosphine-stabilized ferrous acyl thiolato carbonyls via the reaction of phosphine thioesters and sources of Fe(0). The reaction generates both Fe(SPh)(Ph2PC6H4CO)(CO)3 (1) and the diferrous diacyl Fe2(SPh)2(CO)3(Ph2PC6H4CO)2, which carbonylates to give 1. For the extremely bulky arylthioester Ph2PC6H4C(O)SC6H4-2,6-(2,4,6-trimethylphenyl)2, oxidative addition is arrested and the Fe(0) adduct of the phosphine is obtained. Complex 1 reacts with cyanide to give Et4N[Fe(SPh)(Ph2PC6H4CO)(CN)(CO)2] (Et4N[2]). 13C and 31P NMR spectra indicate that substitution is stereospecific and cis to P. The IR spectrum of [2]− in CH2Cl2 solution very closely matches that for HmdCN. XANES and EXAFS measurements also indicate close structural and electronic similarity of Et4N[2] to the active site of wild-type Hmd. Complex 1 also stereospecifically forms a derivative with TsCH2NC, but the adduct is more labile than Et4N[2]. Tricarbonyl 1 was found to reversibly protonate to give a thermally labile derivative, IR measurements of which indicate that the acyl and thiolate ligands are probably not protonated in Hmd. PMID:21062066

  10. Physalin B inhibits Rhodnius prolixus hemocyte phagocytosis and microaggregation by the activation of endogenous PAF-acetyl hydrolase activities.

    PubMed

    Castro, D P; Figueiredo, M B; Genta, F A; Ribeiro, I M; Tomassini, T C B; Azambuja, P; Garcia, E S

    2009-06-01

    The effects of physalin B (a natural secosteroidal chemical from Physalis angulata, Solanaceae) on phagocytosis and microaggregation by hemocytes of 5th-instar larvae of Rhodnius prolixus were investigated. In this insect, hemocyte phagocytosis and microaggregation are known to be induced by the platelet-activating factor (PAF) or arachidonic acid (AA) and regulated by phospholipase A(2) (PLA(2)) and PAF-acetyl hydrolase (PAF-AH) activities. Phagocytic activity and formation of hemocyte microaggregates by Rhodnius hemocytes were strongly blocked by oral treatment of this insect with physalin B (1mug/mL of blood meal). The inhibition induced by physalin B was reversed for both phagocytosis and microaggregation by exogenous arachidonic acid (10microg/insect) or PAF (1microg/insect) applied by hemocelic injection. Following treatment with physalin B there were no significant alterations in PLA(2) activities, but a significant enhancement of PAF-AH was observed. These results show that physalin B inhibits hemocytic activity by depressing insect PAF analogous (iPAF) levels in hemolymph and confirm the role of PAF-AH in the cellular immune reactions in R. prolixus.

  11. Similarities between catalase and cytosolic epoxide hydrolase.

    PubMed

    Guenthner, T M; Qato, M; Whalen, R; Glomb, S

    1989-01-01

    Cytosolic epoxide hydrolase, measured as trans-stilbene oxide hydrolase activity, was isolated and purified from human and guinea pig liver cytosol. Antiserum to the guinea pig liver preparation reacted strongly with bovine liver catalase. We determined that this lack of selectivity of the antiserum was due to catalase contamination of the epoxide hydrolase preparation. We also determined that several commercial catalase preparations are contaminated with cytosolic epoxide hydrolase. Our human epoxide hydrolase preparation contained no detectable catalase contamination, yet antiserum to this protein also cross-reacted slightly with catalase, indicating some intrinsic similarity between the two enzymes. We conclude that catalase and cytosolic epoxide hydrolase contain some similar immunogenic epitopes, and we surmise that similarities between the subunits of these two enzymes may lead to their partial copurification. Functional similarities between the two enzymes are also demonstrated, as several compounds that inhibit catalase are also shown to inhibit cytosolic epoxide hydrolase activity in the same concentration range and rank order.

  12. PehN, a polygalacturonase homologue with a low hydrolase activity, is coregulated with the other Erwinia chrysanthemi polygalacturonases.

    PubMed

    Hugouvieux-Cotte-Pattat, Nicole; Shevchik, Vladimir E; Nasser, William

    2002-05-01

    Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants. E. chrysanthemi also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes, pehV, pehW, and pehX, encoding exo-poly-alpha-D-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the pehN gene does not significantly alter the virulence of E. chrysanthemi. Regulation of pehN transcription was analyzed by using gene fusions. Like other pectinase genes, pehN transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the pel genes but activates that of pehV, pehW, and pehX, also activates transcription of pehN. The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase

  13. Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase.

    PubMed

    Singhal, Anshika; Arora, Gunjan; Virmani, Richa; Kundu, Parijat; Khanna, Tanya; Sajid, Andaleeb; Misra, Richa; Joshi, Jayadev; Yadav, Vikas; Samanta, Sintu; Saini, Neeru; Pandey, Amit K; Visweswariah, Sandhya S; Hentschker, Christian; Becher, Dörte; Gerth, Ulf; Singh, Yogendra

    2015-10-23

    Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins has not been studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to the identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein-tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys-224. This residue is situated in a lid region that covers the enzyme's active site. Consequently, acetylation and succinylation negatively regulate the activity of PtpB.

  14. Relationship between plasma lipids and palmitoyl-CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates.

    PubMed Central

    Alegret, M.; Ferrando, R.; Vázquez, M.; Adzet, T.; Merlos, M.; Laguna, J. C.

    1994-01-01

    1. The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-CoA hydrolase and synthetase activities, as well as the correlations with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats. 2. The administration of the three drugs caused a significant reduction in body weight gain, accompanied with a paradoxical increase in food intake in groups treated with BFB and GFB. 3. Drug treatment produced gross hepatomegaly and increase in peroxisomal beta-oxidation, and these parameters were strongly correlated. The order of potency was BFB > CFB > or = GFB. 4. Both plasma cholesterol (BFB approximately CFB > GFB) and triglyceride (BFB approximately GFB > CFB) levels were reduced in treated animals. There was an inverse correlation between these parameters and peroxisomal beta-oxidation, although the peroxisomal proliferation seemed to explain only a small part of the hypolipidemic effect observed. 5. Cytosolic and microsomal (but not mitochondrial) palmitoyl-CoA hydrolase activities were increased by the three drugs (BFB > CFB > GFB), probably by inducing the hydrolase I isoform, which is insensitive to inhibition by fibrates in vitro. The increased hydrolase activities were directly and strongly correlated with peroxisomal beta-oxidation. 6. Palmitoyl-CoA synthetase activity was also increased by the treatment with fibrates (BFB > CFB > GFB), probably as a consequence of the enhancement of hydrolase activities.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7915611

  15. Production of sophorolipids from whey. II. Product composition, surface active properties, cytotoxicity and stability against hydrolases by enzymatic treatment.

    PubMed

    Otto, R T; Daniel, H J; Pekin, G; Müller-Decker, K; Fürstenberger, G; Reuss, M; Syldatk, C

    1999-10-01

    Sophorolipids, obtained by a two-stage process starting from deproteinized whey concentrate using Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214, were compared to products from one-stage processes, using different lipidic compounds as substrates. Results showed that above all carbon source and not cultivation conditions had a distinct influence on the composition of the crude product mixture and therefore on the physicochemical and biological properties of the sophorolipids, such as, for example, surface activity, cytotoxicity and stability against hydrolases. The results were completed by corresponding data for purified mono- and diacetylated (17-hydroxyoctadecenoic)-1',4"-lactonized sophorolipids. Crude sophorolipid mixtures showed moderate to good surface active properties (SFTmin 39 mN m-1, CMC 130 mg l-1), water solubilities (2-3 g l-1) and low cytotoxicities (LC50 300-700 mg l-1). In contrast, purified sophorolipids were more surface active (SFTmin 36 mN m-1, CMC 10 mg l-1), less water soluble (max. 70 mg l-1) and showed stronger cytotoxic effects (LC50 15 mg l-1). Incubation of crude sophorolipid mixtures with different hydrolases demonstrated that treatment with commercially available lipases such as from Candida rugosa and Mucor miehei distinctly reduced the surface active properties of the sophorolipids, while treatment with porcine liver esterase and glycosidases had no effect.

  16. Membrane anchoring of diacylglycerol lactones substituted with rigid hydrophobic acyl domains correlates with biological activities.

    PubMed

    Raifman, Or; Kolusheva, Sofiya; Comin, Maria J; Kedei, Noemi; Lewin, Nancy E; Blumberg, Peter M; Marquez, Victor E; Jelinek, Raz

    2010-01-01

    Synthetic diacylglycerol lactones (DAG lactones) are effective modulators of critical cellular signaling pathways downstream of the lipophilic second messenger diacylglycerol that activate a host of protein kinase C (PKC) isozymes as well as other non-kinase proteins that share with PKC similar C1 membrane-targeting domains. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study characterizes the membrane interactions and bilayer anchoring of a series of DAG lactones in which the hydrophobic moiety is a 'molecular rod', namely a rigid 4-[2-(R-phenyl)ethynyl]benzoate moiety in the acyl position. Use of assays employing chromatic biomimetic vesicles and biophysical techniques revealed that the mode of membrane anchoring of the DAG lactone derivatives was markedly affected by the presence of the hydrophobic diphenyl rod and by the size of the functional unit at the terminus of the rod. Two primary mechanisms of interaction were observed: surface binding of the DAG lactones at the lipid/water interface and deep insertion of the ligands into the alkyl core of the lipid bilayer. These membrane-insertion properties could explain the different patterns of the PKC translocation from the cytosol to membranes that is induced by the molecular-rod DAG lactones. This investigation emphasizes that the side residues of DAG lactones, rather than simply conferring hydrophobicity, profoundly influence membrane interactions, and thus may further contribute to the diversity of biological actions of these synthetic biomimetic ligands.

  17. α-Ketoheterocycle-Based Inhibitors of Fatty Acid Amide Hydrolase (FAAH)

    PubMed Central

    2011-01-01

    A summary of the initial discovery and characterization of the enzyme fatty acid amide hydrolase (FAAH), and the subsequent advancement of an important class of competitive, reversible, potent, and selective inhibitors is presented. Initially explored using substrate-inspired inhibitors bearing electrophilic carbonyls, the examination of α-ketoheterocyle-based inhibitors of FAAH with the benefit of a unique activity-based protein-profiling (ABPP)-based proteome-wide selectivity assay, a powerful in vivo biomarker-based in vivo screen, and subsequent retrospective X-ray cocrystal structures with the enzyme, is summarized. These efforts defined the impact of the central activating heterocycle and its key substituents, provided key simplifications in the C2 acyl side chain and clear interpretations for the unique role and subsequent optimization of the central activating heterocycle, and established the basis for the recent further conformational constraints in the C2 acyl side chain, providing potent, long-acting, orally active FAAH inhibitors. PMID:22639704

  18. Discovery libraries targeting the major enzyme classes: the serine hydrolases.

    PubMed

    Otrubova, Katerina; Srinivasan, Venkat; Boger, Dale L

    2014-08-15

    Two libraries of modestly reactive ureas containing either electron-deficient acyl anilines or acyl pyrazoles were prepared and are reported as screening libraries for candidate serine hydrolase inhibitors. Within each library is a small but powerful subset of compounds that serve as a chemotype fragment screening library capable of subsequent structural diversification. Elaboration of the pyrazole-based ureas provided remarkably potent irreversible inhibitors of fatty acid amide hydrolase (FAAH, apparent Ki=100-200 pM) complementary to those previously disclosed enlisting electron-deficient aniline-based ureas.

  19. Discovery libraries targeting the major enzyme classes: the serine hydrolases

    PubMed Central

    Otrubova, Katerina; Srinivasan, Venkat; Boger, Dale L.

    2014-01-01

    Two libraries of modestly reactive ureas containing either electron-deficient acyl anilines or acyl pyrazoles were prepared and are reported as screening libraries for candidate serine hydrolase inhibitors. Within each library is a small but powerful subset of compounds that serve as a chemotype fragment screening library capable of subsequent diversification. Elaboration of the pyrazole-based ureas provided remarkably potent irreversible structural inhibitors of fatty acid amide hydrolase (FAAH, apparent Ki = 100-200 pM) complementary to those previously disclosed enlisting electron-deficient aniline-based ureas. PMID:25037918

  20. Some hydrolase activities from the tick Hyalomma lusitanicum koch, 1844 (Ixodoidea: Ixodida).

    PubMed

    Giménez-Pardo, C; Martínez-Grueiro, M M

    2008-12-01

    In this work has been made a detection and preliminary characterization of some hydrolases in whole extracts from unfed adult males and females of Hyalomma lusitanicum, one of the vectors for Theileria annulata that causes Mediterranean theileriosis in cattle. We have elected as targets, proteases as enzymes implicated in the nutritional processes of ticks, esterases that are usually implicated in resistance to organophosphates and phosphatises often implicated in protein phosphorilation and control of ticks salivary gland. The biological role and physiological significance are discussed in terms of the possibility of use these enzymes as possible in future anti-tick vaccination or acaricide resistance.

  1. Synthesis, crystal structure and biological activity of two Mn complexes with 4-acyl pyrazolone derivatives.

    PubMed

    Li, Yue; Zhao, Jing; He, Chuan-Chuan; Zhang, Li; Sun, Su-Rong; Xu, Guan-Cheng

    2015-09-01

    In order to study the biological activities of transitional metal complexes based on 4-acyl pyrazolone derivatives, two Mn complexes [Mn(HLa)(La)]·(CH3CN)1.5·H2O (1) and [Mn2(Lb)2(μ-EtO)2(EtOH)2] (2) (H2La = N-(1-phenyl-3-methyl-4-benzoyl-5-pyrazolone)-2-thiophenecarboxylic acid hydrazide, H2Lb = N-(1-phenyl-3-methyl-4-propenylidene-5-pyrazolone)-2-thiophenecarboxylic acid hydrazide) have been synthesized and characterized. Single crystal X-ray diffraction analysis indicated that 1 is a mononuclear complex and 2 exhibits a dinuclear centrosymmetric structure. Binding of the complexes with Herring Sperm DNA (HS-DNA) showed that complexes 1 and 2 could intercalate to DNA with quenching constant of 5.3×10(4) M(-1) and 4.9×10(4) M(-1), respectively. The interactions of the complexes with bovine serum albumin (BSA) indicated that complexes 1 and 2 could quench the intrinsic fluorescence of BSA in a static quenching process. Further, the inhibitory effects of the complexes on the cell population growth of the human esophageal cancer Eca-109 cells and the cervical cancer HeLa cells were determined by MTT assay, which indicated that both 1 and 2 significantly inhibited the growth of Eca-109 and HeLa cells, the inhibitory activity of complex 1 is stronger than that of 2. We further observed that complex 1 inhibited the growth of HeLa cells through inducing the apoptosis and arresting cell cycle at S phase. Our results suggested that both complexes 1 and 2 have DNA- and protein-binding capacity and antitumor activity.

  2. Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals α-Transglucosylase Activity in Glycoside Hydrolase Family 31*

    PubMed Central

    Larsbrink, Johan; Izumi, Atsushi; Hemsworth, Glyn R.; Davies, Gideon J.; Brumer, Harry

    2012-01-01

    The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these α-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in α-glucan side chain (de)branching, regulation of oligo- and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-α-glucan 4-α-glucosyltransferase from GH31. Distinct from 1,4-α-glucan 6-α-glucosyltransferases (EC 2.4.1.24) and 4-α-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from α(1→4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-β-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme. PMID:23132856

  3. Structural enzymology of Cellvibrio japonicus Agd31B protein reveals α-transglucosylase activity in glycoside hydrolase family 31.

    PubMed

    Larsbrink, Johan; Izumi, Atsushi; Hemsworth, Glyn R; Davies, Gideon J; Brumer, Harry

    2012-12-21

    The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these α-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in α-glucan side chain (de)branching, regulation of oligo- and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-α-glucan 4-α-glucosyltransferase from GH31. Distinct from 1,4-α-glucan 6-α-glucosyltransferases (EC 2.4.1.24) and 4-α-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from α(1→4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-β-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme.

  4. Activities of acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) in microsomal preparations of developing sunflower and safflower seeds.

    PubMed

    Banaś, Walentyna; Sanchez Garcia, Alicia; Banaś, Antoni; Stymne, Sten

    2013-06-01

    The last step in triacylglycerols (TAG) biosynthesis in oil seeds, the acylation of diacylglycerols (DAG), is catalysed by two types of enzymes: the acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). The relative contribution of these enzymes in the synthesis of TAG has not yet been defined in any plant tissue. In the presented work, microsomal preparations were obtained from sunflower and safflower seeds at different stages of development and used in DGAT and PDAT enzyme assays. The ratio between PDAT and DGAT activity differed dramatically between the two different species. DGAT activities were measured with two different acyl acceptors and assay methods using two different acyl-CoAs, and in all cases the ratio of PDAT to DGAT activity was significantly higher in safflower than sunflower. The sunflower DGAT, measured by both methods, showed significant higher activity with 18:2-CoA than with 18:1-CoA, whereas the opposite specificity was seen with the safflower enzyme. The specificities of PDAT on the other hand, were similar in both species with 18:2-phosphatidylcholine being a better acyl donor than 18:1-PC and with acyl groups at the sn-2 position utilised about fourfold the rate of the sn-1 position. No DAG:DAG transacylase activity could be detected in the microsomal preparations.

  5. Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion associated peptidoglycan hydrolase: fusions, deletions and synergy with LysH5

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virion-associated peptidoglycan hydrolases have a potential as antimicrobial agents due to their ability to lyse Gram positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriopha...

  6. Variants of glycoside hydrolases

    DOEpatents

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2013-02-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  7. Variants of glycoside hydrolases

    DOEpatents

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2011-04-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  8. Anti-Tumor Effects of Novel 5-O-Acyl Plumbagins Based on the Inhibition of Mammalian DNA Replicative Polymerase Activity

    PubMed Central

    Kawamura, Moe; Kuriyama, Isoko; Maruo, Sayako; Kuramochi, Kouji; Tsubaki, Kazunori; Yoshida, Hiromi; Mizushina, Yoshiyuki

    2014-01-01

    We previously found that vitamin K3 (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase γ (pol γ). In this study, we focused on plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), and chemically synthesized novel plumbagins conjugated with C2:0 to C22:6 fatty acids (5-O-acyl plumbagins). These chemically modified plumbagins enhanced mammalian pol inhibition and their cytotoxic activity. Plumbagin conjugated with chains consisting of more than C18-unsaturated fatty acids strongly inhibited the activities of calf pol α and human pol γ. Plumbagin conjugated with oleic acid (C18:1-acyl plumbagin) showed the strongest suppression of human colon carcinoma (HCT116) cell proliferation among the ten synthesized 5-O-acyl plumbagins. The inhibitory activity on pol α, a DNA replicative pol, by these compounds showed high correlation with their cancer cell proliferation suppressive activity. C18:1-Acyl plumbagin selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols and DNA metabolic enzymes tested. This compound inhibited the proliferation of various human cancer cell lines, and was the cytotoxic inhibitor showing strongest inhibition towards HT-29 colon cancer cells (LD50 = 2.9 µM) among the nine cell lines tested. In an in vivo anti-tumor assay conducted on nude mice bearing solid tumors of HT-29 cells, C18:1-acyl plumbagin was shown to be a promising tumor suppressor. These data indicate that novel 5-O-acyl plumbagins act as anti-cancer agents based on mammalian DNA replicative pol α inhibition. Moreover, the results suggest that acylation of plumbagin is an effective chemical modification to improve the anti-cancer activity of vitamin K3 derivatives, such as plumbagin. PMID:24520419

  9. Relationship between plasma lipids and palmitoyl-CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates.

    PubMed

    Alegret, M; Ferrando, R; Vázquez, M; Adzet, T; Merlos, M; Laguna, J C

    1994-06-01

    1. The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-CoA hydrolase and synthetase activities, as well as the correlations with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats. 2. The administration of the three drugs caused a significant reduction in body weight gain, accompanied with a paradoxical increase in food intake in groups treated with BFB and GFB. 3. Drug treatment produced gross hepatomegaly and increase in peroxisomal beta-oxidation, and these parameters were strongly correlated. The order of potency was BFB > CFB > or = GFB. 4. Both plasma cholesterol (BFB approximately CFB > GFB) and triglyceride (BFB approximately GFB > CFB) levels were reduced in treated animals. There was an inverse correlation between these parameters and peroxisomal beta-oxidation, although the peroxisomal proliferation seemed to explain only a small part of the hypolipidemic effect observed. 5. Cytosolic and microsomal (but not mitochondrial) palmitoyl-CoA hydrolase activities were increased by the three drugs (BFB > CFB > GFB), probably by inducing the hydrolase I isoform, which is insensitive to inhibition by fibrates in vitro. The increased hydrolase activities were directly and strongly correlated with peroxisomal beta-oxidation. 6. Palmitoyl-CoA synthetase activity was also increased by the treatment with fibrates (BFB > CFB > GFB), probably as a consequence of the enhancement of hydrolase activities. 7. Some of the effects of fibrate treatment can be explained, at least in part, in terms of peroxisomal induction and caution should be exercised in the extrapolation of these results to species, such as man,that are insensitive to peroxisomal proliferation.

  10. Selective inhibition and selective induction of multiple microsomal epoxide hydrolases.

    PubMed

    Guenthner, T M

    1986-03-01

    The inhibition in vitro and induction in vivo of microsomal trans-stilbene oxide hydrolase have been studied. This microsomal epoxide hydrolase activity is distinguishable from the previously well-defined microsomal arene oxide hydrolase by a number of catalytic criteria. Two substituted chalcone oxides, 4-phenylchalcone oxide and 4'-phenylchalcone oxide, are potent inhibitors of microsomal trans-stilbene oxide hydrolase, but have no apparent activity against benzo[a]pyrene 4,5-oxide hydrolase. Conversely, compounds that are potent inhibitors of benzo[a]pyrene 4,5-oxide hydrolase, including styrene oxide, cyclohexene oxide, and trichloropropene oxide, inhibit microsomal trans-stilbene oxide hydrolase only at very high (millimolar) concentrations. The chalcone oxides inhibit microsomal trans-stilbene oxide hydrolase noncompetitively, and have micromolar or nanomolar affinity constants for the enzyme. Attempts were made to induce microsomal trans-stilbene oxide hydrolase in vivo. Compounds that induced microsomal benzo[a]pyrene 4,5-oxide hydrolase levels in mice did not simultaneously induce trans-stilbene oxide hydrolase levels. Clofibrate was an exception; it induced levels of both enzymes to a small but statistically significant degree. The two microsomal hydrolase activities have, therefore, very different catalytic sites and appear to be under separate genetic control. 4-Phenylchalcone oxide and 4'-phenylchalcone oxide are selective inhibitors of microsomal trans-stilbene oxide hydrolase and may prove to be very useful in assessing the involvement of this enzyme in the metabolism of endogenous or xenobiotic epoxides.

  11. Hydrolase-like properties of a cofactor-independent dioxygenase.

    PubMed

    Thierbach, Sven; Büldt-Karentzopoulos, Klaudia; Dreiling, Alena; Hennecke, Ulrich; König, Simone; Fetzner, Susanne

    2012-05-29

    Mechanistic promiscuity: The (2-alkyl)-3-hydroxy-4(1H)-quinolone-cleaving dioxygenase Hod has an α/β-hydrolase fold and a Ser/His/Asp triad in its active site. Isatoic anhydride, a suicide substrate of serine hydrolases, inactivates Hod by covalent modification of the active-site serine, thus indicating that the α/β-hydrolase fold can accommodate dioxygenase chemistry without completely abandoning hydrolase-like properties.

  12. Modulating effects of acyl-CoA synthetase 5-derived mitochondrial Wnt2B palmitoylation on intestinal Wnt activity

    PubMed Central

    Klaus, Christina; Schneider, Ursula; Hedberg, Christian; Schütz, Anke K; Bernhagen, Jürgen; Waldmann, Herbert; Gassler, Nikolaus; Kaemmerer, Elke

    2014-01-01

    AIM: To investigate the role of acyl-CoA synthetase 5 (ACSL5) activity in Wnt signaling in intestinal surface epithelia. METHODS: Several cell lines were used to investigate the ACSL5-dependent expression and synthesis of Wnt2B, a mitochondrially expressed protein of the Wnt signaling family. Wnt activity was functionally assessed with a luciferase reporter assay. ACSL5-related biochemical Wnt2B modifications were investigated with a modified acyl-exchange assay. The findings from the cell culture models were verified using an Apcmin/+ mouse model as well as normal and neoplastic diseased human intestinal tissues. RESULTS: In the presence of ACSL5, Wnt2B was unable to translocate into the nucleus and was enriched in mitochondria, which was paralleled by a significant decrease in Wnt activity. ACSL5-dependent S-palmitoylation of Wnt2B was identified as a molecular reason for mitochondrial Wnt2B accumulation. In cell culture systems, a strong relation of ACSL5 expression, Wnt2B palmitoylation, and degree of malignancy were found. Using normal mucosa, the association of ACSL5 and Wnt2B was seen, but in intestinal neoplasias the mechanism was only rudimentarily observed. CONCLUSION: ACSL5 mediates antiproliferative activities via Wnt2B palmitoylation with diminished Wnt activity. The molecular pathway is probably relevant for intestinal homeostasis, overwhelmed by other pathways in carcinogenesis. PMID:25356045

  13. Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

    PubMed Central

    Recuero-Checa, Maria A.; Sharma, Manu; Lau, Constance; Watkins, Paul A.; Gaydos, Charlotte A.; Dean, Deborah

    2016-01-01

    The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3–6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs. PMID:26988341

  14. Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs).

    PubMed

    Recuero-Checa, Maria A; Sharma, Manu; Lau, Constance; Watkins, Paul A; Gaydos, Charlotte A; Dean, Deborah

    2016-03-18

    The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3-6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs.

  15. Crystal structure of human mitochondrial acyl-CoA thioesterase (ACOT2)

    PubMed Central

    Mandel, Corey R.; Tweel, Benjamin; Tong, Liang

    2009-01-01

    Acyl-CoA thioesterases (ACOTs) catalyze the hydrolysis of CoA esters to free CoA and carboxylic acids and have important functions in lipid metabolism and other cellular processes. Type I ACOTs are found only in animals and contain an α/β hydrolase domain, through currently no structural information is available on any of these enzymes. We report here the crystal structure at 2.1 Å resolution of human mitochondrial ACOT2, a type I enzyme. The structure contains two domains, N and C domains. The C domain has the α/β hydrolase fold, with the catalytic triad Ser294-His422-Asp388. The N domain contains a seven-stranded β-sandwich, which has some distant structural homologs in other proteins. The active site is located in a large pocket at the interface between the two domains. The structural information has significant relevance for other type I ACOTs and related enzymes. PMID:19497300

  16. Crystal structure of human mitochondrial acyl-CoA thioesterase (ACOT2).

    PubMed

    Mandel, Corey R; Tweel, Benjamin; Tong, Liang

    2009-08-07

    Acyl-CoA thioesterases (ACOTs) catalyze the hydrolysis of CoA esters to free CoA and carboxylic acids and have important functions in lipid metabolism and other cellular processes. Type I ACOTs are found only in animals and contain an alpha/beta hydrolase domain, through currently no structural information is available on any of these enzymes. We report here the crystal structure at 2.1A resolution of human mitochondrial ACOT2, a type I enzyme. The structure contains two domains, N and C domains. The C domain has the alpha/beta hydrolase fold, with the catalytic triad Ser294-His422-Asp388. The N domain contains a seven-stranded beta-sandwich, which has some distant structural homologs in other proteins. The active site is located in a large pocket at the interface between the two domains. The structural information has significant relevance for other type I ACOTs and related enzymes.

  17. Structural determination and DPPH radical-scavenging activity of two acylated flavonoid tetraglycosides in oolong tea (Camellia sinensis).

    PubMed

    Lee, Viola Szu-Yuan; Chen, Chiy-Rong; Liao, Yun-Wen; Tzen, Jason Tze-Cheng; Chang, Chi-I

    2008-06-01

    Two major acylated flavonoid tetraglycosides were isolated from the methanol extract of oolong tea. Their structures were elucidated by spectroscopic methods as quercetin 3-O-[2(G)-(E)-coumaroyl-3(G)-O-beta-D-glucosyl-3(R)-O-beta-D-glucosylrutinoside] (1) and kaempferol 3-O-[2(G)-(E)-coumaroyl-3(G)-O-beta-D-glucosyl-3(R)-O-beta-D-glucosylrutinoside] (2). Compounds 1 and 2 exhibited scavenging activity against DPPH radical with EC(50) values of 30.5 and 487.2 microM, respectively.

  18. 3-Acyl dihydroflavonols from poplar resins collected by honey bees are active against the bee pathogens Paenibacillus larvae and Ascosphaera apis.

    PubMed

    Wilson, Michael B; Pawlus, Alison D; Brinkman, Doug; Gardner, Gary; Hegeman, Adrian D; Spivak, Marla; Cohen, Jerry D

    2017-03-01

    Honey bees, Apis mellifera, collect antimicrobial plant resins from the environment and deposit them in their nests as propolis. This behavior is of practical concern to beekeepers since the presence of propolis in the hive has a variety of benefits, including the suppression of disease symptoms. To connect the benefits that bees derive from propolis with particular resinous plants, we determined the identity and botanical origin of propolis compounds active against bee pathogens using bioassay-guided fractionation against the bacterium Paenibacillus larvae, the causative agent of American foulbrood. Eleven dihydroflavonols were isolated from propolis collected in Fallon, NV, including pinobanksin-3-octanoate. This hitherto unknown derivative and five other 3-acyl-dihydroflavonols showed inhibitory activity against both P. larvae (IC50 = 17-68 μM) and Ascosphaera apis (IC50 = 8-23 μM), the fungal agent of chalkbrood. A structure-activity relationship between acyl group size and antimicrobial activity was found, with longer acyl groups increasing activity against P. larvae and shorter acyl groups increasing activity against A. apis. Finally, it was determined that the isolated 3-acyl-dihydroflavonols originated from Populus fremontii, and further analysis showed these compounds can also be found in other North American Populus spp.

  19. Regulation of yeast ESCRT-III membrane scission activity by the Doa4 ubiquitin hydrolase.

    PubMed

    Johnson, Natalie; West, Matt; Odorizzi, Greg

    2017-03-01

    ESCRT-III executes membrane scission during the budding of intralumenal vesicles (ILVs) at endosomes. The scission mechanism is unknown but appears to be linked to the cycle of assembly and disassembly of ESCRT-III complexes at membranes. Regulating this cycle is therefore expected to be important for determining the timing of ESCRT-III-mediated membrane scission. We show that in Saccharomyces cerevisiae, ESCRT-III complexes are stabilized and ILV membrane scission is delayed by Doa4, which is the ubiquitin hydrolase that deubiquitinates transmembrane proteins sorted as cargoes into ILVs. These results suggest a mechanism to delay ILV budding while cargoes undergo deubiquitination. We further show that deubiquitination of ILV cargoes is inhibited via Doa4 binding to Vps20, which is the subunit of ESCRT-III that initiates assembly of the complex. Current models suggest that ESCRT-III complexes surround ubiquitinated cargoes to trap them at the site of ILV budding while the cargoes undergo deubiquitination. Thus our results also propose a mechanism to prevent the onset of ILV cargo deubiquitination at the initiation of ESCRT-III complex assembly.

  20. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

    PubMed

    Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  1. Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

    PubMed Central

    Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I.; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity. PMID:24278284

  2. Structural and Functional Analyses of a Glycoside Hydrolase Family 5 Enzyme with an Unexpected [beta]-Fucosidase Activity

    SciTech Connect

    Yoshida, Shosuke; Park, David S.; Bae, Brian; Mackie, Roderick; Cann, Isaac K.O.; Nair, Satish K.

    2012-02-15

    We present characterization of PbFucA, a family 5 glycoside hydrolase (GH5) from Prevotella bryantii B{sub 1}4. While GH5 members typically are xylanases, PbFucA shows no activity toward xylan polysaccharides. A screen against a panel of p-nitrophenol coupled sugars identifies PbFucA as a {beta}-D-fucosidase. We also present the 2.2 {angstrom} resolution structure of PbFucA and use structure-based mutational analysis to confirm the role of catalytically essential residues. A comparison of the active sites of PbFucA with those of family 5 and 51 glycosidases reveals that while the essential catalytic framework is identical between these enzymes, the steric contours of the respective active site clefts are distinct and likely account for substrate discrimination. Our results show that members of this cluster of orthologous group (COG) 5520 have {beta}-D-fucosidase activities, despite showing an overall sequence and structural similarity to GH-5 xylanases.

  3. Structure and activity of Paenibacillus polymyxa xyloglucanase from glycoside hydrolase family 44.

    PubMed

    Ariza, Antonio; Eklöf, Jens M; Spadiut, Oliver; Offen, Wendy A; Roberts, Shirley M; Besenmatter, Werner; Friis, Esben P; Skjøt, Michael; Wilson, Keith S; Brumer, Harry; Davies, Gideon

    2011-09-30

    The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the "accessory enzymes," including xyloglucanases, that may aid biomass degradation through removal of "hemicellulose" polysaccharides. Here, we report the biochemical characterization of the endo-β-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the -4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the -3 to -1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the -4' subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.

  4. Ubiquitin C-terminal hydrolase37 regulates Tcf7 DNA binding for the activation of Wnt signalling

    PubMed Central

    Han, Wonhee; Lee, Hyeyoon; Han, Jin-Kwan

    2017-01-01

    The Tcf/Lef family of transcription factors mediates the Wnt/β-catenin pathway that is involved in a wide range of biological processes, including vertebrate embryogenesis and diverse pathogenesis. Post-translational modifications, including phosphorylation, sumoylation and acetylation, are known to be important for the regulation of Tcf/Lef proteins. However, the importance of ubiquitination and ubiquitin-mediated regulatory mechanisms for Tcf/Lef activity are still unclear. Here, we newly show that ubiquitin C-terminal hydrolase 37 (Uch37), a deubiquitinase, interacts with Tcf7 (formerly named Tcf1) to activate Wnt signalling. Biochemical analyses demonstrated that deubiquitinating activity of Uch37 is not involved in Tcf7 protein stability but is required for the association of Tcf7 to target gene promoter in both Xenopus embryo and human liver cancer cells. In vivo analyses further revealed that Uch37 functions as a positive regulator of the Wnt/β-catenin pathway downstream of β-catenin stabilization that is required for the expression of ventrolateral mesoderm genes during Xenopus gastrulation. Our study provides a new mechanism for chromatin occupancy of Tcf7 and uncovers the physiological significance of Uch37 during early vertebrate development by regulating the Wnt/β-catenin pathway. PMID:28198400

  5. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    PubMed

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

  6. Ubiquitin C-terminal hydrolase37 regulates Tcf7 DNA binding for the activation of Wnt signalling.

    PubMed

    Han, Wonhee; Lee, Hyeyoon; Han, Jin-Kwan

    2017-02-15

    The Tcf/Lef family of transcription factors mediates the Wnt/β-catenin pathway that is involved in a wide range of biological processes, including vertebrate embryogenesis and diverse pathogenesis. Post-translational modifications, including phosphorylation, sumoylation and acetylation, are known to be important for the regulation of Tcf/Lef proteins. However, the importance of ubiquitination and ubiquitin-mediated regulatory mechanisms for Tcf/Lef activity are still unclear. Here, we newly show that ubiquitin C-terminal hydrolase 37 (Uch37), a deubiquitinase, interacts with Tcf7 (formerly named Tcf1) to activate Wnt signalling. Biochemical analyses demonstrated that deubiquitinating activity of Uch37 is not involved in Tcf7 protein stability but is required for the association of Tcf7 to target gene promoter in both Xenopus embryo and human liver cancer cells. In vivo analyses further revealed that Uch37 functions as a positive regulator of the Wnt/β-catenin pathway downstream of β-catenin stabilization that is required for the expression of ventrolateral mesoderm genes during Xenopus gastrulation. Our study provides a new mechanism for chromatin occupancy of Tcf7 and uncovers the physiological significance of Uch37 during early vertebrate development by regulating the Wnt/β-catenin pathway.

  7. Characterization of a glycoside hydrolase family 1 β-galactosidase from hot spring metagenome with transglycosylation activity.

    PubMed

    Gupta, Richa; Govil, Tanvi; Capalash, Neena; Sharma, Prince

    2012-11-01

    A novel, thermostable, alkalophilic β-D-galactosidase (Mbgl) was isolated from a metagenome of geothermal springs in northern Himalayan region of India. Mbgl was 447 amino acids in size and had conserved catalytic residues E170 and E358, indicating that it belonged to family 1 of glycosyl hydrolases showing maximum homology (89 %) with uncharacterized β-galactosidase of Eubacterium, Meiothermus ruber DSM1279. Temperature and pH optima of Mbgl were 65 °C and 8.0 respectively, and it retained 80 % activity even at pH 10.0. Mbgl was active as a homotetramer, recognized β-(1,4)-D-galactoside as the preferred glycosidic bond, and preferentially hydrolyzed pNPgal with K(m) 3.33 mM and k(cat) 2,000 s(-1). It displayed high transglycosylation activity with wide acceptor specificity including hexoses and pentoses leading to the formation of prebiotic galacto-oligosaccharides whereas its lactose hydrolysis potential was low.

  8. Activity-Based Probes linked with Laser-Cleavable Mass Tags for Signal Amplification in Imaging Mass Spectrometry: Analysis of Serine Hydrolase Enzymes in Mammalian Tissue

    PubMed Central

    Yang, Junhai; Chaurand, Pierre; Norris, Jeremy L.; Porter, Ned A.; Caprioli, Richard M.

    2012-01-01

    A novel functional Imaging Mass Spectrometry technology is described that utilizes activity-based probes for imaging enzyme active sites in tissue sections. We demonstrate this technology using an activity-based probe (fluorophosphate) that is specific for serine hydrolases. A dendrimer containing multiple mass tags that is attached to the activity-based probe is used to analyze the binding sites of the probe through release and measurement of the mass tags on laser irradiation. A generation 8 Poly(amido amine) dendrimer with 1024 amino groups was labeled with an azide group and then more than 900 mass tags were attached in order to achieve signal amplification of nearly three orders of magnitude. The experimental protocol first involves binding of the activity-based probe containing an alkyne group to serine hydrolases in the tissue section followed by attachment of the dendrimer labeled with mass tags to the bound probe by Click chemistry. On irradiation of the labeled tissue by the laser beam in a raster pattern, the mass tags are liberated and recorded by the mass analyzer, consequently, the ion image of the mass tag reveals the distribution of serine hydrolases in the tissue. This process was shown using rat brain and mouse embryo sections. Targeted imaging has the advantage of providing high spatial resolution and high sensitivity through the use of signal amplification chemistry with high target specificity through the use of an enzyme activity probe. PMID:22424244

  9. Isolation, Identification and Partial Characterization of a Lactobacillus casei Strain with Bile Salt Hydrolase Activity from Pulque.

    PubMed

    González-Vázquez, R; Azaola-Espinosa, A; Mayorga-Reyes, L; Reyes-Nava, L A; Shah, N P; Rivera-Espinoza, Y

    2015-12-01

    The aim of this study was to isolate, from pulque, Lactobacillus spp. capable of survival in simulated gastrointestinal stress conditions. Nine Gram-positive rods were isolated; however, only one strain (J57) shared identity with Lactobacillus and was registered as Lactobacillus casei J57 (GenBank accession: JN182264). The other strains were identified as Bacillus spp. The most significant observation during the test of tolerance to simulated gastrointestinal conditions (acidity, gastric juice and bile salts) was that L. casei J57 showed a rapid decrease (p ≤ 0.05) in the viable population at 0 h. Bile salts were the stress condition that most affected its survival, from which deoxycholic acid and the mix of bile salts (oxgall) were the most toxic. L. casei J57 showed bile salt hydrolase activity over primary and secondary bile salts as follows: 44.91, 671.72, 45.27 and 61.57 U/mg to glycocholate, taurocholate, glycodeoxycholate and taurodeoxycholate. In contrast, the control strain (L. casei Shirota) only showed activity over tauroconjugates. These results suggest that L. casei J57 shows potential for probiotic applications.

  10. Subsite-specific contributions of different aromatic residues in the active site architecture of glycoside hydrolase family 12

    PubMed Central

    Zhang, Xiaomei; Wang, Shuai; Wu, Xiuyun; Liu, Shijia; Li, Dandan; Xu, Hao; Gao, Peiji; Chen, Guanjun; Wang, Lushan

    2015-01-01

    The active site architecture of glycoside hydrolase (GH) is a contiguous subregion of the enzyme constituted by residues clustered in the three-dimensional space, recognizing the monomeric unit of ligand through hydrogen bonds and hydrophobic interactions. Mutations of the key residues in the active site architecture of the GH12 family exerted different impacts on catalytic efficiency. Binding affinities between the aromatic amino acids and carbohydrate rings were quantitatively determined by isothermal titration calorimetry (ITC) and the quantum mechanical (QM) method, showing that the binding capacity order of Tyr>Trp>His (and Phe) was determined by their side-chain properties. The results also revealed that the binding constant of a certain residue remained unchanged when altering its location, while the catalytic efficiency changed dramatically. Increased binding affinity at a relatively distant subsite, such as the mutant of W7Y at the −4 subsite, resulted in a marked increase in the intermediate product of cellotetraose and enhanced the reactivity of endoglucanase by 144%; while tighter binding near the catalytic center, i.e. W22Y at the −2 subsite, enabled the enzyme to bind and hydrolyze smaller oligosaccharides. Clarification of the specific roles of the aromatics at different subsites may pave the way for a more rational design of GHs. PMID:26670009

  11. Computational study of phosphatase activity in soluble epoxide hydrolase: high efficiency through a water bridge mediated proton shuttle.

    PubMed

    De Vivo, Marco; Ensing, Bernd; Klein, Michael L

    2005-08-17

    Recently, a new branch of fatty acid metabolism has been opened by the novel phosphatase activity found in the N-terminal domain of the, hence bifunctional, soluble epoxide hydrolase (sEH). Importantly, this finding has also provided a new site for drug targeting in sEH's activity regulation. Classical MD and hybrid Car-Parrinello QM/MM calculations have been performed to investigate the reaction mechanism of the phosphoenzyme intermediate formation in the first step of the catalysis. The results support a concerted multi-event reaction mechanism: (1) a dissociative in-line nucleophilic substitution for the phosphoryl transfer reaction; (2) a double proton transfer involved in the formation of a good leaving group in the transition state. The presence of a water bridge in the substrate/enzyme complex allowed an efficient proton shuttle, showing its key role in speeding up the catalysis. The calculated free energy of the favored catalytic pathway is approximately 19 kcal/mol, in excellent agreement with experimental data.

  12. Chemical Reporters for Exploring Protein Acylation

    PubMed Central

    Thinon, Emmanuelle; Hang, Howard C.

    2015-01-01

    Proteins are acylated by a variety of metabolites that regulates many important cellular pathways in all kingdoms of life. Acyl groups in cells can vary in structure from the smallest unit, acetate, to modified long chain fatty acids, all of which can be activated and covalently attached to diverse amino acid side chains and consequently modulate protein function. For example, acetylation of Lys residues can alter the charge state of proteins and generate new recognition elements for protein–protein interactions. Alternatively, long chain fatty-acylation targets proteins to membranes and enables spatial control of cell signalling. To facilitate the analysis of protein acylation in biology, acyl analogues bearing alkyne or azide tags have been developed that enable fluorescent imaging and proteomic profiling of modified proteins using bioorthogonal ligation methods. Herein, we summarize the currently available acylation chemical reporters and highlight their utility to discover and quantify the roles of protein acylation in biology. PMID:25849926

  13. Activation of LXR increases acyl-CoA synthetase activity through direct regulation of ACSL3 in human placental trophoblast cells.

    PubMed

    Weedon-Fekjaer, M Susanne; Dalen, Knut Tomas; Solaas, Karianne; Staff, Anne Cathrine; Duttaroy, Asim K; Nebb, Hilde Irene

    2010-07-01

    Placental fatty acid transport and metabolism are important for proper growth and development of the feto-placental unit. The nuclear receptors, liver X receptors alpha and beta (LXRalpha and LXRbeta), are key regulators of lipid metabolism in many tissues, but little is known about their role in fatty acid transport and metabolism in placenta. The current study investigates the LXR-mediated regulation of long-chain acyl-CoA synthetase 3 (ACSL3) and its functions in human placental trophoblast cells. We demonstrate that activation of LXR increases ACSL3 expression, acyl-CoA synthetase activity, and fatty acid uptake in human tropholast cells. Silencing of ACSL3 in these cells attenuates the LXR-mediated increase in acyl-CoA synthetase activity. Furthermore, we show that ACSL3 is directly regulated by LXR through a conserved LXR responsive element in the ACSL3 promoter. Our results suggest that LXR plays a regulatory role in fatty acid metabolism by direct regulation of ACSL3 in human placental trophoblast cells.

  14. Crystal structure of the cystic fibrosis transmembrane conductance regulator inhibitory factor Cif reveals novel active-site features of an epoxide hydrolase virulence factor.

    PubMed

    Bahl, Christopher D; Morisseau, Christophe; Bomberger, Jennifer M; Stanton, Bruce A; Hammock, Bruce D; O'Toole, George A; Madden, Dean R

    2010-04-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other alpha/beta hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-A resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of alpha/beta hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.

  15. Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor

    SciTech Connect

    Bahl, C.; Morisseau, C; Bomberger, J; Stanton, B; Hammock, B; O' Toole, G; Madden, D

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other {alpha}/{beta} hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-{angstrom} resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of {alpha}/{beta} hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.

  16. Mechanistic studies of ubiquitin C-terminal hydrolase L1.

    PubMed

    Case, April; Stein, Ross L

    2006-02-21

    Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.

  17. Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation

    SciTech Connect

    Boudreaux, David A.; Maiti, Tushar K.; Davies, Christopher W.; Das, Chittaranjan

    2010-07-06

    Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 {angstrom} from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarily at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal {beta}-hairpin at the distal site - a surface-exposed hydrophobic crevice 17 {angstrom} away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 {angstrom} of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.

  18. Activation of TRPA1 channels by the fatty acid amide hydrolase inhibitor 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597).

    PubMed

    Niforatos, Wende; Zhang, Xu-Feng; Lake, Marc R; Walter, Karl A; Neelands, Torben; Holzman, Thomas F; Scott, Victoria E; Faltynek, Connie R; Moreland, Robert B; Chen, Jun

    2007-05-01

    As a member of the transient receptor potential (TRP) ion channel superfamily, the ligand-gated ion channel TRPA1 has been implicated in nociceptive function and pain states. The endogenous ligands that activate TRPA1 remain unknown. However, various agonists have been identified, including environmental irritants (e.g., acrolein) and ingredients of pungent natural products [e.g., allyl isothiocyanate (ITC), cinnamaldehyde, allicin, and gingerol]. In general, these agents are either highly reactive, nonselective, or not potent or efficacious, significantly limiting their utilities in the study of TRPA1 channel properties and biological functions. In a search for novel TRPA1 agonists, we identified 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597), a potent and systemically active inhibitor of fatty acid amide hydrolase (FAAH). This enzyme is responsible for anandamide degradation and therefore has been pursued as an antinociceptive and antiepileptic drug target. Using Ca(2+) influx assays and patch-clamp techniques, we demonstrated that URB597 could activate heterologously expressed human and rat TRPA1 channels, whereas two other FAAH inhibitors (i.e., URB532 and Compound 7) had no effect. When applied to inside-out membrane patches expressing rat TRPA1, URB597 elicited single-channel activities with a unitary conductance of 40 pS. Furthermore, URB597 activated TRPA1 channels endogenously expressed in a population of rat dorsal root ganglion neurons that also responded to ITC. In contrast to its effect on TRPA1, URB597 inhibited TRPM8 and had no effects on TRPV1 or TRPV4. Thus, we conclude that URB597 is a novel agonist of TRPA1 and probably activates the channel through a direct gating mechanism.

  19. Soluble epoxide hydrolase activity regulates inflammatory responses and seizure generation in two mouse models of temporal lobe epilepsy.

    PubMed

    Hung, Yu-Wen; Hung, Shao-Wen; Wu, Yi-Chen; Wong, Lin-King; Lai, Ming-Tsong; Shih, Yang-Hsin; Lee, Tzong-Shyuan; Lin, Yung-Yang

    2015-01-01

    Neuroinflammation is known to be involved in epileptogenesis with unclear mechanisms. Inhibition of soluble epoxide hydrolase (sEH) seems to offer anti-inflammatory protection to ischemic brain injury in rodents. Thus, it is hypothesized that sEH inhibition might also affect the neuroinflammatory responses caused by epileptic seizures. In the present study, we investigated the involvement of sEH in neuroinflammation, seizure generation and subsequent epileptogenesis using two mouse models of temporal lobe epilepsy. Experimental epileptic seizures were induced by either pilocarpine or electrical amygdala kindling in both wild-type (WT) C57BL/6 mice and sEH knockout (sEH KO) mice. The sEH expression in the hippocampus was detected by immunohistochemistry and Western blot analysis. The effects of the sEH hydrolase inhibitors, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy) phenyl)-urea (TPPU), and of the genetic deletion of sEH on seizure-induced neuroinflammatory responses and the development of epilepsy were evaluated. In the hippocampus of WT mice, sEH was mainly expressed in astrocytes (GFAP(+)), neurons (NeuN(+)) and scattered microglia (Iba-1(+)) in the regions of CA1, CA3 and dentate gyrus. Expression of sEH was significantly increased on day 7, 14, 21 and 28 after pilocarpine-induced status epilepticus (SE). Administration with sEH inhibitors attenuated the SE-induced up-regulation of interleukin-1β (IL-1β) and interleukin-6 (IL-6), the degradation of EETs, as well as IκB phosphorylation. Following treatment with AUDA, the frequency and duration of spontaneous motor seizures in the pilocarpine-SE mice were decreased and the seizure-induction threshold of the fully kindled mice was increased. Up-regulation of hippocampal IL-1β and IL-6 was found in both WT and sEH KO mice after successful induction of SE. Notably, sEH KO mice were more susceptible to seizures than WT mice. Seizure related

  20. Identification and characterization of a cold-active phthalate esters hydrolase by screening a metagenomic library derived from biofilms of a wastewater treatment plant.

    PubMed

    Jiao, Yiying; Chen, Xu; Wang, Xin; Liao, Xuewei; Xiao, Lin; Miao, Aijun; Wu, Jun; Yang, Liuyan

    2013-01-01

    A cold-active phthalate esters hydrolase gene (designated dphB) was identified through functional screening of a metagenomic library derived from biofilms of a wastewater treatment plant. The enzyme specifically catalyzed the hydrolysis of dipropyl phthalate, dibutyl phthalate, and dipentyl phthalate to the corresponding monoalkyl phthalate esters at low temperatures. The catalytic triad residues of DphB were proposed to be Ser159, Asp251, and His281.

  1. Antimicrobial activity of human α-defensin 5 and its linear analogs: N-terminal fatty acylation results in enhanced antimicrobial activity of the linear analogs.

    PubMed

    Mathew, Basil; Nagaraj, Ramakrishnan

    2015-09-01

    Human α-defensin 5 (HD5) exhibits broad spectrum antimicrobial activity and plays an important role in mucosal immunity of the small intestine. Although there have been several studies, the structural requirements for activity and mechanism of bacterial killing is yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD5 and linear analogs. Cysteine deletions attenuated the antibacterial activity considerably. Candidacidal activity was affected to a lesser extent. Fatty acid conjugated linear analogs showed antimicrobial activity comparable activity to HD5. Effective surface charge neutralization of bacteria was observed for HD5 as compared to the non-fatty acylated linear analogs. Our results show that HD5 and non-fatty acylated linear analogs enter the bacterial cytoplasm without causing damage to the bacterial inner membrane. Although fatty acylated peptides exhibited antimicrobial activity comparable to HD5, their mechanism of action involved permeabilization of the Escherichia coli inner membrane. HD5 and analogs had the ability to bind plasmid DNA. HD5 had greater binding affinity to plasmid DNA as compared to the analogs. The three dimensional structure of HD5 favors greater interaction with the bacterial cell surface and also with DNA. Antibacterial activity of HD5 involves entry into bacterial cytoplasm and binding to DNA which would result in shut down of the bacterial metabolism leading to cell death. We show how a moderately active linear peptide derived from the α-defensin HD5 can be engineered to enhance antimicrobial activity almost comparable to the native peptide.

  2. The multiple acyl-coenzyme A dehydrogenation disorders, glutaric aciduria type II and ethylmalonic-adipic aciduria. Mitochondrial fatty acid oxidation, acyl-coenzyme A dehydrogenase, and electron transfer flavoprotein activities in fibroblasts.

    PubMed Central

    Amendt, B A; Rhead, W J

    1986-01-01

    The multiple acyl-coenzyme A (CoA) dehydrogenation disorders (MAD) include severe (S) and mild (M) variants, glutaric aciduria type II (MAD:S) and ethylmalonic-adipic aciduria (MAD:M). Intact MAD:M mitochondria oxidized [1-14C]octanoate, [1-14C]palmityl-CoA, and [1,5-14C]glutarate at 20-46% of control levels; MAD:S mitochondria oxidized these three substrates at 0.4-18% of control levels. In MAD:M mitochondria, acyl-CoA dehydrogenase (ADH) activities were similar to control, whereas MAD:S ADH activities ranged from 38% to 73% of control. Electron transfer flavoprotein (ETF) activities in five MAD:M cell lines ranged from 29 to 51% of control (P less than 0.01); ETF deficiency was the primary enzymatic defect in two MAD:M lines. In four MAD:S patients, ETF activities ranged from 3% to 6% of control (P less than 0.001); flavin adenine dinucleotide addition increased residual ETF activity from 4% to 21% of control in a single MAD:S line (P less than 0.01). Three MAD:S patients had ETF activities ranging from 33 to 53% of control; other investigators found deficient ETF-dehydrogenase activity in these MAD:S and three of our MAD:M cell lines. PMID:3722376

  3. Hydrolase and fructosyltransferase activities implicated in the accumulation of different chain size fructans in three Asteraceae species.

    PubMed

    Itaya, Nair M; Asega, Amanda F; Carvalho, Maria Angela M; Figueiredo-Ribeiro, Rita de Cássia L

    2007-09-01

    Fructans are widely distributed in Asteraceae from floras with seasonal growth and are thought to be involved in drought and freezing tolerance, in addition to storage function. Reserve organs of Vernonia herbacea and Viguiera discolor, from the cerrado, and of the perennial herb Smallanthus sonchifolius, endemic to Andean region, store over 80% inulin, with different DP (35, 150, and 15, respectively). The fructan pattern in Asteraceae species could be explained by characteristics of their respective 1-FFTs. Hydrolases and fructosyltransferases from S. sonchifolius, V. herbacea and V. discolor were analyzed in plants at the same environmental conditions. The higher 1-FEH activities found in the species with lower DP, S. sonchifolius and V. herbacea reinforce the hypothesis of the involvement of 1-FEH in fructan profile and suggest that the high DP fructan of V. discolor is a consequence of the low affinity of its 1-FEH to the native long chain inulin. Long term incubation with sucrose suggested that the affinity of 1-FFT of V. discolor for 1-kestose is low when compared to that of V. herbacea. Indeed 1-FFT from V. discolor was shown to be an hDP 1-FFT, preferring longer inulins as acceptors. Conversely, 1-FFT from V. herbacea seems to have a higher affinity for short fructo-oligosaccharides, including 1-kestose, as acceptor substrates. Differences in fructan enzymes of the three Asteraceae provide new information towards the understanding of fructan metabolism and control of carbon flow between low and high DP fructans.

  4. Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis

    PubMed Central

    2016-01-01

    Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb “Hydrolase important for pathogenesis 1” (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections. PMID:27739665

  5. Neonatal Fc receptor stimulation induces ubiquitin c-terminal hydrolase-1 overexpression in podocytes through activation of p38 mitogen-activated protein kinase.

    PubMed

    Gan, Hualei; Feng, Songtao; Wu, Huijuan; Sun, Yu; Hu, Ruimin; Zhao, Zhonghua; Zhang, Zhigang

    2012-09-01

    Ubiquitin c-terminal hydrolase-1 is overexpressed in renal podocytes in some immune complex-mediated glomerulonephritides, an effect closely related to extensive podocyte injury. Neonatal Fc receptor is newly recognized to be present on human renal podocytes. It is presumed that neonatal Fc receptor serves as a sensor for immune stimulation transduction and is involved in the pathogenesis of podocyte injury. In our current study, we found that neonatal Fc receptor was constitutively expressed in normal podocytes and up-regulated by immune stimulation induced by antithymocyte serum. An increase in neonatal Fc receptor expression was observed in human podocytes within diseased glomeruli in 97 cases of various glomerulonephritides. The expression percentage was significantly higher in immune-mediated disease, including membranous nephropathy (46.7%), immunoglobin A nephropathy (66.7%), lupus nephritis (87.5%), and acute proliferative glomerulonephritis (100%), than in normal kidney samples (16.7%) (P < .05), whereas there was no significant difference between minimal-change disease and normal kidney. Further study showed that neonatal Fc receptor up-regulated the expression of ubiquitin c-terminal hydrolase-1 via activation of p38 in podocytes subjected to immune stimulation in vitro. These data suggest that neonatal Fc receptor acts as an immune sensor that evokes an inflammatory response, which may lead to functional and morphological changes in podocytes in glomerulonephritides.

  6. The Antioxidant and Starch Hydrolase Inhibitory Activity of Ten Spices in an In Vitro Model of Digestion: Bioaccessibility of Anthocyanins and Carotenoids

    PubMed Central

    Jayawardena, Nilakshi; Watawana, Mindani I.; Jayathilaka, Ruchini T.; Waisundara, Viduranga Y.

    2015-01-01

    The antioxidant and starch hydrolase inhibitory activities of cardamom, cloves, coriander, cumin seeds, curry leaves, fenugreek, mustard seeds, nutmeg, sweet cumin, and star anise extracts were investigated in an in vitro model of digestion mimicking the gastric and duodenal conditions. The total phenolic contents in all spice extracts had statistically significantly (P < 0.05) increased following both gastric and duodenal digestion. This was also in correlation with the antioxidant assays quantifying the water-soluble antioxidant capacity of the extracts. The lipophilic Oxygen Radical Absorbance Capacity assay did not indicate a statistically significant change in the values during any of the digestion phases. Statistically significant (P < 0.05) reductions in the anthocyanin contents were observed during the digestion phases in contrast to the carotenoid contents. With the exception of the cumin seed extract, none of the spice extracts showed statistically significant changes in the initial starch hydrolase enzyme inhibitory values prior to gastric and duodenal digestion. In conclusion, this study was able to prove that the 10 spices were a significant source of total phenolics, antioxidant, and starch hydrolase inhibitory activities. PMID:26693245

  7. The Antioxidant and Starch Hydrolase Inhibitory Activity of Ten Spices in an In Vitro Model of Digestion: Bioaccessibility of Anthocyanins and Carotenoids.

    PubMed

    Jayawardena, Nilakshi; Watawana, Mindani I; Jayathilaka, Ruchini T; Waisundara, Viduranga Y

    2015-01-01

    The antioxidant and starch hydrolase inhibitory activities of cardamom, cloves, coriander, cumin seeds, curry leaves, fenugreek, mustard seeds, nutmeg, sweet cumin, and star anise extracts were investigated in an in vitro model of digestion mimicking the gastric and duodenal conditions. The total phenolic contents in all spice extracts had statistically significantly (P < 0.05) increased following both gastric and duodenal digestion. This was also in correlation with the antioxidant assays quantifying the water-soluble antioxidant capacity of the extracts. The lipophilic Oxygen Radical Absorbance Capacity assay did not indicate a statistically significant change in the values during any of the digestion phases. Statistically significant (P < 0.05) reductions in the anthocyanin contents were observed during the digestion phases in contrast to the carotenoid contents. With the exception of the cumin seed extract, none of the spice extracts showed statistically significant changes in the initial starch hydrolase enzyme inhibitory values prior to gastric and duodenal digestion. In conclusion, this study was able to prove that the 10 spices were a significant source of total phenolics, antioxidant, and starch hydrolase inhibitory activities.

  8. High-level expression of active recombinant ubiquitin carboxyl-terminal hydrolase of Drosophila melanogaster in Pichia pastoris.

    PubMed

    Jin, Feng-liang; Xu, Xiao-xia; Yu, Xiao-qiang; Ren, Shun-xiang

    2009-06-01

    Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin. The high specificity for the recognition site makes UCHs useful enzymes for in vitro cleavage of ubiquitin fusion proteins. In this work, an active C-terminal His-tagged UCH from Drosophila melanogaster (DmUCH) was produced as a secretory form in a recombinant strain of the methylotrophic yeast Pichia pastoris. The production of recombinant DmUCH by Mut(s) strain was much higher than that by Mut(+) strain, which was confirmed by Western blot analysis. When expression was induced at pH 6.0 in a BMMY/methanol medium, the concentration of recombinant DmUCH reached 210 mg l(-1). With the (His)(6)-tag, the recombinant DmUCH was easily purified by Ni-NTA chromatography and 18 mg pure active DmUCH were obtained from 100ml culture broth supernatant. Ubiquitin-magainin fusion protein was efficiently cleaved by DmUCH, yielding recombinant magainin with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant magainin was purified to homogeneity easily by reversed-phase HPLC. Analysis of the recombinant magainin by ESI-MS showed that the molecular weight of the purified recombinant magainin was 2465 Da, which perfectly matches the mass calculated from the amino acid sequence. The result of mass spectrometry confirmed that the purified His-tagged DmUCH can recognize the ubiquitin-magainin fusion protein and cleave it at the carboxyl terminus of ubiquitin precisely. Our results showed that P. pastoris is a robust system to express the secreted form of DmUCH.

  9. Rational Design of Fatty Acid Amide Hydrolase Inhibitors that Act by Covalently Bonding to Two Active Site Residues

    PubMed Central

    Otrubova, Katerina; Brown, Monica; McCormick, Michael S.; Han, Gye W.; O’Neal, Scott T.; Cravatt, Benjamin F.; Stevens, Raymond C.; Lichtman, Aron H.; Boger, Dale L.

    2013-01-01

    The design and characterization of α-ketoheterocycle fatty acid amide hydrolase (FAAH) inhibitors are disclosed that additionally and irreversibly target a cysteine (Cys269) found in the enzyme cytosolic port while maintaining the reversible covalent Ser241 attachment responsible for their rapid and initially reversible enzyme inhibition. Two α-ketooxazoles (3 and 4) containing strategically placed electrophiles at the C5 position of the pyridyl substituent of 2 (OL-135) were prepared and examined as inhibitors of FAAH. Consistent with the observed time-dependent non-competitive inhibition, the co-crystal X-ray structure of 3 bound to a humanized variant of rat FAAH revealed that 3 was not only covalently bound to the active site catalytic nucleophile Ser241 as a deprotonated hemiketal, but also to Cys269 through the pyridyl C5-substituent, thus providing an inhibitor with dual covalent attachment in the enzyme active site. In vivo characterization of the prototypical inhibitors in mice demonstrate that they raise endogenous brain levels of FAAH substrates to a greater extent and for a much longer duration (>6 h) than the reversible inhibitor 2, indicating that the inhibitors accumulate and persist in the brain to completely inhibit FAAH for a prolonged period. Consistent with this behavior and the targeted irreversible enzyme inhibition, 3 reversed cold allodynia in the chronic constriction injury model of neuropathic pain in mice for a sustained period (>6 h) beyond that observed with the reversible inhibitor 2, providing effects that were unchanged over the 1–6 h time course monitored. PMID:23581831

  10. Changes in bile acids, FGF-19 and sterol absorption in response to bile salt hydrolase active L. reuteri NCIMB 30242

    PubMed Central

    Martoni, Christopher J; Labbé, Alain; Ganopolsky, Jorge G; Prakash, Satya; Jones, Mitchell L

    2015-01-01

    The size and composition of the circulating bile acid (BA) pool are important factors in regulating the human gut microbiota. Disrupted regulation of BA metabolism is implicated in several chronic diseases. Bile salt hydrolase (BSH)-active Lactobacillus reuteri NCIMB 30242, previously shown to decrease LDL-cholesterol and increase circulating BA, was investigated for its dose response effect on BA profile in a pilot clinical study. Ten otherwise healthy hypercholesterolemic adults, recruited from a clinical trial site in London, ON, were randomized to consume delayed release or standard release capsules containing L. reuteri NCIMB 30242 in escalating dose over 4 weeks. In another aspect, 4 healthy normocholesterolemic subjects with LDL-C below 3.4 mmol/l received delayed release L. reuteri NCIMB 30242 at a constant dose over 4 weeks. The primary outcome measure was the change in plasma BA profile over the intervention period. Additional outcomes included circulating fibroblast growth factor (FGF)-19, plant sterols and LDL-cholesterol as well as fecal microbiota and bsh gene presence. After one week of intervention subjects receiving delayed release L. reuteri NCIMB 30242 increased total BA by 1.13 ± 0.67 μmol/l (P = 0.02), conjugated BA by 0.67 ± 0.39 μmol/l (P = 0.02) and unconjugated BA by 0.46 ± 0.43 μmol/l (P = 0.07), which represented a greater than 2-fold change relative to baseline. Increases in BA were largely maintained post-week 1 and were generally correlated with FGF-19 and inversely correlated with plant sterols. This is the first clinical support showing that a BSH-active probiotic can significantly and rapidly influence BA metabolism and may prove useful in chronic diseases beyond hypercholesterolemia. PMID:25612224

  11. Silica Gel for Enhanced Activity and Hypochlorite Protection of Cyanuric Acid Hydrolase in Recombinant Escherichia coli

    PubMed Central

    Radian, Adi; Aukema, Kelly G.; Aksan, Alptekin

    2015-01-01

    ABSTRACT Chlorinated isocyanuric acids are widely used water disinfectants that generate hypochlorite, but with repeated application, they build up cyanuric acid (CYA) that must be removed to maintain disinfection. 3-Aminopropyltriethoxysilane (APTES)-treated Escherichia coli cells expressing cyanuric acid hydrolase (CAH) from Moorella thermoacetica exhibited significantly high CYA degradation rates and provided protection against enzyme inactivation by hypochlorite (chlorine). APTES coating or encapsulation of cells had two benefits: (i) overcoming diffusion limitations imposed by the cell wall and (ii) protecting against hypochlorite inactivation of CAH activity. Cells encapsulated in APTES gels degraded CYA three times faster than nonfunctionalized tetraethoxysilane (TEOS) gels, and cells coated with APTES degraded CYA at a rate of 29 µmol/min per mg of CAH protein, similar to the rate with purified enzyme. UV spectroscopy, fluorescence spectroscopy, and scanning electron microscopy showed that the higher rates were due to APTES increasing membrane permeability and enhancing cyanuric acid diffusion into the cytoplasm to reach the CAH enzyme. Purified CAH enzyme was shown to be rapidly inactivated by hypochlorite. APTES aggregates surrounding cells protected via the amine groups reacting with hypochlorite as shown by pH changes, zeta potential measurements, and infrared spectroscopy. APTES-encapsulated E. coli cells expressing CAH degraded cyanuric acid at high rates in the presence of 1 to 10 ppm hypochlorite, showing effectiveness under swimming pool conditions. In contrast, CAH activity in TEOS gels or free cells was completely inactivated by hypochlorite. These studies show that commercially available silica materials can selectively enhance, protect, and immobilize whole-cell biocatalysts for specialized applications. PMID:26530383

  12. Characterisation of the antibacterial properties of a bacterial derived peptidoglycan hydrolase (LysCs4), active against C. sakazakii and other Gram-negative food-related pathogens.

    PubMed

    Endersen, Lorraine; Coffey, Aidan; Ross, R Paul; McAuliffe, Olivia; Hill, Colin; O'Mahony, Jim

    2015-12-23

    Illness caused by the consumption of contaminated food products continues to represent one of the main challenges facing food manufacturers worldwide. Even with current intervention technologies and increased hygiene measures, foodborne illness remains a significant threat to public health. This coupled with the increasing emergence of multidrug resistant pathogens has increased the need for the development of novel technologies for pathogen control. Bacterial derived peptidoglycan hydrolases represent a vast and highly diverse group of enzymes with potential for biocontrol of a range of Gram-positive and Gram-negative foodborne pathogens. In this study, we describe the identification, cloning, expression and purification of a peptidoglycan hydrolase (LysCs4) derived from Cronobacter sakazakii for biocontrol of the aforementioned infant formula pathogen itself. In silico analysis of LysCs4 revealed the gene to display greatest sequence similarity to a putative lysozyme encoded by the lytic Cronobacter phage ES2. Conserved domain analysis of LysCs4 revealed the presence of a single catalytic domain predicted to display O-Glycosyl hydrolase activity and to be a member of the GH24 family. The ability of this enzyme to hydrolyse the peptidoglycan of 25 Gram-negative strains, across 4 different genera, highlights its potential as a novel candidate for biocontrol of C. sakazakii and other Gram-negative food related pathogens.

  13. Impact of self-assembly properties on antibacterial activity of short acyl-lysine oligomers.

    PubMed

    Sarig, Hadar; Rotem, Shahar; Ziserman, Lior; Danino, Dganit; Mor, Amram

    2008-12-01

    We investigated both the structural and functional consequences of modifying the hydrophobic, lipopeptide-mimetic oligo-acyl-lysine (OAK) N(alpha)-hexadecanoyl-l-lysyl-l-lysyl-aminododecanoyl-l-lysyl-amide (c(16)KKc(12)K) to its unsaturated analog hexadecenoyl-KKc(12)K [c(16(omega7))KKc(12)K]. Despite similar tendencies for self-assembly in solution (critical aggregation concentrations, approximately 10 muM), the analogous OAKs displayed dissimilar antibacterial properties (e.g., bactericidal kinetics taking minutes versus hours). Diverse experimental evidence provided insight into these discrepancies: whereas c(16(omega7))KKc(12)K created wiry interconnected nanofiber networks, c(16)KKc(12)K formed both wider and stiffer fibers which displayed distinct binding properties to phospholipid membranes. Unsaturation also shifted their gel-to-liquid transition temperatures and altered their light-scattering properties, suggesting the disassembly of c(16(omega7))KKc(12)K in the presence of bacteria. Collectively, the data indicated that the higher efficiency in interfering with bacterial viability emanated from a wobbly packing imposed by a single double bond. This suggests that similar strategies might improve hydrophobic OAKs and related lipopeptide antibiotics.

  14. Modulation of hexa-acyl pyrophosphate lipid A population under Escherichia coli phosphate (Pho) regulon activation.

    PubMed

    Lamarche, Martin G; Kim, Sang-Hyun; Crépin, Sébastien; Mourez, Michael; Bertrand, Nicolas; Bishop, Russell E; Dubreuil, J Daniel; Harel, Josée

    2008-08-01

    Environmental phosphate is an important signal for microorganism gene regulation, and it has recently been shown to trigger some key bacterial virulence mechanisms. In many bacteria, the Pho regulon is the major circuit involved in adaptation to phosphate limitation. The Pho regulon is controlled jointly by the two-component regulatory system PhoR/PhoB and by the phosphate-specific transport (Pst) system, which both belong to the Pho regulon. We showed that a pst mutation results in virulence attenuation in extraintestinal pathogenic Escherichia coli (ExPEC) strains. Our results indicate that the bacterial cell surface of the pst mutants is altered. In this study, we show that pst mutants of ExPEC strains display an increased sensitivity to different cationic antimicrobial peptides and vancomycin. Remarkably, the hexa-acylated 1-pyrophosphate form of lipid A is significantly less abundant in pst mutants. Among differentially expressed genes in the pst mutant, lpxT coding for an enzyme that transfers a phosphoryl group to lipid A, forming the 1-diphosphate species, was found to be downregulated. Our results strongly suggest that the Pho regulon is involved in lipid A modifications, which could contribute to bacterial surface perturbations. Since the Pho regulon and the Pst system are conserved in many bacteria, such a lipid A modification mechanism could be widely distributed among gram-negative bacterial species.

  15. Suppression of long chain acyl-CoA synthetase 3 decreases hepatic de novo fatty acid synthesis through decreased transcriptional activity.

    PubMed

    Bu, So Young; Mashek, Mara T; Mashek, Douglas G

    2009-10-30

    Long chain acyl-CoA synthetases (ACSL) and fatty acid transport proteins (FATP) activate fatty acids to acyl-CoAs in the initial step of fatty acid metabolism. Numerous isoforms of ACSL and FATP exist with different tissue distribution patterns, intracellular locations, and substrate preferences, suggesting that each isoform has distinct functions in channeling fatty acids into different metabolic pathways. Because fatty acids, acyl-CoAs, and downstream lipid metabolites regulate various transcription factors that control hepatic energy metabolism, we hypothesized that ACSL or FATP isoforms differentially regulate hepatic gene expression. Using small interference RNA (siRNA), we knocked down each liver-specific ACSL and FATP isoform in rat primary hepatocyte cultures and subsequently analyzed reporter gene activity of numerous transcription factors and performed quantitative mRNA analysis of their target genes. Compared with control cells, which were transfected with control siRNA, knockdown of acyl-CoA synthetase 3 (ACSL3) significantly decreased reporter gene activity of several lipogenic transcription factors such as peroxisome proliferator activation receptor-gamma, carbohydrate-responsive element-binding protein, sterol regulatory element-binding protein-1c, and liver X receptor-alpha and the expression of their target genes. These findings were further supported by metabolic labeling studies that showed [1-(14)C]acetate incorporation into lipid extracts was decreased in cells treated with ACSL3 siRNAs and that ACSL3 expression is up-regulated in ob/ob mice and mice fed a high sucrose diet. ACSL3 knockdown decreased total acyl-CoA synthetase activity without substantially altering the expression of other ACSL isoforms. In summary, these results identify a novel role for ACSL3 in mediating transcriptional control of hepatic lipogenesis.

  16. Structural insights into the γ-lactamase activity and substrate enantioselectivity of an isochorismatase-like hydrolase from Microbacterium hydrocarbonoxydans

    PubMed Central

    Gao, Shuaihua; Zhou, Yu; Zhang, Weiwei; Wang, Wenhe; Yu, You; Mu, Yajuan; Wang, Hao; Gong, Xinqi; Zheng, Guojun; Feng, Yue

    2017-01-01

    (+)-γ-lactamase catalyzes the specific hydrolysis of (+)-γ-lactam out of the racemic γ-lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one) to leave optically pure (−)-γ-lactam, which is the key building block of antiviral drugs such as carbovir and abacavir. However, no structural data has been reported on how the enzymes bind the γ-lactams and achieve their enantioselectivities. We previously identified an isochorismatase-like hydrolase (IHL, Mh33H4-5540) with (+)-γ-lactamase activity, which constitutes a novel family of γ-lactamase. Here, we first discovered that this enzyme actually hydrolyzed both (+)- and (−)-γ-lactam, but with apparently different specificities. We determined the crystal structures of the apo-form, (+)-γ-lactam bound, and (−)-γ-lactam bound forms of the enzyme. The structures showed that the binding sites of both (+) and (−)-γ-lactam resemble those of IHLs, but the “cover” loop conserved in IHLs is lacking in the enzyme, probably resulting in its incomplete enantioselectivity. Structural, biochemical, and molecular dynamics simulation studies demonstrated that the steric clash caused by the binding-site residues, especially the side-chain of Cys111 would reduce the binding affinity of (−)-γ-lactam and possibly the catalytic efficiency, which might explain the different catalytic specificities of the enantiomers of γ-lactam. Our results would facilitate the directed evolution and application of Mh33H4-5540 in antiviral drug synthesis. PMID:28295028

  17. Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

    PubMed Central

    Vandavasi, Venu Gopal; Cooper, Jonathan B.; Ginell, Stephan L.

    2016-01-01

    ABSTRACT The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed. PMID:27795378

  18. Bacterial lipopolysaccharide suppresses the production of catalytically active lysosomal acid hydrolases in human macrophages

    PubMed Central

    1986-01-01

    Sub-microgram quantities of bacterial lipopolysaccharide (LPS) have been found to substantially reduce the intracellular catalytic activities of three representative lysosomal enzymes (namely, acid phosphatase, hexosaminidase, and beta-glucuronidase) in human monocyte- derived macrophages. This response was not associated with a concurrent increase in enzyme catalytic activity in the culture supernatant, and hence, could not be explained by mobilization of preformed material. By conducting experiments in the presence and absence of indomethacin, a cyclooxygenase inhibitor, the reduction in lysosomal enzyme catalytic activities was shown not to be dependent on the ability of LPS to induce prostaglandin E2 production. The response was not found to be the result of a more generalized LPS-dependent reduction in the ability of the cells to synthesize protein, since the presence of LPS in macrophage cultures did not appreciably affect the amount of [35S]methionine incorporated into total cellular proteins. A kinetic analysis of the effect of LPS on the down-regulation of enzyme catalytic activities indicated that this was an early response of the cells to LPS exposure. An investigation of the effects of blockade of enzyme catabolism (using the lysosomotropic weak-base, methylamine) indicated that the reduction of catalytic enzyme activities in response to LPS was probably due to a decreased rate of production of active product, rather than an enhanced rate of enzyme catabolism. This suggestion was confirmed by experiments in which the synthesis of pro- hexosaminidase (measured by biosynthetic labeling with [35S]methionine and specific immunoprecipitation of labeled pro-hexosaminidase) was found to be reduced by 42% after a 24-h exposure to LPS (although the synthesis of complement component C3 was stimulated by a factor of 4.5). It is suggested that the ability of LPS to regulate the functional expression of protein products contributes to changes in the overall

  19. Modification of soil microbial activity and several hydrolases in a forest soil artificially contaminated with copper

    NASA Astrophysics Data System (ADS)

    Bellas, Rosa; Leirós, Mā Carmen; Gil-Sotres, Fernando; Trasar-Cepeda, Carmen

    2010-05-01

    Soils have long been exposed to the adverse effects of human activities, which negatively affect soil biological activity. As a result of their functions and ubiquitous presence microorganisms can serve as environmental indicators of soil pollution. Some features of soil microorganisms, such as the microbial biomass size, respiration rate, and enzyme activity are often used as bioindicators of the ecotoxicity of heavy metals. Although copper is essential for microorganisms, excessive concentrations have a negative influence on processes mediated by microorganisms. In this study we measured the response of some microbial indicators to Cu pollution in a forest soil, with the aim of evaluating their potential for predicting Cu contamination. Samples of an Ah horizon from a forest soil under oakwood vegetation (Quercus robur L.) were contaminated in the laboratory with copper added at different doses (0, 120, 360, 1080 and 3240 mg kg-1) as CuCl2×2H2O. The soil samples were kept for 7 days at 25 °C and at a moisture content corresponding to the water holding capacity, and thereafter were analysed for carbon and nitrogen mineralization capacity, microbial biomass C, seed germination and root elongation tests, and for urease, phosphomonoesterase, catalase and ß-glucosidase activities. In addition, carbon mineralization kinetics were studied, by plotting the log of residual C against incubation time, and the metabolic coefficient, qCO2, was estimated. Both organic carbon and nitrogen mineralization were lower in polluted samples, with the greatest decrease observed in the sample contaminated with 1080 mg kg-1. In all samples carbon mineralization followed first order kinetics; the C mineralization constant was lower in contaminated than in uncontaminated samples and, in general, decreased with increasing doses of copper. Moreover, it appears that copper contamination not only reduced the N mineralization capacity, but also modified the N mineralization process, since in

  20. The adjuvant activity of fatty acid esters. The role of acyl chain length and degree of saturation.

    PubMed Central

    Bomford, R

    1981-01-01

    Water-in-oil emulsions of metabolizable fatty acid esters, with the non-toxic surfactant Pluronic L122 as emulsifying agent, potentiated the humoral response to bovine serum albumin and staphylococcal toxoid in the mouse. Adjuvant activity was increased by changing the chemical nature of the esters as follows: (i) using a series of ethyl esters, adjuvant activity appeared when the acyl chain length of the fatty acid component was 16 or greater; (ii) isobutyl and isopropyl esters of palmitic acid (C16:0) were superior to ethyl; (iii) the ethyl esters of oleic (C18:1) and linoleic (C18:2) acids were better than stearic (C18:0). Since emulsions prepared with longer chain saturated esters are very viscous or solid at room temperature, and unsaturated esters are chemically reactive, emulsions were prepared with differing proportions of ethyl caprate (C10:0) and butyl stearate. At a ratio of 9:1 the emulsions possessed the low viscosity of ethyl caprate, but gained the adjuvant activity of butyl stearate. 125I-labelled BSA was retained in the footpad to a significantly greater extent than with a caprate emulsion, but reasons are given for believing that slow release of antigen is not the only mechanism of adjuvant activity. The ester emulsions caused more acute but less chronic local inflammation (footpad swelling) than Freund's incomplete adjuvant. PMID:7275184

  1. Acyl-CoA-binding domain containing 3 modulates NAD+ metabolism through activating poly(ADP-ribose) polymerase 1.

    PubMed

    Chen, Yong; Bang, Sookhee; Park, Soohyun; Shi, Hanyuan; Kim, Sangwon F

    2015-07-15

    NAD(+) plays essential roles in cellular energy homoeostasis and redox state, functioning as a cofactor along the glycolysis and citric acid cycle pathways. Recent discoveries indicated that, through the NAD(+)-consuming enzymes, this molecule may also be involved in many other cellular and biological outcomes such as chromatin remodelling, gene transcription, genomic integrity, cell division, calcium signalling, circadian clock and pluripotency. Poly(ADP-ribose) polymerase 1 (PARP1) is such an enzyme and dysfunctional PARP1 has been linked with the onset and development of various human diseases, including cancer, aging, traumatic brain injury, atherosclerosis, diabetes and inflammation. In the present study, we showed that overexpressed acyl-CoA-binding domain containing 3 (ACBD3), a Golgi-bound protein, significantly reduced cellular NAD(+) content via enhancing PARP1's polymerase activity and enhancing auto-modification of the enzyme in a DNA damage-independent manner. We identified that extracellular signal-regulated kinase (ERK)1/2 as well as de novo fatty acid biosynthesis pathways are involved in ACBD3-mediated activation of PARP1. Importantly, oxidative stress-induced PARP1 activation is greatly attenuated by knocking down the ACBD3 gene. Taken together, these findings suggest that ACBD3 has prominent impacts on cellular NAD(+) metabolism via regulating PARP1 activation-dependent auto-modification and thus cell metabolism and function.

  2. Activity-based metagenomic screening and biochemical characterization of bovine ruminal protozoan glycoside hydrolases.

    PubMed

    Findley, Seth D; Mormile, Melanie R; Sommer-Hurley, Andrea; Zhang, Xue-Cheng; Tipton, Peter; Arnett, Krista; Porter, James H; Kerley, Monty; Stacey, Gary

    2011-11-01

    The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.

  3. Improving the promiscuous nerve agent hydrolase activity of a thermostable archaeal lactonase.

    PubMed

    Merone, Luigia; Mandrich, Luigi; Porzio, Elena; Rossi, Mosé; Müller, Susanne; Reiter, Georg; Worek, Franz; Manco, Giuseppe

    2010-12-01

    The thermostable Phosphotriesterase-Like Lactonase from Sulfolobus solfataricus (SsoPox) hydrolyzes lactones and, at a lower rate, neurotoxic organophosphorus compounds. The persistent demand of detoxification tools in the field of agricultural wastes and restoring of conditions after terrorist acts prompted us to exploit SsoPox as a "starter" to evolve its ancillary nerve agents hydrolytic capability. A directed evolution strategy yielded, among several variants, the single mutant W263F with k(cat) and specificity constant against paraoxon 16- and 6-fold enhanced, respectively, compared to the wild type. Furthermore, a phenomenon of enzyme activation by SDS has been observed, which allowed to increase those values 150- and 28-fold, respectively. The activity of SsoPox against the deadly nerve gas Cyclosarin has been reported for the first time and proved to be substantially unaffected for variant W263F. Finally, outperforming efficiency of W263F was demonstrated, under severe stressing conditions, with respect to the best known phosphotriesterase PTE from Brevundimonas diminuta.

  4. Ubiquitin C-terminal hydrolase L1 (UCH-L1) acts as a novel potentiator of cyclin-dependent kinases to enhance cell proliferation independently of its hydrolase activity.

    PubMed

    Kabuta, Tomohiro; Mitsui, Takeshi; Takahashi, Masaki; Fujiwara, Yuuki; Kabuta, Chihana; Konya, Chiho; Tsuchiya, Yukihiro; Hatanaka, Yusuke; Uchida, Kenko; Hohjoh, Hirohiko; Wada, Keiji

    2013-05-03

    Dysregulation of cell proliferation and the cell cycle are associated with various diseases, such as cancer. Cyclin-dependent kinases (CDKs) play central roles in cell proliferation and the cell cycle. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed in a restricted range of tissues, including the brain and numerous types of cancer. However, the molecular functions of UCH-L1 remain elusive. In this study, we found that UCH-L1 physically interacts with CDK1, CDK4, and CDK5, enhancing their kinase activity. Using several mutants of UCH-L1, we showed that this enhancement is dependent upon interaction levels between UCH-L1 and CDKs but is independent of the known ubiquitin-related functions of UCH-L1. Gain- and loss-of-function studies revealed that UCH-L1 enhances proliferation of multiple cell types, including human cancer cells. Inhibition of the interaction between UCH-L1 and cell cycle-associated CDK resulted in the abolishment of UCH-L1-induced enhancement of cell proliferation. RNA interference of UCH-L1 reduced the growth of human xenograft tumors in mice. We concluded that UCH-L1 is a novel regulator of the kinase activities of CDKs. We believe that our findings from this study will significantly contribute to our understanding of cell cycle-associated diseases.

  5. Mycobacteria Encode Active and Inactive Classes of TesB Fatty-Acyl CoA Thioesterases Revealed through Structural and Functional Analysis.

    PubMed

    Swarbrick, Crystall M D; Bythrow, Glennon V; Aragao, David; Germain, Gabrielle A; Quadri, Luis E N; Forwood, Jade K

    2017-03-14

    Mycobacteria contain a large number of highly divergent species and exhibit unusual lipid metabolism profiles, believed to play important roles in immune invasion. Thioesterases modulate lipid metabolism through the hydrolysis of activated fatty-acyl CoAs; multiple copies are present in mycobacteria, yet many remain uncharacterized. Here, we undertake a comprehensive structural and functional analysis of a TesB thioesterase from Mycobacterium avium (MaTesB). Structural superposition with other TesB thioesterases reveals that the Asp active site residue, highly conserved across a wide range of TesB thioesterases, is mutated to Ala. Consistent with these structural data, the wild-type enzyme failed to hydrolyze an extensive range of acyl-CoA substrates. Mutation of this residue to an active Asp residue restored activity against a range of medium-chain length fatty-acyl CoA substrates. Interestingly, this Ala mutation is highly conserved across a wide range of Mycobacterium species but not found in any other bacteria or organism. Our structural homology analysis revealed that at least one other TesB acyl-CoA thioesterase also contains an Ala residue at the active site, while two other Mycobacterium TesB thioesterases harbor an Asp residue at the active site. The inactive TesBs display a common quaternary structure that is distinct from that of the active TesB thioesterases. Investigation of the effect of expression of either the catalytically active or inactive MaTesB in Mycobacterium smegmatis exposed, to the best of our knowledge, the first genotype-phenotype association implicating a mycobacterial tesB gene. This is the first report that mycobacteria encode active and inactive forms of thioesterases, the latter of which appear to be unique to mycobacteria.

  6. Rational design of broad spectrum antibacterial activity based on a clinically relevant enoyl-acyl carrier protein (ACP) reductase inhibitor.

    PubMed

    Schiebel, Johannes; Chang, Andrew; Shah, Sonam; Lu, Yang; Liu, Li; Pan, Pan; Hirschbeck, Maria W; Tareilus, Mona; Eltschkner, Sandra; Yu, Weixuan; Cummings, Jason E; Knudson, Susan E; Bommineni, Gopal R; Walker, Stephen G; Slayden, Richard A; Sotriffer, Christoph A; Tonge, Peter J; Kisker, Caroline

    2014-06-06

    Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms.

  7. Inhibitory role of acyl homoserine lactones in hemolytic activity and viability of Streptococcus pyogenes M6 S165

    PubMed Central

    Saroj, Sunil D.; Holmer, Linda; Berengueras, Júlia M.; Jonsson, Ann-Beth

    2017-01-01

    Streptococcus pyogenes an adapted human pathogen asymptomatically colonizes the nasopharynx, among other polymicrobial communities. However, information on the events leading to the colonization and expression of virulence markers subject to interspecies and host-bacteria interactions are limited. The interference of acyl homoserine lactones (AHLs) with the hemolytic activity and viability of S. pyogenes M6 S165 was examined. AHLs, with fatty acid side chains ≥12 carbon atoms, inhibited hemolytic activity by downregulating the expression of the sag operon involved in the production of streptolysin S. Inhibitory AHLs upregulated the expression of transcriptional regulator LuxR. Electrophoretic mobility shift assays revealed the interaction of LuxR with the region upstream of sagA. AHL-mediated bactericidal activity observed at higher concentrations (mM range) was an energy-dependent process, constrained by the requirement of glucose and iron. Ferrichrome transporter FtsABCD facilitated transport of AHLs across the streptococcal membrane. The study demonstrates a previously unreported role for AHLs in S. pyogenes virulence. PMID:28303956

  8. Community dynamics and glycoside hydrolase activities of thermophilic bacterial consortia adapted to switchgrass

    SciTech Connect

    Gladden, J.M.; Allgaier, M.; Miller, C.S.; Hazen, T.C.; VanderGheynst, J.S.; Hugenholtz, P.; Simmons, B.A.; Singer, S.W.

    2011-05-01

    Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60 C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80 C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

  9. Glycoside Hydrolase Activities of Thermophilic Bacterial Consortia Adapted to Switchgrass ▿ †

    PubMed Central

    Gladden, John M.; Allgaier, Martin; Miller, Christopher S.; Hazen, Terry C.; VanderGheynst, Jean S.; Hugenholtz, Philip; Simmons, Blake A.; Singer, Steven W.

    2011-01-01

    Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60°C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80°C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions. PMID:21724886

  10. The Structure and Function of an Arabinan-specific [alpha]-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases

    SciTech Connect

    Cartmell, Alan; McKee, Lauren S.; Pena, Maria J.; Larsbrink, Johan; Brumer, Harry; Kaneko, Satoshi; Ichinose, Hitomi; Lewis, Richard J.; Vikso-Nielsen, Anders; Gilbert, Harry; Marles-Wright, Jon

    2012-03-26

    Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific {alpha}-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave {alpha}-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific {alpha}-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed {beta}-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for {alpha}-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

  11. Lipid sulfates and sulfonates are allosteric competitive inhibitors of the N-terminal phosphatase activity of the mammalian soluble epoxide hydrolase.

    PubMed

    Tran, Katherine L; Aronov, Pavel A; Tanaka, Hiromasa; Newman, John W; Hammock, Bruce D; Morisseau, Christophe

    2005-09-13

    The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), a homodimeric enzyme with each monomer containing two domains with distinct activities. The C-terminal domain, containing the epoxide hydrolase activity (Cterm-EH), is involved in the metabolism of arachidonic acid epoxides, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, and inflammation. We recently demonstrated that the N-terminal domain contains a Mg2+-dependent lipid phosphate phosphatase activity (Nterm-phos). However, the biological role of this activity is unknown. The inability of known phosphatase inhibitors to inhibit the Nterm-phos constitutes a significant barrier to the elucidation of its function. We describe herein sulfate, sulfonate, and phosphonate lipids as novel potent inhibitors of Nterm-phos. These compounds are allosteric competitive inhibitors with K(I) in the hundred nanomolar range. These inhibitors may provide a valuable tool to investigate the biological role of the Nterm-phos. We found that polyisoprenyl phosphates are substrates of Nterm-phos, suggesting a possible role in sterol synthesis or inflammation. Furthermore, some of these compounds inhibit the C-terminal sEH activity through a noncompetitive inhibition mechanism involving a new binding site on the C-terminal domain. This novel site may play a role in the natural in vivo regulation of epoxide hydrolysis by sEH.

  12. Modification of the activity of some C cycle hydrolases in soils afforested with Populus alba L. Preliminary results

    NASA Astrophysics Data System (ADS)

    Zorita, Félix; García-Campos, Elena; Gil-Sotres, Fernando; Leirós, Mā Carmen; Trasar-Cepeda, Carmen

    2010-05-01

    Since 1992 a large part of the agricultural land in Galicia (NW Spain) has disappeared as a result of the EU policy of providing grants and aid for transforming marginal land into forest terrain. In Galicia, this policy (EU Regulation 2080/1992) has mainly been applied to good quality agricultural land rather than to marginal land. As a result, the land has undergone a change in use, so that previously good quality agricultural land is now planted with various species of trees, usually of young age. Despite the large area of land transformed, until now the environmental cost of such changes has not been evaluated. Taking into account that one of the possible environmental effects derived from land transformation is changes in emissions of CO2 (a major greenhouse gas), it is therefore essential to evaluate any possible modifications undergone in such soils, with special attention given to biochemical properties, i.e. the properties that determine edaphic metabolism. With this aim, we are currently investigating the effect of afforestation on diverse biochemical properties, including the activity of hydrolytic enzymes involved in the C, N, P and S cycles, in a large number of afforested soils, planted with different trees and located in different areas throughout Galicia. In each case, an agricultural soil located close to the afforested soil, but under the original land use (usually maize cropped soils or pasture soils), is also collected and analysed, and the results obtained for afforested soils compared with those for the corresponding agricultural soils. Here we report some preliminary results on modifications in the activities of some C cycle hydrolases in six soils now planted with poplars, Populus alba L, but originally cropped with maize. Samples of all soils were collected in autumn, after harvesting and before any other agricultural activities were carried out. In all cases, the upper 10 cm of the soils were collected. The soils were sieved (4 mm) prior to

  13. Synthesis, characterization, anti-inflammatory and anti-proliferative activity against MCF-7 cells of O-alkyl and O-acyl flavonoid derivatives.

    PubMed

    Hoang, T Kim-Dung; Huynh, T Kim-Chi; Nguyen, Thanh-Danh

    2015-12-01

    A series of O-alkyl and O-acyl flavonoid derivatives was synthesized in high efficiency. Alkylation and acylation of 5-hydroxyflavonoids showed that the low reactivity hydroxyl group, 5-OH, well reacted with strong reagents whereas with weaker reagents, the different products were obtained dependently on structural characteristic of ring C of respective flavonoid. In order to evaluate anti-inflammatory activity, all compounds were tested for in vitro inhibition of bovine serum albumin denaturation and in vivo inhibition of carrageenan-induced mouse paw edema. Among them, the compounds 3, 3b, 4b and 4c demonstrated more effective anti-inflammatory activity than standard drugs (diclofenac sodium and ketoprofen) in both tests. Meanwhile, the flavonoids 2, 2c, 3a and 4b displayed anti-proliferative activity against MCF-7 cell lines. Triacetyl derivative of hesperetin 4b inducing degradation of DNA in MCF-7 cells was observed.

  14. Ubiquitin C-terminal hydrolase-l1 activity induces polyubiquitin accumulation in podocytes and increases proteinuria in rat membranous nephropathy.

    PubMed

    Meyer-Schwesinger, Catherine; Meyer, Tobias N; Sievert, Henning; Hoxha, Elion; Sachs, Marlies; Klupp, Eva-Maria; Münster, Silvia; Balabanov, Stefan; Carrier, Lucie; Helmchen, Udo; Thaiss, Friedrich; Stahl, Rolf A K

    2011-05-01

    Ubiquitin C-terminal hydrolase L1 (UCH-L1), a key protease of the ubiquitin-proteasome system (UPS), is associated with neurodegenerative diseases and cancer. Recently, de novo expression of UCH-L1 was described in podocytes in patients with membranous nephropathy (MN), in which UCH-L1 expression correlated with increased ubiquitin content. The objective of the present study was to investigate the role of UCH-L1 in ubiquitin homeostasis and proteasomal degradation in a rat model of MN. After disease induction, UCH-L1 expression increased in podocytes and coincided with decreased glomerular monoubiquitin content. After an initial increase in proteasomal activity, the UPS was impaired. In addition to an increase of ubiquitin in podocytes, aggregates were observed 1 year after disease induction, as in MN in human beings. Inhibition of UCH-L1 hydrolase function in MN reduced UPS impairment and ameliorated proteinuria. In contrast, inhibition of proteasomal activity enhanced UPS impairment, resulting in increased proteinuria. Stable UCH-L1 overexpression in cultured podocytes resulted in accumulation of monoubiquitin and polyubiquitin proteins. In contrast, stable knock-down of UCH-L1 reduced monoubiquitin and polyubiquitin proteins and significantly increased proteasomal activity, indicating that the observed effects in rat MN also occurred in cultured podocytes. These data demonstrate that UCH-L1 activity results in polyubiquitin accumulation, proteasome inhibition, and disease aggravation in experimental models of MN.

  15. The Role of the β5-α11 Loop in the Active-Site Dynamics of Acylated Penicillin-Binding Protein A from Mycobacterium tuberculosis

    SciTech Connect

    Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher

    2013-04-22

    Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constants for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5–α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5–α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.

  16. Opaque7 Encodes an Acyl-Activating Enzyme-Like Protein That Affects Storage Protein Synthesis in Maize Endosperm

    PubMed Central

    Wang, Gang; Sun, Xiaoliang; Wang, Guifeng; Wang, Fei; Gao, Qiang; Sun, Xin; Tang, Yuanping; Chang, Chong; Lai, Jinsheng; Zhu, Lihuang; Xu, Zhengkai; Song, Rentao

    2011-01-01

    In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds. PMID:21954158

  17. Exogenous N-acyl-homoserine lactones enhance the expression of flagella of Pseudomonas syringae and activate defence responses in plants.

    PubMed

    Cheng, Feifei; Ma, Anzhou; Zhuang, Guoqiang; Fray, Rupert G

    2016-10-18

    In order to cope with pathogens, plants have evolved sophisticated mechanisms to sense pathogenic attacks and to induce defence responses. The N-acyl-homoserine lactone (AHL)-mediated quorum sensing in bacteria regulates diverse physiological processes, including those involved in pathogenicity. In this work, we study the interactions between AHL-producing transgenic tobacco plants and Pseudomonas syringae pv. tabaci 11528 (P. syringae 11528). Both a reduced incidence of disease and decrease in the growth of P. syringae 11528 were observed in AHL-producing plants compared with wild-type plants. The present data indicate that plant-produced AHLs enhance disease resistance against this pathogen. Subsequent RNA-sequencing analysis showed that the exogenous addition of AHLs up-regulated the expression of P. syringae 11528 genes for flagella production. Expression levels of plant defence genes in AHL-producing and wild-type plants were determined by quantitative real-time polymerase chain reaction. These data showed that plant-produced AHLs activated a wide spectrum of defence responses in plants following inoculation, including the oxidative burst, hypersensitive response, cell wall strengthening, and the production of certain metabolites. These results demonstrate that exogenous AHLs alter the gene expression patterns of pathogens, and plant-produced AHLs either directly or indirectly enhance plant local immunity during the early stage of plant infection.

  18. Surface active molecules: preparation and properties of long chain n-acyl-l-alpha-amino-omega-guanidine alkyl acid derivatives.

    PubMed

    Infante, R; Dominguez, J G; Erra, P; Julia, R; Prats, M

    1984-12-01

    Synopsis A new route for the synthesis of long chain N(alpha)-acyl-l-alpha-amino-omega-guamdine alkyl acid derivatives, with cationic or amphoteric character has been established. The general formula of these compounds is shown below. A physico-chemical and antimicrobial study of these products as a function of the alkyl ester or sodium salt (R), the straight chain length of the fatty acid residue (x) and the number of carbons between the omega-guanidine and omega-carboxyl group (n) has been investigated. The water solubility, surface tension, critical micelle concentration (c.m.c.) and minimum inhibitory concentration (MIC) against Gram-positive and Gram-negative bacteria (including Pseudomonas) has been determined. Dicyclohexylcarbodiimide has been used to condense fatty acids and alpha-amino-omega-guanidine alkyl acids. In these conditions protection of the omega-guanidine group is not necessary. The main characteristic of this synthetic procedure is the use of very mild experimental conditions (temperature, pH) to form the amide linkage which leads to pure optical compounds in high yield in the absence of electrolytes. The results show that some structural modifications, particularly the protection of the carboxyl group, promote variations of the surfactant and antimicrobial properties. Only those molecules with the blocked carboxyl group (cationic molecules, where R = Me, Et or Pr) showed a good surfactant and antimicrobial activity. When the carboxyl group was unprotected (amphoteric molecules, where R = Na(+)) the resulting compounds were inactive.

  19. Molecular orbital studies of enzyme activity: I: Charge relay system and tetrahedral intermediate in acylation of serine proteinases.

    PubMed Central

    Scheiner, S; Kleier, D A; Lipscomb, W N

    1975-01-01

    The charge relay ststem and its role in the acylation of serine proteinases is studied using the partial retention of diatomic differential overlap (PRDDO) technique to perform approximate ab initio molecular orbital calculations on a model of the enzyme-substrate complex. The aspartate in the charge relay system is seen to act as the ultimate proton acceptor during the charging of the serine nucleophile. A projection of the potential energy surface is obtained in a subspace corresponding to this charge transfer and to the coupled motions of active site residues and the substrate. These results together with extended basis set results for cruder models suggest that a concerted transfer of protons from Ser-195 to His-57 and from His-57 to Asp-102 occurs with an energy barrier of 20-25 kcal/mole (84-105 kJ/mole). The subsequent nucleophilic attack on the scissile peptide linkage by the charged serine is then seen to proceed energetically downhill to the tetrahedral intermediate. The formation of the tetrahedral intermediate from the Michaelis complex is calculated to be nearly thermoneutral. PMID:1058476

  20. Dienelactone hydrolase from Pseudomonas sp. strain B13.

    PubMed Central

    Ngai, K L; Schlömann, M; Knackmuss, H J; Ornston, L N

    1987-01-01

    Dienelactone hydrolase (EC 3.1.1.45) catalyzes the conversion of cis- or trans-4-carboxymethylenebut-2-en-4-olide (dienelactone) to maleylacetate. An approximately 24-fold purification from extracts of 3-chlorobenzoate-grown Pseudomonas sp. strain B13 yielded a homogeneous preparation of the enzyme. The purified enzyme crystallized readily and proved to be a monomer with a molecular weight of about 30,000. Each dienelactone hydrolase molecule contains two cysteinyl side chains. One of these was readily titrated by stoichiometric amounts of p-chloromercuribenzoate, resulting in inactivation of the enzyme; the inactivation could be reversed by the addition of dithiothreitol. The other cysteinyl side chain appeared to be protected in the native protein against chemical reaction with p-chloromercuribenzoate. The properties of sulfhydryl side chains in dienelactone hydrolase resembled those that have been characterized for bacterial 4-carboxymethylbut-3-en-4-olide (enol-lactone) hydrolases (EC 3.1.1.24), which also are monomers with molecular weights of about 30,000. The amino acid composition of the dienelactone hydrolase resembled the amino acid composition of enol-lactone hydrolase from Pseudomonas putida, and alignment of the NH2-terminal amino acid sequence of the dienelactone hydrolase with the corresponding sequence of an Acinetobacter calcoaceticus enol-lactone hydrolase revealed sequence identity at 8 of the 28 positions. These observations foster the hypothesis that the lactone hydrolases share a common ancestor. The lactone hydrolases differed in one significant property: the kcat of dienelactone hydrolase was 1,800 min-1, an order of magnitude below the kcat observed with enol-lactone hydrolases. The relatively low catalytic activity of dienelactone hydrolase may demand its production at the high levels observed for induced cultures of Pseudomonas sp. strain B13. PMID:3804973

  1. PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro

    PubMed Central

    Kan, Chin Fung Kelvin; Singh, Amar Bahadur; Dong, Bin; Shende, Vikram Ravindra; Liu, Jingwen

    2017-01-01

    The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells. PMID:25645621

  2. PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro.

    PubMed

    Kan, Chin Fung Kelvin; Singh, Amar Bahadur; Dong, Bin; Shende, Vikram Ravindra; Liu, Jingwen

    2015-05-01

    The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells.

  3. Methyl jasmonate induces lauric acid omega-hydroxylase activity and accumulation of CYP94A1 transcripts but does not affect epoxide hydrolase activities in vicia sativa seedlings

    PubMed

    Pinot; Benveniste; Sala n JP; Durst

    1998-12-01

    Treatment of etiolated Vicia sativa seedlings by the plant hormone methyl jasmonate (MetJA) led to an increase of cytochrome P450 content. Seedlings that were treated for 48 h in a 1 mM solution of MetJA stimulated omega-hydroxylation of 12:0 (lauric acid) 14-fold compared with the control (153 versus 11 pmol min-1 mg-1 protein, respectively). Induction was dose dependent. The increase of activity (2.7-fold) was already detectable after 3 h of treatment. Activity increased as a function of time and reached a steady level after 24 h. Northern-blot analysis revealed that the transcripts coding for CYP94A1, a fatty acid omega-hydroxylase, had already accumulated after 1 h of exposure to MetJA and was maximal between 3 and 6 h. Under the same conditions, a study of the enzymatic hydrolysis of 9,10-epoxystearic acid showed that both microsomal and soluble epoxide hydrolase activities were not affected by MetJA treatment.

  4. Activation of AMP-activated protein kinase signaling pathway by adiponectin and insulin in mouse adipocytes: requirement of acyl-CoA synthetases FATP1 and Acsl1 and association with an elevation in AMP/ATP ratio.

    PubMed

    Liu, Qingqing; Gauthier, Marie-Soleil; Sun, Lei; Ruderman, Neil; Lodish, Harvey

    2010-11-01

    Adiponectin activates AMP-activated protein kinase (AMPK) in adipocytes, but the underlying mechanism remains unclear. Here we tested the hypothesis that AMP, generated in activating fatty acids to their CoA derivatives, catalyzed by acyl-CoA synthetases, is involved in AMPK activation by adiponectin. Moreover, in adipocytes, insulin affects the subcellular localization of acyl-CoA synthetase FATP1. Thus, we also tested whether insulin activates AMPK in these cells and, if so, whether it activates through a similar mechanism. We examined these hypotheses by measuring the AMP/ATP ratio and AMPK activation on adiponectin and insulin stimulation and after knocking down acyl-CoA synthetases in adipocytes. We show that adiponectin activation of AMPK is accompanied by an ∼2-fold increase in the cellular AMP/ATP ratio. Moreover, FATP1 and Acsl1, the 2 major acyl-CoA synthetase isoforms in adipocytes, are essential for AMPK activation by adiponectin. We also show that after 40 min. insulin activated AMPK in adipocytes, which was coupled with a 5-fold increase in the cellular AMP/ATP ratio. Knockdown studies show that FATP1 and Acsl1 are required for these processes, as well as for stimulation of long-chain fatty acid uptake by adiponection and insulin. These studies demonstrate that a change in cellular energy state is associated with AMPK activation by both adiponectin and insulin, which requires the activity of FATP1 and Acsl1.

  5. Synthesis of coenzyme A thioesters using methyl acyl phosphates in an aqueous medium.

    PubMed

    Pal, Mohan; Bearne, Stephen L

    2014-12-28

    Regioselective S-acylation of coenzyme A (CoA) is achieved under aqueous conditions using various aliphatic and aromatic carboxylic acids activated as their methyl acyl phosphate monoesters. Unlike many hydrophobic activating groups, the anionic methyl acyl phosphate mixed anhydride is more compatible with aqueous solvents, making it useful for conducting acylation reactions in an aqueous medium.

  6. Enzymatic Synthesis of Sorboyl-Polydatin Prodrug in Biomass-Derived 2-Methyltetrahydrofuran and Antiradical Activity of the Unsaturated Acylated Derivatives

    PubMed Central

    Yang, Rongling; Zhao, Xiangjie; Jiang, Ling; Zhu, Chun; Zhao, Yuping; Jia, Jianbo

    2016-01-01

    Efficient and highly regioselective synthesis of the potential 6′′-O-sorboyl-polydatin prodrug in biomass-derived 2-methyltetrahydrofuran (2-MeTHF) was achieved using Candida antarctica lipase B for the first time. Under the optimal conditions, the initial reaction rate, maximum substrate conversion, and 6′′-regioselectivity were as high as 8.65 mM/h, 100%, and 100%, respectively. Kinetic and operational stability investigations evidently demonstrated excellent enzyme compatibility of the 2-MeTHF compared to the traditional organic solvents. With respect to the antioxidant properties, three unsaturated ester derivatives showed slightly lower DPPH radical scavenging activities than the parent agent. Interestingly, further studies also revealed that the antiradical capacities of the acylates decreased with the elongation of the unsaturated aliphatic chain length from C4 to C11. The reason might be attributed to the increased steric hindrance derived from the acyl residues in derivatives. PMID:27668253

  7. The Escherichia coli orthologue of the Salmonella ushB gene (ushB(c)) produces neither UDP-sugar hydrolase activity nor detectable protein, but has an identical sequence to that of Escherichia coli cdh.

    PubMed

    Schroder, W; Burger, M; Edwards, C; Douglas, M; Innes, D; Beacham, I R; Burns, D M

    2001-09-11

    Salmonella ushB, which encodes a membrane-bound UDP-sugar hydrolase, has an Escherichia coli orthologue (ushB(c)) which does not detectably produce this activity. In this report, we show that ushB(c) does not produce any detectable protein either, despite being transcribed normally. Remarkably, ushB(c) is shown to have 100% sequence identity with E. coli cdh, previously characterised as encoding an active CDP-diglyceride hydrolase, an apparent contradiction with implications regarding enzyme evolution. We suggest that a useful gene designation is cdh (ushB(c)) rather than either ushB(c) or cdh, alone.

  8. Effect of various eicosanoid products of arachidonic acid on the acyl CoA: Cholesterol acyl transferase activity in three different mammalian cell lines

    SciTech Connect

    Malo, P.El.

    1988-01-01

    Acylcoenzyme A:cholesterol acyltransferase (ACAT) catalyzes cholesterol ester synthesis intracellularly and has been implicated in the development of atherosclerosis. An in vitro assay has been adapted for determining ACAT activity from rat FU5AH hepatoma, Chinese hamster ovary (CHO) and rat thoracic aortic smooth muscle (RSM) cells. Formation of {sup 14}C-labelled cholesteryl oleate at 0 to 60 min {plus minus} cholesterol was determined; in the presence of exogenous cholesterol, ACAT activity was approximately linear and surpassed the plateau observed in ACAT activity without cholesterol. Increasing exogenous cholesterol concentration, the amount of oleoyl CoA or the amount of microsomal protein produced a corresponding increase in ACAT activity, while ester formation was slightly increased by decreasing the ratio of Triton WR-1339 to cholesterol. Both the thromboxane A{sub 2} (TxA{sub 2}) mimic, U-44069, and the inflammatory lipoxygenase product, LTB{sub 4}, decreased optimal in vitro microsomal ACAT activity from RSM, but not form FU5AH, while CHO ACAT activity was suppressed by LTB{sub r} only. PGI{sub 2}, PGE{sub 2} and PGF{sub 2{alpha}} had minimal effects for each cell type.

  9. Impact of subdermal norgestrel on hepatic acyl-coenzyme A:cholesterol- acyltransferase (ACAT) activity: possible antiatherogenic effect.

    PubMed

    Letterie, G S

    2000-06-01

    The impact of subdermally placed ethinyl estradiol, norgestrel, and the combination of the two on cholesterol metabolism as measured by hepatic acyl:cholesterol-acyltransferase (ACAT) activity was examined in the rat model. A total of 48 rats were assigned to one of 6 groups, receiving either 0.1 mg or 1.0 mg of ethinyl estradiol daily, 1.0 or 10 mg of norgestrel daily, and combinations of either 0.1 mg ethinyl estradiol/1.0 mg norgestrel or 1.0 mg ethinyl estradiol/10 mg norgestrel daily. All drugs were administered through subdermally placed time release capsules. The administration of norgestrel only in either 1.0 mg or 10 mg resulted in significantly lower rates of ACAT activity (0.77 +/- 0.566 and 0.91 +/- 0.239 pmol/mg/min, respectively). The combination of 1.0 ethinyl estradiol and 10 mg norgestrel resulted in a significant increase in ACAT activity to 2.17 +/- 0.873. This combination also resulted in significantly greater weight loss at the conclusion of treatment [247.83 +/- 6.2 g (pre) vs. 205.50 +/- 10.6 (post)]. There were no other differences in ACAT activity between groups and no other differences in weight, both between groups and pre- and post-treatment within groups. In summary, subdermally placed norgestrel resulted in a significant lowering of ACAT activity not seen with either administration of ethinyl estradiol alone or the combination of ethinyl estradiol and norgestrel in doses ranging from 0.1 to 1.0 mg of ethinyl estradiol and 1.0 to 10.0 mg of norgestrel. Significantly increased ACAT activity for the combination of 1.0 ethinyl estradiol and 10 mg norgestrel over either ethinyl estradiol or norgestrel alone or a lower dose combination suggests a dose-related threshold and drug-drug interaction for this effect. These results suggest that subdermally placed norgestrel may result in significantly lower ACAT activity and may have a potential role as an antiatherogenic treatment.

  10. High fat fed heart failure animals have enhanced mitochondrial function and acyl-coa dehydrogenase activities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously shown that administration of high fat in heart failure (HF) increased mitochondrial respiration and did not alter left ventricular (LV) function. PPARalpha is a nuclear transcription factor that activates expression of genes involved in fatty acid uptake and utilization. We hypoth...

  11. Synthesis, characterization, stereochemistry and antibacterial activity of N-acyl-2,4,6,8-tetraphenyl-3,7-diazabicyclo[3.3.1]nonanes

    NASA Astrophysics Data System (ADS)

    Ponnuswamy, S.; Pushpalatha, S.; Akila, A.; Raghuvarman, B.; Aravindhan, S.

    2016-12-01

    Three new N-acyl-2,4,6,8-tetraphenyl-3,7-diazabicyclo[3.3.1]nonanes 3-5 have been synthesized. The structural characterization and the conformational preferences of the compounds 3-5 have been carried out using IR, 1D and 2D NMR spectral data. The NMR spectral data indicate that the N-acyl-2,4,6,8-tetraphenyl-3,7-diazabicyclo[3.3.1]nonanes 3-5 prefer to exist in twin-chair conformation with partial flattening at amide nitrogen end. In order to avoid A1,3-strain with coplanar acyl groups, the phenyl groups at the amide nitrogen end are forced to occupy axial orientation. X-ray crystal structure of the N-dichloroacetyl-2,4,6,8-tetraphenyl-3,7-diazabicyclo[3.3.1]nonane 4 also supports the twin-chair conformation in the solid state. Furthermore, the antibacterial activity for the compounds 2-5 has been carried out.

  12. Ricinus communis contains and acyl-CoA synthetase that preferentially activates ricinoleate to its CoA thioester

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As part of our effort to identify enzymes that are critical for producing large amounts of ricinoleate in castor oil, we have isolated three cDNAs encoding acyl-CoA synthetase (ACS) in the castor plant. Analysis of the cDNA sequences reveals that two of them, designated RcACS 2 and RcACS 4, contain...

  13. Dissecting the Structural Elements for the Activation of β-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae

    PubMed Central

    Hou, Jing; Zheng, Heping; Chruszcz, Maksymilian; Zimmerman, Matthew D.; Shumilin, Igor A.; Osinski, Tomasz; Demas, Matt; Grimshaw, Sarah

    2015-01-01

    ABSTRACT β-Ketoacyl-(acyl carrier protein) reductase (FabG) catalyzes the key reductive reaction in the elongation cycle of fatty acid synthesis (FAS), which is a vital metabolic pathway in bacteria and a promising target for new antibiotic development. The activation of the enzyme is usually linked to the formation of a catalytic triad and cofactor binding, and crystal structures of FabG from different organisms have been captured in either the active or inactive conformation. However, the structural elements which enable activation of FabG require further exploration. Here we report the findings of structural, enzymatic, and binding studies of the FabG protein found in the causative agent of cholera, Vibrio cholerae (vcFabG). vcFabG exists predominantly as a dimer in solution and is able to self-associate to form tetramers, which is the state seen in the crystal structure. The formation of the tetramer may be promoted by the presence of the cofactor NADP(H). The transition between the dimeric and tetrameric states of vcFabG is related to changes in the conformations of the α4/α5 helices on the dimer-dimer interface. Two glycine residues adjacent to the dimer interface (G92 and G141) are identified to be the hinge for the conformational changes, while the catalytic tyrosine (Y155) and a glutamine residue that forms hydrogen bonds to both loop β4-α4 and loop β5-α5 (Q152) stabilize the active conformation. The functions of the aforementioned residues were confirmed by binding and enzymatic assays for the corresponding mutants. IMPORTANCE This paper describes the results of structural, enzymatic, and binding studies of FabG from Vibrio cholerae (vcFabG). In this work, we dissected the structural elements responsible for the activation of vcFabG. The structural information provided here is essential for the development of antibiotics specifically targeting bacterial FabG, especially for the multidrug-resistant strains of V. cholerae. PMID:26553852

  14. Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): Enzymes with multiple sterols as substrates and as activators.

    PubMed

    Rogers, Maximillian A; Liu, Jay; Song, Bao-Liang; Li, Bo-Liang; Chang, Catherine C Y; Chang, Ta-Yuan

    2015-07-01

    Cholesterol is essential to the growth and viability of cells. The metabolites of cholesterol include: steroids, oxysterols, and bile acids, all of which play important physiological functions. Cholesterol and its metabolites have been implicated in the pathogenesis of multiple human diseases, including: atherosclerosis, cancer, neurodegenerative diseases, and diabetes. Thus, understanding how cells maintain the homeostasis of cholesterol and its metabolites is an important area of study. Acyl-coenzyme A:cholesterol acyltransferases (ACATs, also abbreviated as SOATs) converts cholesterol to cholesteryl esters and play key roles in the regulation of cellular cholesterol homeostasis. ACATs are most unusual enzymes because (i) they metabolize diverse substrates including both sterols and certain steroids; (ii) they contain two different binding sites for steroidal molecules. In mammals, there are two ACAT genes that encode two different enzymes, ACAT1 and ACAT2. Both are allosteric enzymes that can be activated by a variety of sterols. In addition to cholesterol, other sterols that possess the 3-beta OH at C-3, including PREG, oxysterols (such as 24(S)-hydroxycholesterol and 27-hydroxycholesterol, etc.), and various plant sterols, could all be ACAT substrates. All sterols that possess the iso-octyl side chain including cholesterol, oxysterols, various plant sterols could all be activators of ACAT. PREG can only be an ACAT substrate because it lacks the iso-octyl side chain required to be an ACAT activator. The unnatural cholesterol analogs epi-cholesterol (with 3-alpha OH in steroid ring B) and ent-cholesterol (the mirror image of cholesterol) contain the iso-octyl side chain but do not have the 3-beta OH at C-3. Thus, they can only serve as activators and cannot serve as substrates. Thus, within the ACAT holoenzyme, there are site(s) that bind sterol as substrate and site(s) that bind sterol as activator; these sites are distinct from each other. These features form

  15. Enzymatic activation of double-targeted 5'-O-L-valyl-decitabine prodrug by biphenyl hydrolase-like protein and its molecular design basis.

    PubMed

    Tao, Wenhui; Zhao, Dongyang; Sun, Mengchi; Li, Meng; Zhang, Xiangyu; He, Zhonggui; Sun, Yinghua; Sun, Jin

    2017-04-01

    A primary focus of this research was to explore the activation process and mechanism of decitabine (5-aza-2'-deoxycytidine, DAC) prodrug. Recently, it has been reported that biphenyl hydrolase-like protein (BPHL) can play an important role in the activation of some amino acid nucleoside prodrugs with a general preference for hydrophobic amino acids and 5'-esters. Therefore, we put forward a bold hypothesis that this novel enzyme may be primarily responsible for the activation process of DAC prodrug as well. 5'-O-L-valyl-decitabine (L-val-DAC) was synthesized before and can be transported across biological membranes by the oligopeptide transporter (PEPT1), granting it much greater utility in vivo. In this report, L-val-DAC was found to be a good substrate of BPHL protein (K m 0.59 mM; k cat/K m 553.69 mM(-1) s(-1)). After intestinal absorption, L-val-DAC was rapidly and almost completely hydrolyzed to DAC and L-valine. The catalysis was mainly mediated by the BPHL hydrolase and resulted in the intestinal first-pass effect of L-val-DAC after oral administration in Sprague-Dawley rats with cannulated jugular and portal veins. The structural insights using computational molecular docking showed that BPHL had a unique binding mode for L-val-DAC. As a fundamental basis, the simulation was employed to explain the catalytic mechanism in molecular level. In conclusion, BPHL was at least one of the primary candidate enzymes for L-val-DAC prodrug activation. This promising double-targeted prodrug approach have more advantages than the traditional targeted designs due to its higher transport and more predictable activation, thereby leading to a favorable property for oral delivery.

  16. Reversible Cysteine Acylation Regulates the Activity of Human Palmitoyl-Protein Thioesterase 1 (PPT1)

    PubMed Central

    Segal-Salto, Michal; Sapir, Tamar; Reiner, Orly

    2016-01-01

    Mutations in the depalmitoylating enzyme gene, PPT1, cause the infantile form of Neuronal Ceroid Lipofuscinosis (NCL), an early onset neurodegenerative disease. During recent years there have been different therapeutic attempts including enzyme replacement. Here we show that PPT1 is palmitoylated in vivo and is a substrate for two palmitoylating enzymes, DHHC3 and DHHC7. The palmitoylated protein is detected in both cell lysates and medium. The presence of PPT1 with palmitoylated signal peptide in the cell medium suggests that a subset of the protein is secreted by a nonconventional mechanism. Using a mutant form of PPT1, C6S, which was not palmitoylated, we further demonstrate that palmitoylation does not affect intracellular localization but rather that the unpalmitoylated form enhanced the depalmitoylation activity of the protein. The calculated Vmax of the enzyme was significantly affected by the palmitoylation, suggesting that the addition of a palmitate group is reminiscent of adding a noncompetitive inhibitor. Thus, we reveal the existence of a positive feedback loop, where palmitoylation of PPT1 results in decreased activity and subsequent elevation in the amount of palmitoylated proteins. This positive feedback loop is likely to initiate a vicious cycle, which will enhance disease progression. The understanding of this process may facilitate enzyme replacement strategies. PMID:26731412

  17. Ghrelin acylation and metabolic control.

    PubMed

    Al Massadi, O; Tschöp, M H; Tong, J

    2011-11-01

    Since its discovery, many physiologic functions have been ascribed to ghrelin, a gut derived hormone. The presence of a median fatty acid side chain on the ghrelin peptide is required for the binding and activation of the classical ghrelin receptor, the growth hormone secretagogue receptor (GHSR)-1a. Ghrelin O-acyl transferase (GOAT) was recently discovered as the enzyme responsible for this acylation process. GOAT is expressed in all tissues that have been found to express ghrelin and has demonstrated actions on several complex endocrine organ systems such as the hypothalamus-pituitary-gonadal, insular and adrenal axis as well as the gastrointestinal (GI) tract, bone and gustatory system. Ghrelin acylation is dependent on the function of GOAT and the availability of substrates such as proghrelin and short- to medium-chain fatty acids (MCFAs). This process is governed by GOAT activity and has been shown to be modified by dietary lipids. In this review, we provided evidence that support an important role of GOAT in the regulation of energy homeostasis and glucose metabolism by modulating acyl ghrelin (AG) production. The relevance of GOAT and AG during periods of starvation remains to be defined. In addition, we summarized the recent literature on the metabolic effects of GOAT specific inhibitors and shared our view on the potential of targeting GOAT for the treatment of metabolic disorders such as obesity and type 2 diabetes.

  18. A new phenolic derivative with soluble epoxide hydrolase and nuclear factor-kappaB inhibitory activity from the aqueous extract of Acacia catechu.

    PubMed

    Sun, Ya Nan; Li, Wei; Song, Seok Bean; Yan, Xi Tao; Zhao, Yan; Jo, A Reum; Kang, Jong Seong; Young Ho, Kim

    2016-09-01

    One novel phenolic compound, (4S,5R)-4-(3,4-dihydroxyphenyl)-5-(3-oxobutyl)dihydrofuran-2(3H)-one (1), as well as 12 known compounds (2-13) was obtained from the aqueous extract of Acacia catechu and their chemical structures were determined by spectroscopic analysis. Compounds 8 and 9 exhibited significant soluble epoxide hydrolase (sEH) inhibitory activities with IC50 values of 26.6 ± 0.5 and 24.4 ± 5.6 μM, respectively. Compounds 7-10 showed significant inhibitory effects on TNFα-induced nuclear factor kappa B (NF-κB) transcriptional activity in a dose-dependent manner, with IC50 values ranging from 11.15 to 19.45 μM.

  19. DNA-binding studies and biological activities of new nitrosubstituted acyl thioureas

    NASA Astrophysics Data System (ADS)

    Tahir, Shaista; Badshah, Amin; Hussain, Raja Azadar; Tahir, Muhammad Nawaz; Tabassum, Saira; Patujo, Jahangir Ali; Rauf, Muhammad Khawar

    2015-11-01

    Four new nitrosubstituted acylthioureas i.e. 1-acetyl-3-(4-nitrophenyl)thiourea (TU1), 1-acetyl-3-(2-methyl-4-nitrophenyl)thiourea (TU2), 1-acetyl-3-(2-methoxy-4-nitrophenyl)thiourea (TU3) and 1-acetyl-3-(4-chloro-3-nitrophenyl)thiourea (TU4) have been synthesized and characterized (by C13 and H1 nuclear magnetic resonance, Fourier transform infrared spectroscopy and single crystal X-ray diffraction). As a preliminary investigation of the anti-cancer potencies of the said compounds, DNA interaction studies have been carried out using cyclic voltammetry and UV-vis spectroscopy along with verification from computational studies. The drug-DNA binding constants are found to be in the order, KTU3 9.04 × 106 M-1 > KTU4 8.57 × 106 M-1 > KTU2 6.05 × 106 M-1 > KTU1 1.16 × 106 M-1. Furthermore, the antioxidant, cytotoxic, antibacterial and antifungal activities have been carried out against DPPH (1,1-diphenyl-2-dipicrylhydrazyl), Brine shrimp eggs, gram positive (Micrococcus luteus, Staphylococcus aureus) and gram negative (Bordetella bronchiseptica, Salmonella typhimurium, Enterobacter aerogens) and fungal cultures (Aspergillus fumigatus, Mucor species, Aspergillus niger, Aspergillus flavus) respectively.

  20. Biological activity of acyl glucose esters from Datura wrightii glandular trichomes against three native insect herbivores.

    PubMed

    Hare, J Daniel

    2005-07-01

    Datura wrightii is dimorphic for leaf trichome type in southern California. "Sticky" plants produce glandular trichomes that secrete acylsugars, whereas velvety plants produce nonglandular trichomes. Glandular trichomes confer resistance to some potential insect herbivores and are associated with reduced feeding in the field by two native coleopteran herbivores: the tobacco flea beetle, Epitrix hirtipennis, and a weevil, Trichobaris compacta. In contrast, another native beetle, Lema daturaphila, damages sticky and velvety plants similarly in the field. A series of choice and no-choice "ester removal" and "ester addition" feeding experiments were performed in the laboratory to evaluate the role of acylsugars in feeding by all three insect species. Consumption of sticky leaves after their esters were removed by washing was compared to consumption of unwashed sticky leaves and velvety leaves in ester removal experiments. Consumption of velvety leaves was measured after acylsugars were applied to those leaves in controlled amounts in the ester addition experiments. Consumption by E. hirtipennis was reduced by acylsugars in all experiments. Consumption by T. compacta was reduced by acylsugars in the ester removal experiments, but not in the ester addition experiments. The location of the acylsugars at the tip of a long trichome, rather than simply on the leaf surface, may be an important component of the biological activity of acylsugars against T. compacta in nature. Consumption by L. daturaphila was not significantly reduced by acylsugars in any experiment. The acylsugars caused no significant mortality of any of the three insect species.

  1. A novel glucosylation reaction on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium.

    PubMed

    Matsuba, Yuki; Sasaki, Nobuhiro; Tera, Masayuki; Okamura, Masachika; Abe, Yutaka; Okamoto, Emi; Nakamura, Haruka; Funabashi, Hisakage; Takatsu, Makoto; Saito, Mikako; Matsuoka, Hideaki; Nagasawa, Kazuo; Ozeki, Yoshihiro

    2010-10-01

    Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose-dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as β-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose-dependent glucosyltransferases.

  2. Abnormal relationships between the neural response to high- and low-calorie foods and endogenous acylated ghrelin in women with active and weight-recovered anorexia nervosa

    PubMed Central

    Holsen, Laura M.; Lawson, Elizabeth A.; Christensen, Kara; Klibanski, Anne; Goldstein, Jill M.

    2014-01-01

    Evidence contributing to the understanding of neurobiological mechanisms underlying appetite dysregulation in anorexia nervosa draws heavily on separate lines of research into neuroendocrine and neural circuitry functioning. In particular, studies consistently cite elevated ghrelin and abnormal activation patterns in homeostatic (hypothalamus) and hedonic (striatum, amygdala, insula) regions governing appetite. The current preliminary study examined the interaction of these systems, based on research demonstrating associations between circulating ghrelin levels and activity in these regions in healthy individuals. In a cross-sectional design, we studied 13 women with active anorexia nervosa (AN), 9 women weight-recovered from AN (AN-WR), and 12 healthy-weight control women using a food cue functional magnetic resonance imaging paradigm, with assessment of fasting levels of acylated ghrelin. Healthy-weight control women exhibited significant positive associations between fasting acylated ghrelin and activity in the right amygdala, hippocampus, insula, and orbitofrontal cortex in response to high-calorie foods, associations which were absent in the AN and AN-WR groups. Women with AN-WR demonstrated a negative relationship between ghrelin and activity in the left hippocampus in response to high-calorie foods, while women with AN showed a positive association between ghrelin and activity in the right orbitofrontal cortex in response to low-calorie foods. Findings suggest a breakdown in the interaction between ghrelin signaling and neural activity in relation to reward responsivity in AN, a phenomenon that may be further characterized using pharmacogenetic studies. PMID:24862390

  3. Loss of long-chain acyl-CoA synthetase isoform 1 impairs cardiac autophagy and mitochondrial structure through mechanistic target of rapamycin complex 1 activation

    PubMed Central

    Grevengoed, Trisha J.; Cooper, Daniel E.; Young, Pamela A.; Ellis, Jessica M.; Coleman, Rosalind A.

    2015-01-01

    Because hearts with a temporally induced knockout of acyl-CoA synthetase 1 (Acsl1T−/−) are virtually unable to oxidize fatty acids, glucose use increases 8-fold to compensate. This metabolic switch activates mechanistic target of rapamycin complex 1 (mTORC1), which initiates growth by increasing protein and RNA synthesis and fatty acid metabolism, while decreasing autophagy. Compared with controls, Acsl1T−/− hearts contained 3 times more mitochondria with abnormal structure and displayed a 35–43% lower respiratory function. To study the effects of mTORC1 activation on mitochondrial structure and function, mTORC1 was inhibited by treating Acsl1T−/− and littermate control mice with rapamycin or vehicle alone for 2 wk. Rapamycin treatment normalized mitochondrial structure, number, and the maximal respiration rate in Acsl1T−/− hearts, but did not improve ADP-stimulated oxygen consumption, which was likely caused by the 33–51% lower ATP synthase activity present in both vehicle- and rapamycin-treated Acsl1T−/− hearts. The turnover of microtubule associated protein light chain 3b in Acsl1T−/− hearts was 88% lower than controls, indicating a diminished rate of autophagy. Rapamycin treatment increased autophagy to a rate that was 3.1-fold higher than in controls, allowing the formation of autophagolysosomes and the clearance of damaged mitochondria. Thus, long-chain acyl-CoA synthetase isoform 1 (ACSL1) deficiency in the heart activated mTORC1, thereby inhibiting autophagy and increasing the number of damaged mitochondria.—Grevengoed, T. J., Cooper, D. E., Young, P. A., Ellis, J. M., Coleman, R. A. Loss of long-chain acyl-CoA synthetase isoform 1 impairs cardiac autophagy and mitochondrial structure through mechanistic target of rapamycin complex 1 activation. PMID:26220174

  4. Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth.

    PubMed

    Travers, Marie-Agnès; Sow, Cissé; Zirah, Séverine; Deregnaucourt, Christiane; Chaouch, Soraya; Queiroz, Rayner M L; Charneau, Sébastien; Allain, Thibault; Florent, Isabelle; Grellier, Philippe

    2016-01-01

    Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.

  5. Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth

    PubMed Central

    Travers, Marie-Agnès; Sow, Cissé; Zirah, Séverine; Deregnaucourt, Christiane; Chaouch, Soraya; Queiroz, Rayner M. L.; Charneau, Sébastien; Allain, Thibault; Florent, Isabelle; Grellier, Philippe

    2016-01-01

    Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present. PMID:27729900

  6. Structure-activity relationship studies on 1-heteroaryl-3-phenoxypropan-2-ones acting as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: replacement of the activated ketone group by other serine traps.

    PubMed

    Sundermann, Tom; Hanekamp, Walburga; Lehr, Matthias

    2016-08-01

    Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are serine hydrolases. cPLA2α is involved in the generation of pro-inflammatory lipid mediators, FAAH terminates the anti-inflammatory effects of endocannabinoids. Therefore, inhibitors of these enzymes may represent new drug candidates for the treatment of inflammation. We have reported that certain 1-heteroarylpropan-2-ones are potent inhibitors of cPLA2α and FAAH. The serine reactive ketone group of these compounds, which is crucial for enzyme inhibition, is readily metabolized resulting in inactive alcohol derivatives. In order to obtain metabolically more stable inhibitors, we replaced this moiety by α-ketoheterocyle, cyanamide and nitrile serine traps. Investigations on activity and metabolic stability of these substances revealed that in all cases an increased metabolic stability was accompanied by a loss of inhibitory potency against cPLA2α and FAAH, respectively.

  7. Reaction Mechanisms in Carbohydrate-Active Enzymes: Glycoside Hydrolases and Glycosyltransferases. Insights from ab Initio Quantum Mechanics/Molecular Mechanics Dynamic Simulations.

    PubMed

    Ardèvol, Albert; Rovira, Carme

    2015-06-24

    Carbohydrate-active enzymes such as glycoside hydrolases (GHs) and glycosyltransferases (GTs) are of growing importance as drug targets. The development of efficient competitive inhibitors and chaperones to treat diseases related to these enzymes requires a detailed knowledge of their mechanisms of action. In recent years, sophisticated first-principles modeling approaches have significantly advanced in our understanding of the catalytic mechanisms of GHs and GTs, not only the molecular details of chemical reactions but also the significant implications that just the conformational dynamics of a sugar ring can have on these mechanisms. Here we provide an overview of the progress that has been made in the past decade, combining molecular dynamics simulations with density functional theory to solve these sweet mysteries of nature.

  8. Fast, Continuous, and High-Throughput (Bio)Chemical Activity Assay for N-Acyl-l-Homoserine Lactone Quorum-Quenching Enzymes

    PubMed Central

    Last, Daniel; Krüger, Georg H. E.; Dörr, Mark

    2016-01-01

    ABSTRACT Quorum sensing, the bacterial cell-cell communication by small molecules, controls important processes such as infection and biofilm formation. Therefore, it is a promising target with several therapeutic and technical applications besides its significant ecological relevance. Enzymes inactivating N-acyl-l-homoserine lactones, the most common class of communication molecules among Gram-negative proteobacteria, mainly belong to the groups of quorum-quenching lactonases or quorum-quenching acylases. However, identification, characterization, and optimization of these valuable biocatalysts are based on a very limited number of fundamentally different methods with their respective strengths and weaknesses. Here, a (bio)chemical activity assay is described, which perfectly complements the other methods in this field. It enables continuous and high-throughput activity measurements of purified and unpurified quorum-quenching enzymes within several minutes. For this, the reaction products released by quorum-quenching lactonases and quorum-quenching acylases are converted either by a secondary enzyme or by autohydrolysis to l-homoserine. In turn, l-homoserine is detected by the previously described calcein assay, which is sensitive to α-amino acids with free N and C termini. Besides its establishment, the method was applied to the characterization of three previously undescribed quorum-quenching lactonases and variants thereof and to the identification of quorum-quenching acylase-expressing Escherichia coli clones in an artificial library. Furthermore, this study indicates that porcine aminoacylase 1 is not active toward N-acyl-l-homoserine lactones as published previously but instead converts the autohydrolysis product N-acyl-l-homoserine. IMPORTANCE In this study, a novel method is presented for the identification, characterization, and optimization of quorum-quenching enzymes that are active toward N-acyl-l-homoserine lactones. These are the most common

  9. Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure

    PubMed Central

    O'Farrell, Paul A.; Joshua-Tor, Leemor

    2006-01-01

    Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes. PMID:17007609

  10. In vitro cytotoxic effects of gold nanoparticles coated with functional acyl homoserine lactone lactonase protein from Bacillus licheniformis and their antibiofilm activity against Proteus species.

    PubMed

    Vinoj, Gopalakrishnan; Pati, Rashmirekha; Sonawane, Avinash; Vaseeharan, Baskaralingam

    2015-02-01

    N-acylated homoserine lactonases are known to inhibit the signaling molecules of the biofilm-forming pathogens. In this study, gold nanoparticles were coated with N-acylated homoserine lactonase proteins (AiiA AuNPs) purified from Bacillus licheniformis. The AiiA AuNPs were characterized by UV-visible spectra, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and X-ray diffraction (XRD). The synthesized AiiA AuNPs were found to be spherical in shape and 10 to 30 nm in size. Treatment with AiiA protein-coated AuNPs showed maximum reduction in exopolysaccharide production, metabolic activities, and cell surface hydrophobicity and potent antibiofilm activity against multidrug-resistant Proteus species compared to treatment with AiiA protein alone. AiiA AuNPs exhibited potent antibiofilm activity at 2 to 8 μM concentrations without being harmful to the macrophages. We conclude that at a specific dose, AuNPs coated with AiiA can kill bacteria without harming the host cells, thus representing a potential template for the design of novel antibiofilm and antibacterial protein drugs to decrease bacterial colonization and to overcome the problem of drug resistance. In summary, our data suggest that the combined effect of the lactonase and the gold nanoparticles of the AiiA AuNPs has promising antibiofilm activity against biofilm-forming and multidrug-resistant Proteus species.

  11. Roles of tryptophan residue and disulfide bond in the variable lid region of oxidized polyvinyl alcohol hydrolase.

    PubMed

    Yang, Yu; Ko, Tzu-Ping; Liu, Long; Li, Jianghua; Huang, Chun-Hsiang; Chen, Jian; Guo, Rey-Ting; Du, Guocheng

    2014-09-26

    Oxidized polyvinyl alcohol hydrolase (OPH) catalyzes the cleavage of C-C bond in β-diketone. It belongs to the α/β-hydrolase family and contains a unique lid region that covers the active site. The lid is the most variable region when pOPH from Pseudomonas sp. VM15C and sOPH from Sphingopyxis sp. 113P3 are compared. The wild-type enzymes and the pOPH mutants W255A, W255Y and W255F were analyzed for lipase activity by using p-nitrophenyl (pNP) esters as the substrates. The wild-type enzymes showed increased Km and decreased kcat/Km with the acyl chain length, and the mutants showed reduced kcat/Km for pNP acetate, indicating the importance of Trp255 in sequestering the active site from solvent. The significantly lower activity for pNP butyrate can be a result of product inhibition, as suggested by the complex crystal structures, in which butyric acid, DMSO or PEG occupied the same substrate-binding cleft. The mutant activity was retained with pNP caprylate and pNP laurate as the substrates, reflecting the amphipathic nature of the cleft. Moreover, the disulfide bond formation of Cys257/267 is important for the activity of pOPH, but it is not essential for sOPH, which has a shorter lid structure.

  12. Fructan:fructan 1-fructosyltransferase and inulin hydrolase activities relating to inulin and soluble sugars in Jerusalem artichoke (Helianthus tuberosus Linn.) tubers during storage.

    PubMed

    Maicaurkaew, Sukanya; Jogloy, Sanun; Hamaker, Bruce R; Ningsanond, Suwayd

    2017-03-01

    Influences of harvest time and storage conditions on activities of fructan:fructan1-fructosyltransferase (1-FFT) and inulin hydrolase (InH) in relation to inulin and soluble sugars of Jerusalem artichoke (Helianthus tuberosus L.) tubers were investigated. Maturity affected 1-FFT-activity, inulin contents, and inulin profiles of the tubers harvested between 30 and 70 days after flowering (DAF). Decreases in 1-FFT activity, high molecular weight inulin, and inulin content were observed in late-harvested tubers. The tubers harvested at 50 DAF had the highest inulin content (734.9 ± 20.5 g kg(-1) DW) with a high degree of polymerization (28% of DP >30). During storage of the tubers, increases in InH activity (reached its peak at 15 days of storage) and gradual decreases in 1-FFT activity took placed. These changes were associated with inulin depolymerization, causing decreases in inulin content and increases in soluble sugars. As well, decreasing storage temperatures would retain high inulin content and keep low soluble sugars; and freezing at -18 °C would best retard 1-FFT, InH, and inulin changes.

  13. Short communication: Improving the activity of bile salt hydrolases in Lactobacillus casei based on in silico molecular docking and heterologous expression.

    PubMed

    Xiong, Zhi-Qiang; Wang, Qiao-Hui; Kong, Ling-Hui; Song, Xin; Wang, Guang-Qiang; Xia, Yong-Jun; Zhang, Hui; Sun, Yong; Ai, Lian-Zhong

    2017-02-01

    Bile salt hydrolase (BSH) plays an essential role in the cholesterol-removing effect of lactic acid bacteria, which hydrolyze conjugated bile salts to amino acid and deconjugated bile salts. However, Lactobacillus casei lacks the bsh gene, which may make it highly sensitive to bile salt stress. We wanted to improve the BSH activity of L. casei for various food-industry applications (e.g., milk fermentation). Plate assay testing indicated that Lactobacillus plantarum AR113 has the highest BSH activity. We cloned and sequenced 4 bsh genes from the genome of L. plantarum AR113. Structure modeling and molecular docking of BSH indicated that BSH1 and BSH3 could react efficiently with bile salts, so we selected BSH1 and BSH3 for heterologous expression in L. casei. Compared with single expression of BSH1 or BSH3, co-expression of both protein sequences showed the highest hydrolysis activity by HPLC analysis. Our results suggested that heterologous expression of BSH in L. casei can significantly improve host activity against bile salts, and in silico molecular docking could be an efficient method of rapid screening for BSH with high activity.

  14. Maturation and Activity of Sterol Regulatory Element Binding Protein 1 Is Inhibited by Acyl-CoA Binding Domain Containing 3

    PubMed Central

    Chen, Yong; Patel, Vishala; Bang, Sookhee; Cohen, Natalie; Millar, John; Kim, Sangwon F.

    2012-01-01

    Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis. PMID:23166793

  15. Inhibition of soluble epoxide hydrolase after cardiac arrest/cardiopulmonary resuscitation induces a neuroprotective phenotype in activated microglia and improves neuronal survival.

    PubMed

    Wang, Jianming; Fujiyoshi, Tetsuhiro; Kosaka, Yasuharu; Raybuck, Jonathan D; Lattal, K Matthew; Ikeda, Mizuko; Herson, Paco S; Koerner, Ines P

    2013-10-01

    Cardiac arrest (CA) causes hippocampal neuronal death that frequently leads to severe loss of memory function in survivors. No specific treatment is available to reduce neuronal death and improve functional outcome. The brain's inflammatory response to ischemia can exacerbate injury and provides a potential treatment target. We hypothesized that microglia are activated by CA and contribute to neuronal loss. We used a mouse model to determine whether pharmacologic inhibition of the proinflammatory microglial enzyme soluble epoxide hydrolase (sEH) after CA alters microglial activation and neuronal death. The sEH inhibitor 4-phenylchalcone oxide (4-PCO) was administered after successful cardiopulmonary resuscitation (CPR). The 4-PCO treatment significantly reduced neuronal death and improved memory function after CA/CPR. We found early activation of microglia and increased expression of inflammatory tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the hippocampus after CA/CPR, which was unchanged after 4-PCO treatment, while expression of antiinflammatory IL-10 increased significantly. We conclude that sEH inhibition after CA/CPR can alter the transcription profile in activated microglia to selectively induce antiinflammatory and neuroprotective IL-10 and reduce subsequent neuronal death. Switching microglial gene expression toward a neuroprotective phenotype is a promising new therapeutic approach for ischemic brain injury.

  16. [Protective effect of tiacalix[4]arene-tetrasulphonate on heavy metal inhibition of myometrium myosin subfragment-1 ATP-hydrolase activity].

    PubMed

    Labyntseva, R D; Bevza, O V; Bevza, A A; Liul'ko, A O; Kharchenko, S H; Kal'chenko, V I; Kosterin, S O

    2014-01-01

    Heavy metals have a negative effect on the contractility of uterine smooth muscles (myometrium), these effects can lead to various pathologies of a women reproductive system. To overcome these effects the methods for correcting the myometrium contractile activity are to be developed. Catalyzed by myosin ATPase ATP hydrolysis is the most important reaction in the molecular mechanism of myometrium contraction. We have found an inhibitory effect of 0.03-0.3 mM Ni2+, Pb2+ and Cd2+ on enzymatic hydrolysis of ATP by myosin subfragment-1 obtained from swine uterine smooth muscles. We have demonstrated that 100 μM thiacalix[4]arene-tetrasulphonate (C-798) recovered to the control level of ATPase activity of myosin subfragment-1 in the presence of heavy metal cations. One of the most probable mechanisms of C-798 corrective activity is based on its ability to chelate heavy metals, thus cations Pb, Cd and Ni can be removed from the incubation medium. Computer simulation has demonstrated that the protective effect of C-798 may also be the result of weakening the interaction of heavy metal ions with amino acid residues of the myosin molecule near the active site of ATP hydrolase. The obtained results can be used for further research aimed at assessing the prospects of thiacalix[4]arene-tetrasulfonate as pharmacological compounds.

  17. Endogenous N-acyl taurines regulate skin wound healing

    PubMed Central

    Sasso, Oscar; Pontis, Silvia; Armirotti, Andrea; Cardinali, Giorgia; Kovacs, Daniela; Migliore, Marco; Summa, Maria; Moreno-Sanz, Guillermo; Picardo, Mauro; Piomelli, Daniele

    2016-01-01

    The intracellular serine amidase, fatty acid amide hydrolase (FAAH), degrades a heterogeneous family of lipid-derived bioactive molecules that include amides of long-chain fatty acids with taurine [N-acyl-taurines (NATs)]. The physiological functions of the NATs are unknown. Here we show that genetic or pharmacological disruption of FAAH activity accelerates skin wound healing in mice and stimulates motogenesis of human keratinocytes and differentiation of human fibroblasts in primary cultures. Using untargeted and targeted lipidomics strategies, we identify two long-chain saturated NATs—N-tetracosanoyl-taurine [NAT(24:0)] and N-eicosanoyl-taurine [NAT(20:0)]—as primary substrates for FAAH in mouse skin, and show that the levels of these substances sharply decrease at the margins of a freshly inflicted wound to increase again as healing begins. Additionally, we demonstrate that local administration of synthetic NATs accelerates wound closure in mice and stimulates repair-associated responses in primary cultures of human keratinocytes and fibroblasts, through a mechanism that involves tyrosine phosphorylation of the epidermal growth factor receptor and an increase in intracellular calcium levels, under the permissive control of transient receptor potential vanilloid-1 receptors. The results point to FAAH-regulated NAT signaling as an unprecedented lipid-based mechanism of wound-healing control in mammalian skin, which might be targeted for chronic wound therapy. PMID:27412859

  18. Xanthomonas campestris RpfB is a fatty Acyl-CoA ligase required to counteract the thioesterase activity of the RpfF diffusible signal factor (DSF) synthase.

    PubMed

    Bi, Hongkai; Yu, Yonghong; Dong, Huijuan; Wang, Haihong; Cronan, John E

    2014-07-01

    In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)-11-methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl-CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl-CoA ligase, Escherichia coli FadD, in the E. coli ß-oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3-hydroxyacyl-acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl-ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl-CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl-ACP synthetase, replaced RpfB in counteracting the effects of high

  19. N-acyl-dopamines: novel synthetic CB(1) cannabinoid-receptor ligands and inhibitors of anandamide inactivation with cannabimimetic activity in vitro and in vivo.

    PubMed Central

    Bisogno, T; Melck, D; Bobrov MYu; Gretskaya, N M; Bezuglov, V V; De Petrocellis, L; Di Marzo, V

    2000-01-01

    We reported previously that synthetic amides of polyunsaturated fatty acids with bioactive amines can result in substances that interact with proteins of the endogenous cannabinoid system (ECS). Here we synthesized a series of N-acyl-dopamines (NADAs) and studied their effects on the anandamide membrane transporter, the anandamide amidohydrolase (fatty acid amide hydrolase, FAAH) and the two cannabinoid receptor subtypes, CB(1) and CB(2). NADAs competitively inhibited FAAH from N18TG2 cells (IC(50)=19-100 microM), as well as the binding of the selective CB(1) receptor ligand, [(3)H]SR141716A, to rat brain membranes (K(i)=250-3900 nM). The arachidonoyl (20:4 omega 6), eicosapentaenoyl (20:5 omega 3), docosapentaenoyl (22:5 omega 3), alpha-linolenoyl (18:3 omega 3) and pinolenoyl (5c,9c,12c 18:3 omega 6) homologues were also found to inhibit the anandamide membrane transporter in RBL-2H3 basophilic leukaemia and C6 glioma cells (IC(50)=17.5-33 microM). NADAs did not inhibit the binding of the CB(1)/CB(2) receptor ligand, [(3)H]WIN55,212-2, to rat spleen membranes (K(i)>10 microM). N-arachidonyl-dopamine (AA-DA) exhibited 40-fold selectivity for CB(1) (K(i)=250 nM) over CB(2) receptors, and N-docosapentaenoyl-dopamine exhibited 4-fold selectivity for the anandamide transporter over FAAH. AA-DA (0.1-10 microM) did not displace D1 and D2 dopamine-receptor high-affinity ligands from rat brain membranes, thus suggesting that this compound has little affinity for these receptors. AA-DA was more potent and efficacious than anandamide as a CB(1) agonist, as assessed by measuring the stimulatory effect on intracellular Ca(2+) mobilization in undifferentiated N18TG2 neuroblastoma cells. This effect of AA-DA was counteracted by the CB(1) antagonist SR141716A. AA-DA behaved as a CB(1) agonist in vivo by inducing hypothermia, hypo-locomotion, catalepsy and analgesia in mice (1-10 mg/kg). Finally, AA-DA potently inhibited (IC(50)=0.25 microM) the proliferation of human breast MCF

  20. Cholesterol-Lowering Potentials of Lactic Acid Bacteria Based on Bile-Salt Hydrolase Activity and Effect of Potent Strains on Cholesterol Metabolism In Vitro and In Vivo

    PubMed Central

    Lin, Pei-Pei; Hsieh, You-Miin; Zhang, Zi-yi; Wu, Hui-Ching; Huang, Chun-Chih

    2014-01-01

    This study collected different probiotic isolates from animal and plant sources to evaluate the bile-salt hydrolase activity of probiotics in vitro. The deconjugation potential of bile acid was determined using high-performance liquid chromatography. HepG2 cells were cultured with probiotic strains with high BSH activity. The triglyceride (TG) and apolipoprotein B (apo B) secretion by HepG2 cells were evaluated. Our results show that the BSH activity and bile-acid deconjugation abilities of Pediococcus acidilactici NBHK002, Bifidobacterium adolescentis NBHK006, Lactobacillus rhamnosus NBHK007, and Lactobacillus acidophilus NBHK008 were higher than those of the other probiotic strains. The cholesterol concentration in cholesterol micelles was reduced within 24 h. NBHK007 reduced the TG secretion by 100% after 48 h of incubation. NBHK002, NBHK006, and NBHK007 could reduce apo B secretion by 33%, 38%, and 39%, respectively, after 24 h of incubation. The product PROBIO S-23 produced a greater decrease in the total concentration of cholesterol, low-density lipoprotein, TG, and thiobarbituric acid reactive substance in the serum or livers of hamsters with hypercholesterolemia compared with that of hamsters fed with a high-fat and high-cholesterol diet. These results show that the three probiotic strains of lactic acid bacteria are better candidates for reducing the risk of cardiovascular disease. PMID:25538960

  1. Ubiquitin carboxyl terminal hydrolase L1 negatively regulates TNF{alpha}-mediated vascular smooth muscle cell proliferation via suppressing ERK activation

    SciTech Connect

    Ichikawa, Tomonaga; Li, Jinqing; Dong, Xiaoyu; Potts, Jay D.; Tang, Dong-Qi; Li, Dong-Sheng; Cui, Taixing

    2010-01-01

    Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNF{alpha})-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNF{alpha}-induced activation of ERK and DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNF{alpha} hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.

  2. X-ray Crystallographic Analysis of α-Ketoheterocycle Inhibitors Bound to a Humanized Variant of Fatty Acid Amide Hydrolase

    PubMed Central

    Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine; Kimball, F. Scott; Cravatt, Benjamin F.; Stevens, Raymond C.; Boger, Dale L.

    2009-01-01

    Three cocrystal X-ray structures of the α-ketoheterocycle inhibitors 3–5 bound to a humanized variant of fatty acid amide hydrolase (FAAH) are disclosed and comparatively discussed alongside those of 1 (OL-135) and its isomer 2. These five X-ray structures systematically probe each of the three active site regions key to substrate or inhibitor binding: (1) the conformationally mobile acyl chain-binding pocket and membrane access channel responsible for fatty acid amide substrate and inhibitor acyl chain binding, (2) the atypical active site catalytic residues and surrounding oxyanion hole that covalently binds the core of the α-ketoheterocycle inhibitors captured as deprotonated hemiketals mimicking the tetrahedral intermediate of the enzyme catalyzed reaction, and (3) the cytosolic port and its uniquely important imbedded ordered water molecules and a newly identified anion binding site. The detailed analysis of their key active site interactions and their implications on the interpretation of the available structure–activity relationships are discussed providing important insights for future design. PMID:19924997

  3. Increased long chain acyl-Coa synthetase activity and fatty acid import is linked to membrane synthesis for development of picornavirus replication organelles.

    PubMed

    Nchoutmboube, Jules A; Viktorova, Ekaterina G; Scott, Alison J; Ford, Lauren A; Pei, Zhengtong; Watkins, Paul A; Ernst, Robert K; Belov, George A

    2013-01-01

    All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be

  4. Discovery of the first dual inhibitor of the 5-lipoxygenase-activating protein and soluble epoxide hydrolase using pharmacophore-based virtual screening

    PubMed Central

    Temml, Veronika; Garscha, Ulrike; Romp, Erik; Schubert, Gregor; Gerstmeier, Jana; Kutil, Zsofia; Matuszczak, Barbara; Waltenberger, Birgit; Stuppner, Hermann; Werz, Oliver; Schuster, Daniela

    2017-01-01

    Leukotrienes (LTs) are pro-inflammatory lipid mediators derived from arachidonic acid (AA) with roles in inflammatory and allergic diseases. The biosynthesis of LTs is initiated by transfer of AA via the 5-lipoxygenase-activating protein (FLAP) to 5-lipoxygenase (5-LO). FLAP inhibition abolishes LT formation exerting anti-inflammatory effects. The soluble epoxide hydrolase (sEH) converts AA-derived anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids (di-HETEs). Its inhibition consequently also counteracts inflammation. Targeting both LT biosynthesis and the conversion of EETs with a dual inhibitor of FLAP and sEH may represent a novel, powerful anti-inflammatory strategy. We present a pharmacophore-based virtual screening campaign that led to 20 hit compounds of which 4 targeted FLAP and 4 were sEH inhibitors. Among them, the first dual inhibitor for sEH and FLAP was identified, N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N’-(3,4-dichlorophenyl)urea with IC50 values of 200 nM in a cell-based FLAP test system and 20 nM for sEH activity in a cell-free assay. PMID:28218273

  5. Discovery of the first dual inhibitor of the 5-lipoxygenase-activating protein and soluble epoxide hydrolase using pharmacophore-based virtual screening

    NASA Astrophysics Data System (ADS)

    Temml, Veronika; Garscha, Ulrike; Romp, Erik; Schubert, Gregor; Gerstmeier, Jana; Kutil, Zsofia; Matuszczak, Barbara; Waltenberger, Birgit; Stuppner, Hermann; Werz, Oliver; Schuster, Daniela

    2017-02-01

    Leukotrienes (LTs) are pro-inflammatory lipid mediators derived from arachidonic acid (AA) with roles in inflammatory and allergic diseases. The biosynthesis of LTs is initiated by transfer of AA via the 5-lipoxygenase-activating protein (FLAP) to 5-lipoxygenase (5-LO). FLAP inhibition abolishes LT formation exerting anti-inflammatory effects. The soluble epoxide hydrolase (sEH) converts AA-derived anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids (di-HETEs). Its inhibition consequently also counteracts inflammation. Targeting both LT biosynthesis and the conversion of EETs with a dual inhibitor of FLAP and sEH may represent a novel, powerful anti-inflammatory strategy. We present a pharmacophore-based virtual screening campaign that led to 20 hit compounds of which 4 targeted FLAP and 4 were sEH inhibitors. Among them, the first dual inhibitor for sEH and FLAP was identified, N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N’-(3,4-dichlorophenyl)urea with IC50 values of 200 nM in a cell-based FLAP test system and 20 nM for sEH activity in a cell-free assay.

  6. Blocking of fatty acid amide hydrolase activity with PF-04457845 in human brain: a positron emission tomography study with the novel radioligand [(11)C]CURB.

    PubMed

    Boileau, Isabelle; Rusjan, Pablo M; Williams, Belinda; Mansouri, Esmaeil; Mizrahi, Romina; De Luca, Vincenzo; Johnson, Douglas S; Wilson, Alan A; Houle, Sylvain; Kish, Stephen J; Tong, Junchao

    2015-11-01

    Positron emission tomography with [(11)C]CURB was recently developed to quantify fatty acid amide hydrolase (FAAH), the enzyme responsible for hydrolyzing the endocannabinoid anandamide. This study investigated the test-retest reliability of [(11)C]CURB as well as its in vivo specificity and the validity of the kinetic model by using the highly specific FAAH inhibitor, PF-04457845. Five healthy volunteers completed test-retest [(11)C]CURB scans 1 to 2 months apart and six subjects completed baseline and blocking scans on the same day after PF-04457845 (p.o.) administration (1, 4, or 20 mg; n=2 each). The composite parameter λk3 (an index of FAAH activity, λ=K1/k2) was estimated using an irreversible two-tissue compartment model with plasma input function. There were no clinically observable responses to oral PF-04457845 or [(11)C]CURB injection. Oral administration of PF-04457845 reduced [(11)C]CURB binding to a homogeneous level at all three doses, with λk3 values decreased by ⩾91%. Excellent reproducibility and good reliability (test-retest variability=9%; intraclass correlation coefficient=0.79) were observed across all regions of interest investigated. Our findings suggest that λk3/[(11)C]CURB is a reliable, highly sensitive, and selective tool to measure FAAH activity in human brain in vivo. Moreover, PF-04457845 is a highly potent FAAH inhibitor (>95% inhibition at 1 mg) in living human brain.

  7. [Activation of the bioluminescence of the sensor Escherichia coli strains used for detecting N-acyl-homoserine lactones in the presence of nitrofurans and NO generators].

    PubMed

    Zaĭtseva, Iu V; Granik, V G; Belik, A S; Koksharova, O A; Khmel', I A

    2010-01-01

    Nitrofurans (nitrofurazone, nitrofurantoin, furazidin, nifuroxazide), and nitric oxide generators (sodium nitroprusside and isosorbide mononitrate) in subinhibitory concentrations were shown to significantly increase the bioluminescence of the sensor Escherichia coli strains used for detecting N-acyl-homoserine lactones, signaling molecules of Quorum Sensing (QS) regulatory systems. The highest activation of bioluminescence (up to 250-400 fold) was observed in the presence of nitrofurazone on E. coli DH5alpha biosensors containing lux-reporter plasmids pSB401 or pSB536. However, this activation was not specifically associated with the functioning of QS systems. We suggest that the effect observed results from a direct action of nitrofurans and NO donors on the process of bioluminescence. The data indicate the necessity of using the biosensors that make it possible to detect specific effects of substances tested on QS regulation.

  8. Synthesis and highly potent hypolipidemic activity of alpha-asarone- and fibrate-based 2-acyl and 2-alkyl phenols as HMG-CoA reductase inhibitors.

    PubMed

    Mendieta, Aarón; Jiménez, Fabiola; Garduño-Siciliano, Leticia; Mojica-Villegas, Angélica; Rosales-Acosta, Blanca; Villa-Tanaca, Lourdes; Chamorro-Cevallos, Germán; Medina-Franco, José L; Meurice, Nathalie; Gutiérrez, Rsuini U; Montiel, Luisa E; Cruz, María Del Carmen; Tamariz, Joaquín

    2014-11-01

    In the search for new potential hypolipidemic agents, the present study focused on the synthesis of 2-acyl phenols (6a-c and 7a-c) and their saturated side-chain alkyl phenols (4a-c and 5a-c), and on the evaluation of their hypolipidemic activity using a murine Tyloxapol-induced hyperlipidemic protocol. The whole series of compounds 4-7 greatly and significantly reduced elevated serum levels of total cholesterol, LDL-cholesterol, and triglycerides, with series 6 and 7 showing the greatest potency ever found in our laboratory. At the minimum dose (25mg/kg/day), the latter compounds lowered cholesterol by 68-81%, LDL by 72-86%, and triglycerides by 59-80%. This represents a comparable performance than that shown by simvastatin. Experimental evidence and docking studies suggest that the activity of these derivatives is associated with the inhibition of HMG-CoA reductase.

  9. [Peptide hydrolases with catalytic dyad Ser-Lys. Similarity and distinctions of the active centers of ATP-dependent Lon proteases, LexA repressors, signal peptidases and C-terminal processing proteases].

    PubMed

    Rotanova, T V

    2002-01-01

    It is established that ATP-dependent protease Lon family belongs to the serine-lysine peptide hydrolases clan. Significant similarity of amino acid sequences of proteases Lon and repressors LexA in the regions including the catalytic serine and lysine residues is revealed by comparing primary structures of different families of the enzymes with Ser-Lys catalytic dyad. The both Lon and LexA families are shown to be divided into two subfamilies in accordance with the nature of amino acids in the catalytically active serine environment. Putative DNA binding sites are revealed in proteolytic domains of Lon A subfamily. Similarities and distinctions of the all families peptide hydrolases of the clan in the regions of their active centers are discussed.

  10. Large Scale Structural Rearrangement of a Serine Hydrolase from Francisella tularensis Facilitates Catalysis*

    PubMed Central

    Filippova, Ekaterina V.; Weston, Leigh A.; Kuhn, Misty L.; Geissler, Brett; Gehring, Alexandra M.; Armoush, Nicola; Adkins, Chinessa T.; Minasov, George; Dubrovska, Ievgeniia; Shuvalova, Ludmilla; Winsor, James R.; Lavis, Luke D.; Satchell, Karla J. F.; Becker, Daniel P.; Anderson, Wayne F.; Johnson, R. Jeremy

    2013-01-01

    Tularemia is a deadly, febrile disease caused by infection by the Gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues. PMID:23430251

  11. The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes.

    PubMed

    Stout, Jake M; Boubakir, Zakia; Ambrose, Stephen J; Purves, Randy W; Page, Jonathan E

    2012-08-01

    The psychoactive and analgesic cannabinoids (e.g. Δ(9) -tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g(-1) fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short- and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids.

  12. Physiological Consequences of Compartmentalized Acyl-CoA Metabolism*

    PubMed Central

    Cooper, Daniel E.; Young, Pamela A.; Klett, Eric L.; Coleman, Rosalind A.

    2015-01-01

    Meeting the complex physiological demands of mammalian life requires strict control of the metabolism of long-chain fatty acyl-CoAs because of the multiplicity of their cellular functions. Acyl-CoAs are substrates for energy production; stored within lipid droplets as triacylglycerol, cholesterol esters, and retinol esters; esterified to form membrane phospholipids; or used to activate transcriptional and signaling pathways. Indirect evidence suggests that acyl-CoAs do not wander freely within cells, but instead, are channeled into specific pathways. In this review, we will discuss the evidence for acyl-CoA compartmentalization, highlight the key modes of acyl-CoA regulation, and diagram potential mechanisms for controlling acyl-CoA partitioning. PMID:26124277

  13. The Wood Rot Ascomycete Xylaria polymorpha Produces a Novel GH78 Glycoside Hydrolase That Exhibits α-l-Rhamnosidase and Feruloyl Esterase Activities and Releases Hydroxycinnamic Acids from Lignocelluloses

    PubMed Central

    Nghi, Do Huu; Bittner, Britta; Kellner, Harald; Jehmlich, Nico; Ullrich, René; Pecyna, Marek J.; Nousiainen, Paula; Sipilä, Jussi; Huong, Le Mai; Hofrichter, Martin

    2012-01-01

    Soft rot (type II) fungi belonging to the family Xylariaceae are known to substantially degrade hardwood by means of their poorly understood lignocellulolytic system, which comprises various hydrolases, including feruloyl esterases and laccase. In the present study, several members of the Xylariaceae were found to exhibit high feruloyl esterase activity during growth on lignocellulosic materials such as wheat straw (up to 1,675 mU g−1) or beech wood (up to 80 mU g−1). Following the ester-cleaving activity toward methyl ferulate, a hydrolase of Xylaria polymorpha was produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography to apparent homogeneity (specific activity, 2.2 U mg−1). The peptide sequence of the purified protein deduced from the gene sequence and verified by de novo peptide sequencing shows high similarity to putative α-l-rhamnosidase sequences belonging to the glycoside hydrolase family 78 (GH78; classified under EC 3.2.1.40). The purified enzyme (98 kDa by SDS-PAGE, 103 kDa by size-exclusion chromatography; pI 3.7) converted diverse glycosides (e.g., α-l-rhamnopyranoside and α-l-arabinofuranoside) but also natural and synthetic esters (e.g., chlorogenic acid, hydroxycinnamic acid glycoside esters, veratric acid esters, or p-nitrophenyl acetate) and released free hydroxycinnamic acids (ferulic and coumaric acid) from arabinoxylan and milled wheat straw. These catalytic properties strongly suggest that X. polymorpha GH78 is a multifunctional enzyme. It is the first fungal enzyme that combines glycosyl hydrolase with esterase activities and may help this soft rot fungus to degrade lignocelluloses. PMID:22544251

  14. A Novel Glucosylation Reaction on Anthocyanins Catalyzed by Acyl-Glucose–Dependent Glucosyltransferase in the Petals of Carnation and Delphinium[C][W

    PubMed Central

    Matsuba, Yuki; Sasaki, Nobuhiro; Tera, Masayuki; Okamura, Masachika; Abe, Yutaka; Okamoto, Emi; Nakamura, Haruka; Funabashi, Hisakage; Takatsu, Makoto; Saito, Mikako; Matsuoka, Hideaki; Nagasawa, Kazuo; Ozeki, Yoshihiro

    2010-01-01

    Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose–dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as β-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose–dependent glucosyltransferases. PMID:20971893

  15. Creation of active TIM barrel enzymes through genetic fusion of half-barrel domain constructs derived from two distantly related glycosyl hydrolases.

    PubMed

    Sharma, Prerna; Kaila, Pallavi; Guptasarma, Purnananda

    2016-12-01

    Diverse unrelated enzymes that adopt the beta/alpha (or TIM) barrel topology display similar arrangements of beta/alpha units placed in a radial eight-fold symmetry around the barrel's axis. The TIM barrel was originally thought to be a single structural domain; however, it is now thought that TIM barrels arose from duplication and fusion of smaller half-barrels consisting of four beta/alpha units. We describe here the design, expression and purification, as well as characterization of folding, activity and stability, of chimeras of two TIM barrel glycosyl hydrolases, made by fusing different half-barrel domains derived from an endoglucanase from Clostridium cellulolyticum, CelCCA and a beta-glucosidase from Pyrococcus furiosus, CelB. We show that after refolding following purification from inclusion bodies, the two half-barrel fusion chimeras (CelCCACelB and CelBCelCCA) display catalytic activity although they assemble into large soluble oligomeric aggregated species containing chains of mixed beta and alpha structure. CelBCelCCA displays hyperthermophile-like structural stability as well as significant stability to chemical denaturation (Cm of 2.6 m guanidinium hydrochloride), whereas CelCCACelB displays mesophile-like stability (Tm of ~ 71 °C). The endoglucanase activities of both chimeras are an order of magnitude lower than those of CelB or CelCCA, whereas the beta-glucosidase activity of CelBCelCCA is about two orders of magnitude lower than that of CelB. The chimera CelCCACelB shows no beta-glucosidase activity. Our results demonstrate that half-barrel domains from unrelated sources can fold, assemble and function, with scope for improvement.

  16. Potent Natural Soluble Epoxide Hydrolase Inhibitors from Pentadiplandra brazzeana Baillon: Synthesis, Quantification, and Measurement of Biological Activities In Vitro and In Vivo

    PubMed Central

    Kitamura, Seiya; Morisseau, Christophe; Inceoglu, Bora; Kamita, Shizuo G.; De Nicola, Gina R.; Nyegue, Maximilienne; Hammock, Bruce D.

    2015-01-01

    We describe here three urea-based soluble epoxide hydrolase (sEH) inhibitors from the root of the plant Pentadiplandra brazzeana. The concentration of these ureas in the root was quantified by LC-MS/MS, showing that 1, 3-bis (4-methoxybenzyl) urea (MMU) is the most abundant (42.3 μg/g dry root weight). All of the ureas were chemically synthesized, and their inhibitory activity toward recombinant human and recombinant rat sEH was measured. The most potent compound, MMU, showed an IC50 of 92 nM via fluorescent assay and a Ki of 54 nM via radioactivity-based assay on human sEH. MMU effectively reduced inflammatory pain in a rat nociceptive pain assay. These compounds are among the most potent sEH inhibitors derived from natural sources. Moreover, inhibition of sEH by these compounds may mechanistically explain some of the therapeutic effects of P. brazzeana. PMID:25659109

  17. Elucidation of exo-beta-D-glucosaminidase activity of a family 9 glycoside hydrolase (PBPRA0520) from Photobacterium profundum SS9.

    PubMed

    Honda, Yuji; Shimaya, Nozomi; Ishisaki, Kana; Ebihara, Mitsuru; Taniguchi, Hajime

    2011-04-01

    A glycoside hydrolase (GH) gene from Photobacterium profundum SS9 (PBPRA0520) belonging to GH family 9 was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it is an intracellular enzyme. The recombinant protein showed hydrolytic activity toward chitobiose [(GlcN)(2)] and cellobiose (CG(2)) in various disaccharides. This protein also released 4-nitrophenol (PNP) from both 4-nitrophenyl-β-D-glucosaminide (GlcN-PNP) and 4-nitrophenyl-β-D-glucoside (Glc-PNP). The hydrolytic pattern observed in chitooligosaccharides and cellooligosaccharides suggested that the reaction proceeded from the nonreducing end in an exo-type manner. Time-dependent (1)H-nuclear magnetic resonance (NMR) analysis of the anomeric form of the enzymatic reaction products indicated that the protein is an inverting enzyme. k(cat)/K(m) of (GlcN)(2) hydrolysis was 14 times greater than that of CG(2) hydrolysis. These results suggested that the protein is an exo-β-D-glucosaminidase (EC 3.2.1.165) rather than a glucan 1,4-β-D-glucosidase (EC 3.2.1.74). Based on the results, we suggest that the function of conserved GH9 proteins in the chitin catabolic operon is to cleave a (GlcN)(2)-phosphate derivative by hydrolysis during intracellular chitooligosaccharide catabolism in Vibrionaceae.

  18. The Immunoreactive Exo-1,3-β-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity

    PubMed Central

    Keeratijarut, Angsana; Lohnoo, Tassanee; Rujirawat, Thidarat; Yingyong, Wanta; Kalambaheti, Thareerat; Miller, Shannon; Phuntumart, Vipaporn; Krajaejun, Theerapong

    2015-01-01

    The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called “pythiosis”. Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1). This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases) was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5). Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen. PMID:26263509

  19. Structural characterization of a beta-diketone hydrolase from the cyanobacterium Anabaena sp. PCC 7120 in native and product-bound forms, a coenzyme A-independent member of the crotonase suprafamily.

    PubMed

    Bennett, Joseph P; Whittingham, Jean L; Brzozowski, A Marek; Leonard, Philip M; Grogan, Gideon

    2007-01-09

    The gene alr4455 from the well-studied cyanobacterium Anabaena sp. PCC 7120 encodes a crotonase orthologue that displays beta-diketone hydrolase activity. Anabaena beta-diketone hydrolase (ABDH), in common with 6-oxocamphor hydrolase (OCH) from Rhodococcus sp. NCIMB 9784, catalyzes the desymmetrization of bicyclo[2.2.2]octane-2,6-dione to yield [(S)-3-oxocyclohexyl]acetic acid, a reaction unusual among the crotonase superfamily as the substrate is not an acyl-CoA thioester. The structure of ABDH has been determined to a resolution of 1.5 A in both native and ligand-bound forms. ABDH forms a hexamer similar to OCH and features one active site per enzyme monomer. The arrangement of side chains in the active site indicates that while the catalytic chemistry may be conserved in OCH orthologues, the structural determinants of substrate specificity are different. In the active site of ligand-bound forms that had been cocrystallized with the bicyclic diketone substrate bicyclo[2.2.2]octane-2,6-dione was found the product of the asymmetric enzymatic retro-Claisen reaction [(S)-3-oxocyclohexyl]acetic acid. The structures of ABDH in both native and ligand-bound forms reveal further details about structural variation and modes of coenzyme A-independent activity within the crotonases and provide further evidence of a wider suprafamily of enzymes that have recruited the crotonase fold for the catalysis of reactions other than those regularly attributed to canonical superfamily members.

  20. N-Acyl-Homoserine Lactone Confers Resistance toward Biotrophic and Hemibiotrophic Pathogens via Altered Activation of AtMPK61[C][W

    PubMed Central

    Schikora, Adam; Schenk, Sebastian T.; Stein, Elke; Molitor, Alexandra; Zuccaro, Alga; Kogel, Karl-Heinz

    2011-01-01

    Pathogenic and symbiotic bacteria rely on quorum sensing to coordinate the collective behavior during the interactions with their eukaryotic hosts. Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as signals in such communication. Here we show that plants have evolved means to perceive AHLs and that the length of acyl moiety and the functional group at the γ position specify the plant’s response. Root treatment with the N-3-oxo-tetradecanoyl-L-homoserine lactone (oxo-C14-HSL) reinforced the systemic resistance to the obligate biotrophic fungi Golovinomyces orontii in Arabidopsis (Arabidopsis thaliana) and Blumeria graminis f. sp. hordei in barley (Hordeum vulgare) plants. In addition, oxo-C14-HSL-treated Arabidopsis plants were more resistant toward the hemibiotrophic bacterial pathogen Pseudomonas syringae pv tomato DC3000. Oxo-C14-HSL promoted a stronger activation of mitogen-activated protein kinases AtMPK3 and AtMPK6 when challenged with flg22, followed by a higher expression of the defense-related transcription factors WRKY22 and WRKY29, as well as the PATHOGENESIS-RELATED1 gene. In contrast to wild-type Arabidopsis and mpk3 mutant, the mpk6 mutant is compromised in the AHL effect, suggesting that AtMPK6 is required for AHL-induced resistance. Results of this study show that AHLs commonly produced in the rhizosphere are crucial factors in plant pathology and could be an agronomic issue whose full impact has to be elucidated in future analyses. PMID:21940998

  1. Modulation of cellulase activity by charged lipid bilayers with different acyl chain properties for efficient hydrolysis of ionic liquid-pretreated cellulose.

    PubMed

    Mihono, Kai; Ohtsu, Takeshi; Ohtani, Mai; Yoshimoto, Makoto; Kamimura, Akio

    2016-10-01

    The stability of cellulase activity in the presence of ionic liquids (ILs) is critical for the enzymatic hydrolysis of insoluble cellulose pretreated with ILs. In this work, cellulase was incorporated in the liposomes composed of negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and zwitterionic phosphatidylcholines (PCs) with different length and degree of unsaturation of the acyl chains. The liposomal cellulase-catalyzed reaction was performed at 45°C in the acetate buffer solution (pH 4.8) with 2.0g/L CC31 as cellulosic substrate. The crystallinity of CC31 was reduced by treating with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) at 120°C for 30min. The liposomal cellulase continuously catalyzed hydrolysis of the pretreated CC31 for 48h producing glucose in the presence of 15wt% [Bmim]Cl. The charged lipid membranes were interactive with [Bmim](+), as elucidated by the [Bmim]Cl-induced alterations in fluorescence polarization of the membrane-embedded 1,6-diphenyl-1,3,5-hexatriene (DPH) molecules. The charged membranes offered the microenvironment where inhibitory effects of [Bmim]Cl on the cellulase activity was relieved. The maximum glucose productivity GP of 10.8 mmol-glucose/(hmol-lipid) was obtained at the reaction time of 48h with the cellulase incorporated in the liposomes ([lipid]=5.0mM) composed of 50mol% POPG and 1,2-dilauroyl-sn-glycero-3-phosohocholine (DLPC) with relatively short and saturated acyl chains.

  2. Discovery and Optimization of Piperidyl-1,2,3-Triazole Ureas as Potent, Selective, and In Vivo-Active Inhibitors of Alpha/Beta-Hydrolase Domain Containing 6 (ABHD6)

    PubMed Central

    Hsu, Ku-Lung; Tsuboi, Katsunori; Chang, Jae Won; Whitby, Landon R.; Speers, Anna E.; Pugh, Holly; Cravatt, Benjamin F.

    2014-01-01

    Alpha/beta-hydrolase domain containing 6 (ABHD6) is a transmembrane serine hydrolase that hydrolyzes the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) to regulate certain forms of cannabinoid receptor-dependent signaling in the nervous system. The full spectrum of ABHD6 metabolic activities and functions is currently unknown and would benefit from selective, in vivo-active inhibitors. Here, we report the development and characterization of an advanced series of irreversible (2-substituted)-piperidyl-1,2,3-triazole urea inhibitors of ABHD6, including compounds KT182 and KT203, which show exceptional potency and selectivity in cells (< 5 nM) and, at equivalent doses in mice (1 mg kg-1), served as systemic and peripherally-restricted ABHD6 inhibitors, respectively. We also describe an orally-bioavailable ABHD6 inhibitor KT185 that displays excellent selectivity against other brain and liver serine hydrolases in vivo. We thus describe several chemical probes for biological studies of ABHD6, including brain-penetrant and peripherally-restricted inhibitors that should prove of value for interrogating ABHD6 function in animal models. PMID:24152295

  3. Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse.

    PubMed

    Guenthner, T M; Hammock, B D; Vogel, U; Oesch, F

    1981-04-10

    Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.

  4. Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis†

    PubMed Central

    van Loo, Bert; Kingma, Jaap; Arand, Michael; Wubbolts, Marcel G.; Janssen, Dick B.

    2006-01-01

    Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative α/β-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts. PMID:16597997

  5. Length of the active-site crossover loop defines the substrate specificity of ubiquitin C-terminal hydrolases for ubiquitin chains.

    PubMed

    Zhou, Zi-Ren; Zhang, Yu-Hang; Liu, Shuai; Song, Ai-Xin; Hu, Hong-Yu

    2012-01-01

    UCHs [Ub (ubiquitin) C-terminal hydrolases] are a family of deubiquitinating enzymes that are often thought to only remove small C-terminal peptide tails from Ub adducts. Among the four UCHs identified to date, neither UCH-L3 nor UCH-L1 can catalyse the hydrolysis of isopeptide Ub chains, but UCH-L5 can when it is present in the PA700 complex of the proteasome. In the present paper, we report that the UCH domain of UCH-L5, different from UCH-L1 and UCH-L3, by itself can process the K48-diUb (Lys48-linked di-ubiquitin) substrate by cleaving the isopeptide bond between two Ub units. The catalytic specificity of the four UCHs is dependent on the length of the active-site crossover loop. The UCH domain with a long crossover loop (usually >14 residues), such as that of UCH-L5 or BAP1 [BRCA1 (breast cancer early-onset 1)-associated protein 1], is able to cleave both small and large Ub derivatives, whereas the one with a short loop can only process small Ub derivatives. We also found that elongation of the crossover loop enables UCH-L1 to have isopeptidase activity for K48-diUb in a length-dependent manner. Thus the loop length of UCHs defines their substrate specificity for diUb chains, suggesting that the chain flexibility of the crossover loop plays an important role in determining its catalytic activity and substrate specificity for cleaving isopeptide Ub chains.

  6. Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

    PubMed

    Schaefer, A L; Val, D L; Hanzelka, B L; Cronan, J E; Greenberg, E P

    1996-09-03

    Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

  7. Synthesis, Structure, and Anticancer Activity of Arene-Ruthenium(II) Complexes with Acylpyrazolones Bearing Aliphatic Groups in the Acyl Moiety.

    PubMed

    Palmucci, Jessica; Marchetti, Fabio; Pettinari, Riccardo; Pettinari, Claudio; Scopelliti, Rosario; Riedel, Tina; Therrien, Bruno; Galindo, Agustin; Dyson, Paul J

    2016-11-21

    A series of neutral ruthenium(II) arene complexes [(arene)Ru(Q(R))Cl] (arene = p-cymene (cym) or hexamethylbenzene (hmb)) containing 4-acyl-5-pyrazolonate Q(R) ligands with different electronic and steric substituents (R = 4-cyclohexyl, 4-stearoyl, or 4-adamantyl) and related ionic complexes [(arene)Ru(Q(R))(PTA)][PF6] (PTA = 1,3,5-triaza-7-phosphaadamantane) were synthesized and characterized by spectroscopy (IR, UV-vis, ESI-MS, and (1)H and (13)C NMR), elemental analysis, X-ray crystallography, and density functional theory studies. The cytotoxicity of the proligands and metal complexes was evaluated in vitro against human ovarian carcinoma cells (A2780 and A2780cisR), as well as against nontumorous human embryonic kidney (HEK293) cells. In general the cationic PTA-containing complexes are more cytotoxic than their neutral precursors with a chloride ligand in place of the PTA. Moreover, the complexes do not show cross-resistance and are essentially equally cytotoxic to both the A2780 and A2780cisR cell lines, although they only show limited selectivity toward the cancer cell lines.

  8. Understanding Acyl Chain and Glycerolipid Metabolism in Plants

    SciTech Connect

    Ohlrogge, John B.

    2013-11-05

    Progress is reported in these areas: acyl-editing in initial eukaryotic lipid assembly in soybean seeds; identification and characterization of two Arabidopsis thaliana lysophosphatidyl acyltransferases with preference for lysophosphatidylethanolamine; and characterization and subcellular distribution of lysolipid acyl transferase activity of pea leaves.

  9. Characterization of the "Escherichia Coli" Acyl Carrier Protein Phosphodiesterase

    ERIC Educational Resources Information Center

    Thomas, Jacob

    2009-01-01

    Acyl carrier protein (ACP) is a small essential protein that functions as a carrier of the acyl intermediates of fatty acid synthesis. ACP requires the posttranslational attachment of a 4'phosphopantetheine functional group, derived from CoA, in order to perform its metabolic function. A Mn[superscript 2+] dependent enzymatic activity that removes…

  10. Modified acyl-ACP desaturase

    DOEpatents

    Cahoon, E.B.; Shanklin, J.; Lindgvist, Y.; Schneider, G.

    1998-01-06

    Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 1 fig.

  11. A 1-year study of the activities of seven hydrolases in a communal wastewater treatment plant: trends and correlations.

    PubMed

    Kreutz, Jennifer Anna; Böckenhüser, Ina; Wacht, Marion; Fischer, Klaus

    2016-08-01

    The activities of seven hydrolytic enzymes (L-alanine aminopeptidase, esterase, α-and β-glucosidase, phosphomonoesterase, phosphodiesterase, sulfatase) were monitored during 1 year in parallel and serial treatment units of the biological stage of a communal wastewater treatment plant. The spatial homogeneity of enzyme activities was high (coefficients of variation <10 % for the entire treatment stage). A significant difference between aerated and stirred tanks was not observed. Temperature seemed not to exert a direct influence. Long periods with comparably constant activities were interrupted by a few strong, short-time rises. The mean enzyme activities followed the sequence sulfatase < α-glucosidase < phosphodiesterase ≈ β-glucosidase≈esterase < phosphomonoesterase < L-alanine aminopeptidase. The enzyme activities correlated among themselves at different levels. Very strong (r > 0.8) and highly significant (p < 0.01) correlations between the activities of both glucosidases, both phosphoesterases, and between phosphomonoesterase and both glucosidases were ascertained, pointing to the importance of substrate specificity and similarity of metabolic functions. Moderate and strong activity correlations with various wastewater constituents and with process parameters, e.g., concentrations, loads and eliminated amounts of phosphorous, TOC concentrations and loads of the plant effluent, dry matter content of activated sludge, and sludge volume, were found. The esterase activity was least correlated with other enzymes and often showed deviating dependencies on process parameters, raising questions concerning its appropriateness as a sum parameter for enzymatic and heterotrophic activity.

  12. Blocking of fatty acid amide hydrolase activity with PF-04457845 in human brain: a positron emission tomography study with the novel radioligand [11C]CURB

    PubMed Central

    Boileau, Isabelle; Rusjan, Pablo M; Williams, Belinda; Mansouri, Esmaeil; Mizrahi, Romina; De Luca, Vincenzo; Johnson, Douglas S; Wilson, Alan A; Houle, Sylvain; Kish, Stephen J; Tong, Junchao

    2015-01-01

    Positron emission tomography with [11C]CURB was recently developed to quantify fatty acid amide hydrolase (FAAH), the enzyme responsible for hydrolyzing the endocannabinoid anandamide. This study investigated the test–retest reliability of [11C]CURB as well as its in vivo specificity and the validity of the kinetic model by using the highly specific FAAH inhibitor, PF-04457845. Five healthy volunteers completed test–retest [11C]CURB scans 1 to 2 months apart and six subjects completed baseline and blocking scans on the same day after PF-04457845 (p.o.) administration (1, 4, or 20 mg; n=2 each). The composite parameter λk3 (an index of FAAH activity, λ=K1/k2) was estimated using an irreversible two-tissue compartment model with plasma input function. There were no clinically observable responses to oral PF-04457845 or [11C]CURB injection. Oral administration of PF-04457845 reduced [11C]CURB binding to a homogeneous level at all three doses, with λk3 values decreased by ⩾91%. Excellent reproducibility and good reliability (test–retest variability=9% intraclass correlation coefficient=0.79) were observed across all regions of interest investigated. Our findings suggest that λk3/[11C]CURB is a reliable, highly sensitive, and selective tool to measure FAAH activity in human brain in vivo. Moreover, PF-04457845 is a highly potent FAAH inhibitor (>95% inhibition at 1 mg) in living human brain. PMID:26082009

  13. Acyl-Lipid Metabolism

    PubMed Central

    Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred; Andersson, Mats X.; Arondel, Vincent; Bates, Philip D.; Baud, Sébastien; Bird, David; DeBono, Allan; Durrett, Timothy P.; Franke, Rochus B.; Graham, Ian A.; Katayama, Kenta; Kelly, Amélie A.; Larson, Tony; Markham, Jonathan E.; Miquel, Martine; Molina, Isabel; Nishida, Ikuo; Rowland, Owen; Samuels, Lacey; Schmid, Katherine M.; Wada, Hajime; Welti, Ruth; Xu, Changcheng; Zallot, Rémi; Ohlrogge, John

    2010-01-01

    Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. PMID:22303259

  14. Acyl-Lipid Metabolism

    PubMed Central

    Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred; Andersson, Mats X.; Arondel, Vincent; Bates, Philip D.; Baud, Sébastien; Bird, David; DeBono, Allan; Durrett, Timothy P.; Franke, Rochus B.; Graham, Ian A.; Katayama, Kenta; Kelly, Amélie A.; Larson, Tony; Markham, Jonathan E.; Miquel, Martine; Molina, Isabel; Nishida, Ikuo; Rowland, Owen; Samuels, Lacey; Schmid, Katherine M.; Wada, Hajime; Welti, Ruth; Xu, Changcheng; Zallot, Rémi; Ohlrogge, John

    2013-01-01

    Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. PMID:23505340

  15. Down-regulation of acyl-CoA oxidase gene expression and increased NF-kappaB activity in etomoxir-induced cardiac hypertrophy.

    PubMed

    Cabrero, Agatha; Merlos, Manuel; Laguna, Juan C; Carrera, Manuel Vázquez

    2003-02-01

    Activation of nuclear factor-kappaB (NF-kappaB) is required for hypertrophic growth of cardiomyocytes. Etomoxir is an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I) that activates peroxisome proliferator-activated receptor alpha (PPARalpha) and induces cardiac hypertrophy through an unknown mechanism. We studied the mRNA expression of genes involved in fatty acid oxidation in the heart of mice treated for 1 or 10 days with etomoxir (100 mg/kg/day). Etomoxir administration for 1 day significantly increased (4.4-fold induction) the mRNA expression of acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in peroxisomal beta-oxidation. In contrast, etomoxir treatment for 10 days dramatically decreased ACO mRNA levels by 96%. The reduction in ACO expression in the hearts of 10-day etomoxir-treated mice was accompanied by an increase in the mRNA expression of the antioxidant enzyme glutathione peroxidase and the cardiac marker of oxidative stress bax. Moreover, the activity of the redox-regulated transcription factor NF-kappaB was increased in heart after 10 days of etomoxir treatment. Overall, the findings here presented show that etomoxir treatment may induce cardiac hypertrophy via increased cellular oxidative stress and NF-kappaB activation.

  16. Prioritization of active antimalarials using structural interaction profile of Plasmodium falciparum enoyl-acyl carrier protein reductase (PfENR)-triclosan derivatives.

    PubMed

    Kumar, S P; George, L B; Jasrai, Y T; Pandya, H A

    2015-01-01

    An empirical relationship between the experimental inhibitory activities of triclosan derivatives and its computationally predicted Plasmodium falciparum enoyl-acyl carrier protein (ACP) reductase (PfENR) dock poses was developed to model activities of known antimalarials. A statistical model was developed using 57 triclosan derivatives with significant measures (r = 0.849, q(2) = 0.619, s = 0.481) and applied on structurally related and structurally diverse external datasets. A substructure-based search on ChEMBL malaria dataset (280 compounds) yielded only two molecules with significant docking energy, whereas eight active antimalarials (EC(50) < 100 nM, tested on 3D7 strain) with better predicted activities (pIC(50) ~ 7) from Open Access Malaria Box (400 compounds) were prioritized. Further, calculations on the structurally diverse rhodanine molecules (known PfENR inhibitors) distinguished actives (experimental IC(50) = 0.035 μM; predicted pIC(50) = 6.568) and inactives (experimental IC(50) = 50 μM; predicted pIC50 = -4.078), which showed that antimalarials possessing dock poses similar to experimental interaction profiles can be used as leads to test experimentally on enzyme assays.

  17. ROMPgel beads in IRORI format: acylations revisited.

    PubMed

    Roberts, Richard S

    2005-01-01

    Functionalized "designer" polymers derived from ring-opening metathesis polymerization (ROMPgels) are attractive for their high loading, high purity, and ease of synthesis. Their physical state may vary from liquid to gel to granular solid, making a general method of handling these polymers difficult. By incorporating a suitable norbornene-substituted linker on standard Wang beads, ROMPgels can be easily grafted onto the resin, adding the convenience of a bead format while still maintaining the high loading and excellent site accessibility. This advantage is demonstrated by the use of an N-hydroxysuccinimide ROMPgel (3.3 mmol g(-1), a 3-fold increase from the parent linker resin) in IRORI Kan format. Conditions for the acylation of these IRORI-formatted ROMPgels are reported, along with the scope and limitations of the choice of acylating reagents. Yields are greatly improved by the use of perfluorinated solvents as a nonparticipating cosolvent in the acylation process. A simple titration method for the quantification of the acylated ROMPgels is also reported. Spent Kans are regenerated after each use without apparent loss of activity or purity after several cycles. Due to the high loading and reduced swelling of the ROMPgel resin, up to 0.39 mmol acyl group has successfully been recovered from a single IRORI miniKan, demonstrating the high capacity of the resin and applicability to both lead discovery and optimization programs.

  18. Structural and functional attributes of malaria parasite diadenosine tetraphosphate hydrolase

    PubMed Central

    Sharma, Arvind; Yogavel, Manickam; Sharma, Amit

    2016-01-01

    Malaria symptoms are driven by periodic multiplication cycles of Plasmodium parasites in human red blood corpuscles (RBCs). Malaria infection still accounts for ~600,000 annual deaths, and hence discovery of both new drug targets and drugs remains vital. In the present study, we have investigated the malaria parasite enzyme diadenosine tetraphosphate (Ap4A) hydrolase that regulates levels of signalling molecules like Ap4A by hydrolyzing them to ATP and AMP. We have tracked the spatial distribution of parasitic Ap4A hydrolase in infected RBCs, and reveal its unusual localization on the infected RBC membrane in subpopulation of infected cells. Interestingly, enzyme activity assays reveal an interaction between Ap4A hydrolase and the parasite growth inhibitor suramin. We also present a high resolution crystal structure of Ap4A hydrolase in apo- and sulphate- bound state, where the sulphate resides in the enzyme active site by mimicking the phosphate of substrates like Ap4A. The unexpected infected erythrocyte localization of the parasitic Ap4A hydrolase hints at a possible role of this enzyme in purinerigic signaling. In addition, atomic structure of Ap4A hydrolase provides insights for selective drug targeting. PMID:26829485

  19. Lytic activity of the virion-associated peptidoglycan hydrolase HydH5 of staphylococcus aureus bacteriophage vB_SauS-phiIPLA88

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 (phiIPLA88) contains a virion-associated muralytic enzyme (HydH5) encoded by orf58, which is located in the morphogenetic module. Comparative bioinformatic analysis revealed that HydH5 significantly resembled other peptidoglycan hydrolases encode...

  20. Changes in the Activity of Some Hydrolases, Peroxidase, and Catalase in the Rice Seed during Germination 1

    PubMed Central

    Palmiano, Evelyn P.; Juliano, Bienvenido O.

    1973-01-01

    A study was made of the changes in activity of enzymes involved in the breakdown of stored phytin, lipid, and hemicellulose in the aleurone layer of rice seed (Oryza sativa L., variety IR8) during the 1st week of germination in the light. Enzyme assays were made on crude extracts from degermed seed, and activities were expressed on a per seed basis. Phytase activity increased within the 1st day of germination. The increase in activity of most other enzymes—phosphomonoesterase, phosphodiesterase, esterase, lipase, peroxidase, catalase, β-glucosidase, and α- and β-galactosidase—closely followed the increase in protein content. Their peak activities occurred by the 5th to the 7th day. Some enzymes, such as β-1, 3-glucanase and α-amylase, continued to increase in activity after the 7th day. Phytase, β-1, 3-glucanase, and α-amylase followed a similar sequence of production in embryoless seed halves incubated in 0.12 μM gibberellin A3, but the production of lipase was delayed. PMID:16658546

  1. Soluble epoxide hydrolase inhibition and peroxisome proliferator activated receptor γ agonist improve vascular function and decrease renal injury in hypertensive obese rats.

    PubMed

    Imig, John D; Walsh, Katie A; Hye Khan, Md Abdul; Nagasawa, Tasuku; Cherian-Shaw, Mary; Shaw, Sean M; Hammock, Bruce D

    2012-12-01

    Cardiometabolic syndrome occurs with obesity and consists of pathophysiological factors that increase the risk for cardiovascular events. Soluble epoxide hydrolase inhibition (sEHi) is a novel therapeutic approach that exerts renal and cardiovascular protection. Although sEHi as a therapeutic approach is promising, it could be more effective for the treatment of cardiometabolic syndrome when combined with peroxisome proliferator activated receptor γ (PPARγ) agonists. We hypothesized that the PPARγ agonist, rosiglitazone in combination with a sEHi (tAUCB) will provide synergistic actions to decrease blood pressure, improve vascular function, decrease inflammation, and prevent renal damage in spontaneously hypertensive obese rats (SHROB). SHROB were treated with rosiglitazone, tAUCB or the combination of tAUCB and rosiglitazone for four-weeks and compared with spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Blood pressure increased in SHROB (164 ± 7 mmHg) and decreased 10 mmHg when treated with rosiglitazone, tAUCB, or tAUCB and rosiglitazone. Mesenteric artery dilation to the K(ATP) channel opener pinacidil was attenuated in SHROB (E(Max) = 77 ± 7%), compared with WKY (E(Max) = 115 ± 19) and SHR (E(Max) = 93 ± 12%). Vasodilation to pinacidil was improved by rosiglitazone (E(Max) = 92 ± 14%) but not tAUCB. Renal macrophage infiltration increased in SHROB and significantly decreased with rosiglitazone or tAUCB and rosiglitazone treatment. Albuminuria was increased in SHROB (90 ± 20 mg/d) and was significantly decreased by the combination of tAUCB and rosiglitazone (37 ± 9 mg/d). Glomerular injury in SHROB was also significantly decreased by tAUCB and rosiglitazone. These results indicate that even though sEHi or PPARγ agonist have benefits when used individually, the combination is more beneficial for the multidisease features in cardiometabolic syndrome.

  2. Soluble epoxide hydrolase inhibition and peroxisome proliferator activated receptor γ agonist improve vascular function and decrease renal injury in hypertensive obese rats

    PubMed Central

    Imig, John D; Walsh, Katie A; Khan, Md Abdul Hye; Nagasawa, Tasuku; Cherian-Shaw, Mary; Shaw, Sean M; Hammock, Bruce D

    2013-01-01

    Cardiometabolic syndrome occurs with obesity and consists of pathophysiological factors that increase the risk for cardiovascular events. Soluble epoxide hydrolase inhibition (sEHi) is a novel therapeutic approach that exerts renal and cardiovascular protection. Although sEHi as a therapeutic approach is promising, it could be more effective for the treatment of cardiometabolic syndrome when combined with peroxisome proliferator activated receptor γ (PPARγ) agonists. We hypothesized that the PPARγ agonist, rosiglitazone in combination with a sEHi (tAUCB) will provide synergistic actions to decrease blood pressure, improve vascular function, decrease inflammation, and prevent renal damage in spontaneously hypertensive obese rats (SHROB). SHROB were treated with rosiglitazone, tAUCB or the combination of tAUCB and rosiglitazone for four-weeks and compared with spontaneously hypertensive (SHR) and Wistar–Kyoto (WKY) rats. Blood pressure increased in SHROB (164 ±7 mmHg) and decreased 10 mmHg when treated with rosiglitazone, tAUCB, or tAUCB and rosiglitazone. Mesenteric artery dilation to the KATP channel opener pinacidil was attenuated in SHROB (EMax = 77 ±7%), compared with WKY (EMax = 115 ±19) and SHR (EMax = 93 ±12%). Vasodilation to pinacidil was improved by rosiglitazone (EMax = 92 ±14%) but not tAUCB. Renal macrophage infiltration increased in SHROB and significantly decreased with rosiglitazone or tAUCB and rosiglitazone treatment. Albuminuria was increased in SHROB (90 ±20 mg/d) and was significantly decreased by the combination of tAUCB and rosiglitazone (37 ±9 mg/d). Glomerular injury in SHROB was also significantly decreased by tAUCB and rosiglitazone. These results indicate that even though sEHi or PPARγ agonist have benefits when used individually, the combination is more beneficial for the multidisease features in cardiometabolic syndrome. PMID:23354399

  3. Long-Term Blockade of Cocaine Self-Administration and Locomotor Activation in Rats by an Adenoviral Vector-Delivered Cocaine Hydrolase.

    PubMed

    Smethells, John R; Swalve, Natashia; Brimijoin, Stephen; Gao, Yang; Parks, Robin J; Greer, Adam; Carroll, Marilyn E

    2016-05-01

    A promising approach in treating cocaine abuse is to metabolize cocaine in the blood using a mutated butyrylcholinesterase (BChE) that functions as a cocaine hydrolase (CocH). In rats, a helper-dependent adenoviral (hdAD) vector-mediated delivery of CocH abolished ongoing cocaine use and cocaine-primed reinstatement of drug-seeking for several months. This enzyme also metabolizes ghrelin, an effect that may be beneficial in maintaining healthy weights. The effect of a single hdAD-CocH vector injection was examined in rats on measures of anxiety, body weight, cocaine self-administration, and cocaine-induced locomotor activity. To examine anxiety, periadolescent rats were tested in an elevated-plus maze. Weight gain was then examined under four rodent diets. Ten months after CocH-injection, adult rats were trained to self-administer cocaine intravenously and, subsequently, cocaine-induced locomotion was tested. Viral gene transfer produced sustained plasma levels of CocH for over 13 months of testing. CocH-treated rats did not differ from controls in measures of anxiety, and only showed a transient reduction in weight gain during the first 3 weeks postinjection. However, CocH-treated rats were insensitive to cocaine. At 10 months postinjection, none of the CocH-treated rats initiated cocaine self-administration, unlike 90% of the control rats. At 13 months postinjection, CocH-treated rats showed no cocaine-induced locomotion, whereas control rats showed a dose-dependent enhancement of locomotion. CocH vector produced a long-term blockade of the rewarding and behavioral effects of cocaine in rats, emphasizing its role as a promising therapeutic intervention in cocaine abuse.

  4. Bacterial Cyanuric Acid Hydrolase for Water Treatment

    PubMed Central

    Yeom, Sujin; Mutlu, Baris R.; Aksan, Alptekin

    2015-01-01

    Di- and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 μM, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 μM cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation. PMID:26187963

  5. Changes of oxidase and hydrolase activities in pecan leaves elicited by black pecan aphid (Hemiptera: Aphididae) feeding.

    PubMed

    Chen, Yigen; Ni, Xinzhi; Cottrell, Ted E; Wood, Bruce W; Buntin, G David

    2009-06-01

    The black pecan aphid, Melanocallis caryaefoliae (Davis) (Hemiptera: Aphididae), is a foliar feeder of pecan, Carya illinoinensis (Wangenh.) K. Koch (Juglandaceae). The pest causes chlorosis of leaflet lamina, physiological damage to foliage and trees, and commonly limits the profitability of commercial pecan orchard enterprises. However, key aspects of this host-pest interaction are poorly understood. We report here the effects of M. caryaefoliae feeding on the foliar activity of oxidative (i.e., catalase, lipoxygenase [LOX]-1 and 3, and peroxidase) and hydrolytic (i.e., esterase) enzymes in relation to the degree of aphid resistance among pecan varieties. The 2-yr study showed that M. caryaefoliae-infested foliage exhibited elevated peroxidase activity only in susceptible ('Desirable', 'Sumner', and 'Schley'), but not in resistant ('Cape Fear', 'Gloria Grande', and 'Money Maker') genotypes. Susceptible genotypes also exhibited more severe leaf chlorosis in response to M. caryaefoliae feeding than the resistant genotypes; however, the aphid feeding did not influence catalase or esterase activity in all varieties, except the increase of esterase activity in Desirable and Gloria Grande. Melanocallis caryaefoliae feeding also influences activity of two lipoxygenase isozymes, with LOX3 being more frequently induced than LOX1. Foliar LOX3 activity was more frequently induced by M. caryaefoliae feeding in the moderately resistant 'Oconee' and highly resistant Money Maker and Cape Fear than in the susceptible genotypes. Therefore, the elevation of peroxidase is likely to be associated with aphid susceptibility and contributed to the severe leaf chlorosis, whereas the increase of LOX3 activity might be associated with aphid resistance in pecan. These findings contribute to our understanding of the etiology of M. caryaefoliae-elicited leaf chlorosis on pecan foliage. Such information may also be used to develop enzyme markers for identifying black pecan aphid resistance

  6. Acylation of the Type 3 Secretion System Translocon Using a Dedicated Acyl Carrier Protein

    PubMed Central

    Agrebi, Rym; Canestrari, Mickaël J.; Mignot, Tâm; Lebrun, Régine; Bouveret, Emmanuelle

    2017-01-01

    Bacterial pathogens often deliver effectors into host cells using type 3 secretion systems (T3SS), the extremity of which forms a translocon that perforates the host plasma membrane. The T3SS encoded by Salmonella pathogenicity island 1 (SPI-1) is genetically associated with an acyl carrier protein, IacP, whose role has remained enigmatic. In this study, using tandem affinity purification, we identify a direct protein-protein interaction between IacP and the translocon protein SipB. We show, by mass spectrometry and radiolabelling, that SipB is acylated, which provides evidence for a modification of the translocon that has not been described before. A unique and conserved cysteine residue of SipB is identified as crucial for this modification. Although acylation of SipB was not essential to virulence, we show that this posttranslational modification promoted SipB insertion into host-cell membranes and pore-forming activity linked to the SPI-1 T3SS. Cooccurrence of acyl carrier and translocon proteins in several γ- and β-proteobacteria suggests that acylation of the translocon is conserved in these other pathogenic bacteria. These results also indicate that acyl carrier proteins, known for their involvement in metabolic pathways, have also evolved as cofactors of new bacterial protein lipidation pathways. PMID:28085879

  7. Ontogeny of mRNA expression and activity of long-chain acyl-CoA synthetase (ACSL) isoforms in Mus musculus heart.

    PubMed

    de Jong, Hendrik; Neal, Andrea C; Coleman, Rosalind A; Lewin, Tal M

    2007-01-01

    Long-chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for virtually every metabolic pathway that catabolizes FA or synthesizes complex lipids. We have hypothesized that each of the five cloned ACSL isoforms partitions FA towards specific downstream pathways. Adult heart expresses all five cloned ACSL isoforms, but their independent functional roles have not been elucidated. Studies implicate ACSL1 in both oxidative and lipid synthetic pathways. To clarify the functional role of ACSL1 and the other ACSL isoforms (3-6), we examined ACS specific activity and Acsl mRNA expression in the developing mouse heart which increases FA oxidative pathways for energy production after birth. Compared to the embryonic heart, ACS specific activity was 14-fold higher on post-natal day 1 (P1). On P1, as compared to the fetus, only Acsl1 mRNA increased, whereas transcripts for the other Acsl isoforms remained the same, suggesting that ACSL1 is the major isoform responsible for activating long-chain FA for myocardial oxidation after birth. In contrast, the mRNA abundance of Acsl3 was highest on E16, and decreased dramatically by P7, suggesting that ACSL3 may play a critical role during the development of the fetal heart. Our data support the hypothesis that each ACSL has a specific role in the channeling of FA towards distinct metabolic fates.

  8. Structural requirements for charged lipid molecules to directly increase or suppress K+ channel activity in smooth muscle cells. Effects of fatty acids, lysophosphatidate, acyl coenzyme A and sphingosine

    PubMed Central

    1994-01-01

    We determined the structural features necessary for fatty acids to exert their action on K+ channels of gastric smooth muscle cells. Examination of the effects of a variety of synthetic and naturally occurring lipid compounds on K+ channel activity in cell-attached and excised membrane patches revealed that negatively charged analogs of medium to long chain fatty acids (but not short chain analogs) as well as certain other negatively charged lipids activate the channels. In contrast, positively charged, medium to long chain analogs suppress activity, and neutral analogs are without effect. The key requirements for effective compounds seem to be a sufficiently hydrophobic domain and the presence of a charged group. Furthermore, those negatively charged compounds unable to "flip" across the bilayer are effective only when applied at the cytosolic surface of the membrane, suggesting that the site of fatty acid action is also located there. Finally, because some of the effective compounds, for example, the fatty acids themselves, lysophosphatidate, acyl Coenzyme A, and sphingosine, are naturally occurring substances and can be liberated by agonist- activated or metabolic enzymes, they may act as second messengers targeting ion channels. PMID:8195783

  9. A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40T and Its Transglycosylase Activity

    PubMed Central

    Wang, Damao; Kim, Do Hyoung; Seo, Nari; Yun, Eun Ju; An, Hyun Joo; Kim, Jae-Han

    2016-01-01

    ABSTRACT In this study, we characterized Gly5M, originating from a marine bacterium, as a novel β-1,3-1,6-endoglucanase in glycoside hydrolase family 5 (GH5) in the Carbohydrate-Active enZyme database. The gly5M gene encodes Gly5M, a newly characterized enzyme from GH5 subfamily 47 (GH5_47) in Saccharophagus degradans 2-40T. The gly5M gene was cloned and overexpressed in Escherichia coli. Through analysis of the enzymatic reaction products by thin-layer chromatography, high-performance liquid chromatography, and matrix-assisted laser desorption ionization–tandem time of flight mass spectrometry, Gly5M was identified as a novel β-1,3-endoglucanase (EC 3.2.1.39) and bacterial β-1,6-glucanase (EC 3.2.1.75) in GH5. The β-1,3-endoglucanase and β-1,6-endoglucanase activities were detected by using laminarin (a β-1,3-glucan with β-1,6-glycosidic linkages derived from brown macroalgae) and pustulan (a β-1,6-glucan derived from fungal cell walls) as the substrates, respectively. This enzyme also showed transglycosylase activity toward β-1,3-oligosaccharides when laminarioligosaccharides were used as the substrates. Since laminarin is the major form of glucan storage in brown macroalgae, Gly5M could be used to produce glucose and laminarioligosaccharides, using brown macroalgae, for industrial purposes. IMPORTANCE In this study, we have discovered a novel β-1,3-1,6-endoglucanase with a unique transglycosylase activity, namely, Gly5M, from a marine bacterium, Saccharophagus degradans 2-40T. Gly5M was identified as the newly found β-1,3-endoglucanase and bacterial β-1,6-glucanase in GH5. Gly5M is capable of cleaving glycosidic linkages of both β-1,3-glucans and β-1,6-glucans. Gly5M also possesses a transglycosylase activity toward β-1,3-oligosacchrides. Due to the broad specificity of Gly5M, this enzyme can be used to produce glucose or high-value β-1,3- and/or β-1,6-oligosaccharides. PMID:27208098

  10. Hydrolase and glycosynthase activity of endo-1,3-β-glucanase from the thermophile Pyrococcus furiosus

    PubMed Central

    Van Lieshout, J.; Faijes, M.; Nieto, J.; Van Der Oost, J.; Planas, A.

    2004-01-01

    Pyrococcus furiosus laminarinase (LamA, PF0076) is an endo-glycosidase that hydrolyzes β-1,3-gluco-oligosaccharides, but not β-1,4-gluco-oligosaccharides. We studied the specificity of LamA towards small saccharides by using 4-methylumbelliferyl β-glucosides with different linkages. Besides endo-activity, wild-type LamA has some exo-activity, and catalyzes the hydrolysis of mixed-linked oligosaccharides (Glcβ4Glcβ3Glcβ-MU (Glc = glucosyl, MU = 4-methylumbelliferyl)) with both β-1,4 and β-1,3 specificities. The LamA mutant E170A had severely reduced hydrolytic activity, which is consistent with Glu170 being the catalytic nucleophile. The E170A mutant was active as a glycosynthase, catalyzing the condensation of α-laminaribiosyl fluoride to different acceptors. The best condensation yields were found at pH 6.5 and 50 °C, but did not exceed 30%. Depending on the acceptor, the synthase generated either a β-1,3 or a β-1,4 linkage. PMID:15810439

  11. The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity

    PubMed Central

    Iordanov, Iordan; Mihályi, Csaba; Tóth, Balázs; Csanády, László

    2016-01-01

    Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca2+-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9. When expressed in E.coli, chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified. In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity (kcat~4-9s-1), that of NUDT9H did not support catalysis. Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating (K1/2~1-5 μM), confirming functionality of chimeric domains. Thus, TRPM2 is not a 'chanzyme'. Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies. DOI: http://dx.doi.org/10.7554/eLife.17600.001 PMID:27383051

  12. Cysteine-286 as the site of acylation of the Lux-specific fatty acyl-CoA reductase.

    PubMed

    Lee, C Y; Meighen, E A

    1997-04-04

    The channelling of fatty acids into the fatty aldehyde substrate for the bacterial bioluminescence reaction is catalyzed by a fatty acid reductase multienzyme complex, which channels fatty acids through the thioesterase (LuxD), synthetase (LuxE) and reductase (LuxC) components. Although all three components can be readily acylated in extracts of different luminescent bacteria, this complex has been successfully purified only from Photobacterium phosphoreum and the sites of acylation identified on LuxD and LuxE. To identify the acylation site on LuxC, the nucleotide sequence of P. phosphoreum luxC has been determined and the gene expressed in a mutant Escherichia coli strain. Even in crude extracts, the acylated reductase intermediate as well as acyl-CoA reductase activity could be readily detected, providing the basis for analysis of mutant reductases. Comparison of the amino-acid sequences of LuxC from P. phosphoreum, P. leiognathi and other luminescent bacteria, showed that only three cysteine residues (C171, C279, and C286) were conserved. As a cysteine residue on LuxC has been implicated in fatty acyl transfer, each of the conserved cysteine residues of the P. phosphoreum and P. leiognathi reductases was converted to a serine residue, and the properties of the mutant proteins examined. Only mutation of C286-blocked reductase activity and prevented formation of the acylated reductase intermediate, showing that C286 is the site of acylation on LuxC.

  13. Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance.

    PubMed

    de Oliveira, Leandro C; da Silva, Viviam M; Colussi, Francieli; Cabral, Aline D; de Oliveira Neto, Mario; Squina, Fabio M; Garcia, Wanius

    2015-01-01

    Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features.

  14. Conformational Changes in a Hyperthermostable Glycoside Hydrolase: Enzymatic Activity Is a Consequence of the Loop Dynamics and Protonation Balance

    PubMed Central

    de Oliveira, Leandro C.; da Silva, Viviam M.; Colussi, Francieli; Cabral, Aline D.; de Oliveira Neto, Mario; Squina, Fabio M.; Garcia, Wanius

    2015-01-01

    Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features. PMID:25723179

  15. The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

    SciTech Connect

    Duong-Ly, Krisna C.; Gabelli, Sandra B.; Xu, WenLian; Dunn, Christopher A.; Schoeffield, Andrew J.; Bessman, Maurice J.; Amzel, L. Mario

    2011-09-06

    A Nudix enzyme from Bacillus cereus catalyzes the hydrolysis of CDP-choline to produce CMP and phosphocholine. Here, we show that in addition, the enzyme has a 3{prime} {yields} 5{prime} RNA exonuclease activity. The structure of the free enzyme, determined to a 1.8-{angstrom} resolution, shows that the enzyme is an asymmetric dimer. Each monomer consists of two domains, an N-terminal helical domain and a C-terminal Nudix domain. The N-terminal domain is placed relative to the C-terminal domain such as to result in an overall asymmetric arrangement with two distinct catalytic sites: one with an 'enclosed' Nudix pyrophosphatase site and the other with a more open, less-defined cavity. Residues that may be important for determining the asymmetry are conserved among a group of uncharacterized Nudix enzymes from Gram-positive bacteria. Our data support a model where CDP-choline hydrolysis is catalyzed by the enclosed Nudix site and RNA exonuclease activity is catalyzed by the open site. CDP-Chase is the first identified member of a novel Nudix family in which structural asymmetry has a profound effect on the recognition of substrates.

  16. Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase

    PubMed Central

    Kawelke, Steffen; Feussner, Ivo

    2015-01-01

    Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2) and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2) was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity. PMID:26714272

  17. Relationships between lipophilicity and biological activities in a series of indoline-based anti-oxidative acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors.

    PubMed

    Takahashi, Kenji; Kunishiro, Kazuyoshi; Kasai, Masayasu; Miike, Tomohiro; Kurahashi, Kazuyoshi; Shirahase, Hiroaki

    2008-01-01

    A novel series of 1-alkyl-7-amido-indoline-based anti-oxidative acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors have been reported and are expected to lower plasma cholesterol levels due to the inhibition of intestinal and hepatic ACAT, and to inhibit cholesterol accumulation in macrophages due to the inhibition of low density lipoprotein (LDL) oxidation. In the present study, relationships between lipophilicity and biological activities were examined in 13 derivatives. Lipophilicity (logP) increased and water solubility decreased with dependence on the number of carbons in the 1-alkyl chain. Inhibitory activity against both in vitro intestinal ACAT and LDL oxidation positively correlated with logP; however, the optimum logP, at which the level of activity is maximal, differed between these two effects. Inhibitory activity against in vitro plasma oxidation was weakly dependent on logP. Plasma concentrations of the derivatives after oral administration at 10 mg/kg correlated negatively with logP and positively with water solubility. Hypocholesterolemic activity in rats fed a high-cholesterol diet, and the ratio of Cmax and IC50 values for ACAT inhibition, an index of effective plasma concentration, positively and highly correlated with logP, while ex vivo inhibitory activity against plasma oxidation in rats, and the ratio of Cmax and IC50 values for the inhibition of plasma oxidation negatively correlated with logP. In conclusion, in vitro ACAT inhibitory and anti-oxidative activity were differently dependent on logP, and intestinal absorption was inversely dependent on lipophilicity in indoline-based anti-oxidative ACAT inhibitors. The hypocholesterolemic effect positively correlated and the ex vivo anti-oxidative effect negatively correlated with lipophilicity. Optimum logP as a bioavailable dual inhibitor against in vivo ACAT and lipid peroxidation was estimated to be 3.8 (1-pentyl and 1-isopentyl derivatives) in the present series of derivatives.

  18. Different effects of fibrates on the microsomal fatty acid chain elongation and the acyl composition of phospholipids in guinea-pigs.

    PubMed

    Vázquez, M; Alegret, M; López, M; Rodríguez, C; Adzet, T; Merlos, M; Laguna, J C

    1995-12-01

    1. The effects in vitro and in vivo of three fibric acid derivatives, clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on some enzyme activities related to fatty acid biosynthesis, namely palmitoyl-CoA synthetase and hydrolases (microsomal and cytosolic), NADH and NADPH cytochrome c reductases and acyl-CoA elongases were investigated in guinea-pigs. 2. The three fibrates inhibited acyl-CoA elongation in vitro, irrespective of the substrate of elongation used (saturated, monounsaturated, polyunsaturated) and with an order of potency GFB > BFB > CFB. In the case of GFB, inhibition occurred at concentrations that can be reached in vivo. 3. Microsomal palmitoyl-CoA hydrolase and synthetase were also inhibited in vitro (GFB > or = BFB > CFB), whereas NADH cytochrome c reductase activity was increased by GFB. Nevertheless, the magnitude of changes were lower than those observed in elongation activities. 4. Treatment with fibrates did not produce peroxisomal proliferation in guinea-pigs, as measured by peroxisomal beta-oxidation activity and liver weight/body weight ratio. Nevertheless, fibrates provoked a reduction in plasma cholesterol and triglycerides, at least in GFB- and BFB-treated animals. 5. Fatty acid elongation was significantly modified by GFB treatment in vivo. The remaining enzyme activities studied were only slightly changed by fibrate treatment. 6. Treatment with BFB and to a lesser extent with CFB, increased the relative proportion of MUFA (palmitoleic and oleic acids) in microsomal phospholipids, whereas PUFA (mainly linoleic acid) decreased. GFB behaved differently, increasing palmitic and linoleic acids and decreasing stearic and oleic acids. The latter changes are attributable to an inhibition of elongation activity by GFB. 7. The changes observed after fibrate treatment in both rats and guinea-pigs, as they are not directly related to peroxisome proliferation, could be more reliably extrapolated to man than those observed only in rats.

  19. Different effects of fibrates on the microsomal fatty acid chain elongation and the acyl composition of phospholipids in guinea-pigs.

    PubMed Central

    Vázquez, M.; Alegret, M.; López, M.; Rodríguez, C.; Adzet, T.; Merlos, M.; Laguna, J. C.

    1995-01-01

    1. The effects in vitro and in vivo of three fibric acid derivatives, clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on some enzyme activities related to fatty acid biosynthesis, namely palmitoyl-CoA synthetase and hydrolases (microsomal and cytosolic), NADH and NADPH cytochrome c reductases and acyl-CoA elongases were investigated in guinea-pigs. 2. The three fibrates inhibited acyl-CoA elongation in vitro, irrespective of the substrate of elongation used (saturated, monounsaturated, polyunsaturated) and with an order of potency GFB > BFB > CFB. In the case of GFB, inhibition occurred at concentrations that can be reached in vivo. 3. Microsomal palmitoyl-CoA hydrolase and synthetase were also inhibited in vitro (GFB > or = BFB > CFB), whereas NADH cytochrome c reductase activity was increased by GFB. Nevertheless, the magnitude of changes were lower than those observed in elongation activities. 4. Treatment with fibrates did not produce peroxisomal proliferation in guinea-pigs, as measured by peroxisomal beta-oxidation activity and liver weight/body weight ratio. Nevertheless, fibrates provoked a reduction in plasma cholesterol and triglycerides, at least in GFB- and BFB-treated animals. 5. Fatty acid elongation was significantly modified by GFB treatment in vivo. The remaining enzyme activities studied were only slightly changed by fibrate treatment. 6. Treatment with BFB and to a lesser extent with CFB, increased the relative proportion of MUFA (palmitoleic and oleic acids) in microsomal phospholipids, whereas PUFA (mainly linoleic acid) decreased. GFB behaved differently, increasing palmitic and linoleic acids and decreasing stearic and oleic acids. The latter changes are attributable to an inhibition of elongation activity by GFB. 7. The changes observed after fibrate treatment in both rats and guinea-pigs, as they are not directly related to peroxisome proliferation, could be more reliably extrapolated to man than those observed only in rats. PMID

  20. Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity.

    PubMed Central

    Thompson, G A; Scherer, D E; Foxall-Van Aken, S; Kenny, J W; Young, H L; Shintani, D K; Kridl, J C; Knauf, V C

    1991-01-01

    Stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) catalyzes the principal conversion of saturated fatty acids to unsaturated fatty acids in the synthesis of vegetable oils. Stearoyl-ACP desaturase was purified from developing embryos of safflower seed, and extensive amino acid sequence was determined. The amino acid sequence was used in conjunction with polymerase chain reactions to clone a full-length cDNA. The primary structure of the protein, as deduced from the nucleotide sequence of the cDNA, includes a 33-amino-acid transit peptide not found in the purified enzyme. Expression in Escherichia coli of a gene encoding the mature form of stearoyl-ACP desaturase did not result in an altered fatty acid composition. However, active enzyme was detected when assayed in vitro with added spinach ferredoxin. The lack of significant activity in vitro without added ferredoxin and the lack of observed change in fatty acid composition indicate that ferredoxin is a required cofactor for the enzyme and that E. coli ferredoxin functions poorly, if at all, as an electron donor for the plant enzyme. Images PMID:2006194

  1. Long chain acyl-CoA synthetase-3 is a molecular target for peroxisome proliferator-activated receptor delta in HepG2 hepatoma cells.

    PubMed

    Cao, Aiqin; Li, Hai; Zhou, Yue; Wu, Minhao; Liu, Jingwen

    2010-05-28

    ACSL3 is a member of the long chain acyl-CoA synthetase (ACSL) family that plays key roles in fatty acid metabolism in various tissues in an isozyme-specific manner. Our previous studies showed that ACSL3 was transcriptionally up-regulated by the cytokine oncostatin M (OSM) in HepG2 cells, accompanied by reduced cellular triglyceride content and enhanced beta-oxidation. In this study, we investigated the molecular mechanism underlying the OSM-induced activation of ACSL3 gene transcription in HepG2 cells. We showed that OSM treatment resulted in a coordinated elevation of mRNA levels of ACSL3 and peroxisome proliferator-activated receptor delta (PPARdelta). The effect of OSM on ACSL3 mRNA expression was inhibited by cellular depletion of PPARdelta. By utilizing a PPARdelta agonist, L165041, we demonstrated that activation of PPARdelta led to increases in ACSL3 promoter activity, mRNA level, and protein level in HepG2 cells. Analysis of the ACSL3 promoter sequence identified two imperfect PPAR-responsive elements (PPRE) located in the ACSL3 promoter region -944 to -915, relative to the transcription start site. The up-regulation of ACSL3 promoter activity by PPARdelta was abolished by deletion of this PPRE-containing region or mutation to disrupt the binding sites. Direct interactions of PPARdelta with ACSL3-PPRE sequences were demonstrated by gel mobility shift and chromatin immunoprecipitation assays. Finally, we provided in vivo evidence showing that activation of PPARdelta by L165041 in hamsters increased ACSL3 mRNA and protein levels in the liver. These new findings define ACSL3 as a novel molecular target of PPARdelta in HepG2 cells and provide a regulatory mechanism for ACSL3 transcription in liver tissue.

  2. A Cerulenin Insensitive Short Chain 3-Ketoacyl-Acyl Carrier Protein Synthase in Spinacia oleracea Leaves

    PubMed Central

    Jaworski, Jan G.; Clough, Richard C.; Barnum, Susan R.

    1989-01-01

    A cerulenin insensitive 3-ketoacyl-acyl carrier protein synthase has been assayed in extracts of spinach (Spinacia oleracea) leaf. The enzyme was active in the 40 to 80% ammonium sulfate precipitate of whole leaf homogenates and catalyzed the synthesis of acetoacetyl-acyl carrier protein. This condensation reaction was five-fold faster than acetyl-CoA:acyl carrier protein transacylase, and the initial rates of acyl-acyl carrier protein synthesis were independent of the presence of cerulenin. In the presence of fatty acid synthase cofactors and 100 micromolar cerulenin, the principal fatty acid product of de novo synthesis was butyric and hexanoic acids. Using conformationally sensitive native polyacrylamide gel electrophoresis for separation, malonyl-, acetyl-, butyryl-, hexanoyl, and long chain acyl-acyl carrier proteins could be detected by immunoblotting and autoradiography. In the presence of 100 micromolar cerulenin, the accumulation of butyryl- and hexanoyl-acyl carrier protein was observed, with no detectable long chain acyl-acyl carrier proteins or fatty acids being produced. In the absence of cerulenin, the long chain acyl-acyl carrier proteins also accumulated. Images Figure 2 Figure 3 PMID:16666765

  3. Structures of the Michaelis Complex (1.2A) and the Covalent Acyl Intermediate (2.0A ) of Cefamandole Bound in the Active Sites of the Mycobacterium tuberculosis beta-Lactamase K72A and E166A Mutants

    SciTech Connect

    L Tremblay; h Xu; J Blanchard

    2011-12-31

    The genome of Mycobacterium tuberculosis (TB) contains a gene that encodes a highly active {beta}-lactamase, BlaC, that imparts TB with resistance to {beta}-lactam chemotherapy. The structure of covalent BlaC-{beta}-lactam complexes suggests that active site residues K73 and E166 are essential for acylation and deacylation, respectively. We have prepared the K73A and E166A mutant forms of BlaC and have determined the structures of the Michaelis complex of cefamandole and the covalently bound acyl intermediate of cefamandole at resolutions of 1.2 and 2.0 {angstrom}, respectively. These structures provide insight into the details of the catalytic mechanism.

  4. Rat long-chain acyl-CoA synthetase mRNA, protein, and activity vary in tissue distribution and in response to diet.

    PubMed

    Mashek, Douglas G; Li, Lei O; Coleman, Rosalind A

    2006-09-01

    Distinct isoforms of long-chain acyl-CoA synthetases (ACSLs) may partition fatty acids toward specific metabolic cellular pathways. For each of the five members of the rat ACSL family, we analyzed tissue mRNA distributions, and we correlated the mRNA, protein, and activity of ACSL1 and ACSL4 after fasting and refeeding a 69% sucrose diet. Not only did quantitative real-time PCR analyses reveal unique tissue expression patterns for each ACSL isoform, but expression varied markedly in different adipose depots. Fasting increased ACSL4 mRNA abundance in liver, muscle, and gonadal and inguinal adipose tissues, and refeeding decreased ACSL4 mRNA. A similar pattern was observed for ACSL1, but both fasting and refeeding decreased ACSL1 mRNA in gonadal adipose. Fasting also decreased ACSL3 and ACSL5 mRNAs in liver and ACSL6 mRNA in muscle. Surprisingly, in nearly every tissue measured, the effects of fasting and refeeding on the mRNA abundance of ACSL1 and ACSL4 were discordant with changes in protein abundance. These data suggest that the individual ACSL isoforms are distinctly regulated across tissues and show that mRNA expression may not provide useful information about isoform function. They further suggest that translational or posttranslational modifications are likely to contribute to the regulation of ACSL isoforms.

  5. Short-term exposures of fish to perfluorooctane sulfonate: acute effects on fatty acyl-coa oxidase activity, oxidative stress, and circulating sex steroids.

    PubMed

    Oakes, Ken D; Sibley, Paul K; Martin, Jon W; MacLean, Dan D; Solomon, Keith R; Mabury, Scott A; Van Der Kraak, Glen J

    2005-05-01

    This study investigated the effects of exposure to waterborne perfluorooctane sulfonate (PFOS) on oxidative stress and reproductive endpoints in fish. Exposures utilized species commonly used in toxicological testing, including the fathead minnow (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss), as well as relatively insensitive taxa such as creek chub (Semotilus atromaculatus), spottail shiner (Notropis hudsonius), and white sucker (Catostomus commersoni). In all fish species, short-term (14-28 d) exposure to PFOS produced only modest mortality at concentrations consistent with environmental spill scenarios. However, PFOS consistently increased hepatic fatty acyl-CoA oxidase activity and increased oxidative damage, as quantified using the 2-thiobarbituric acid-reactive substances assay. Plasma testosterone, 11-ketotestosterone, and 17beta-estradiol titers were often elevated with PFOS exposure. Vitellogenin, the egg yolk precursor protein, was occasionally altered in the plasma with PFOS exposure, but responses varied with maturity. Oviposition frequency and egg deposition in fathead minnow were not significantly impaired with PFOS exposure, despite a trend toward progressive impairment with increasing exposure concentrations. Although short-term PFOS exposure produced significant impacts on biochemical and reproductive endpoints in fish at concentrations consistent with environmental spills, the impact of long-term exposure to environmentally relevant concentrations of PFOS is unclear.

  6. Acyl-CoA binding proteins: multiplicity and function.

    PubMed

    Gossett, R E; Frolov, A A; Roths, J B; Behnke, W D; Kier, A B; Schroeder, F

    1996-09-01

    The physiological role of long-chain fatty acyl-CoA is thought to be primarily in intermediary metabolism of fatty acids. However, recent data show that nM to microM levels of these lipophilic molecules are potent regulators of cell functions in vitro. Although long-chain fatty acyl-CoA are present at several hundred microM concentration in the cell, very little long-chain fatty acyl-CoA actually exists as free or unbound molecules, but rather is bound with high affinity to membrane lipids and/or proteins. Recently, there is growing awareness that cytosol contains nonenzymatic proteins also capable of binding long-chain fatty acyl-CoA with high affinity. Although the identity of the cytosolic long-chain fatty acyl-CoA binding protein(s) has been the subject of some controversy, there is growing evidence that several diverse nonenzymatic cytosolic proteins will bind long-chain fatty acyl-CoA. Not only does acyl-CoA binding protein specifically bind medium and long-chain fatty acyl-CoA (LCFA-CoA), but ubiquitous proteins with multiple ligand specificities such as the fatty acid binding proteins and sterol carrier protein-2 also bind LCFA-CoA with high affinity. The potential of these acyl-CoA binding proteins to influence the level of free LCFA-CoA and thereby the amount of LCFA-CoA bound to regulatory sites in proteins and enzymes is only now being examined in detail. The purpose of this article is to explore the identity, nature, function, and pathobiology of these fascinating newly discovered long-chain fatty acyl-CoA binding proteins. The relative contributions of these three different protein families to LCFA-CoA utilization and/or regulation of cellular activities are the focus of new directions in this field.

  7. Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 HydH5 Virion-Associated Peptidoglycan Hydrolase: Fusions, Deletions, and Synergy with LysH5

    PubMed Central

    Rodríguez-Rubio, Lorena; Martínez, Beatriz; Rodríguez, Ana; Donovan, David M.

    2012-01-01

    Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live S. aureus cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed S. aureus cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human S. aureus strains, the methicillin-resistant S. aureus (MRSA) strain N315, and human Staphylococcus epidermidis strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections. PMID:22267667

  8. Polyglycine hydrolases secreted by pathogenic fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pathogens are known to produce proteases that target host defense proteins. Here we describe polyglycine hydrolases, fungal proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine interdomain linker of targeted plant defense chitinases. Polyglycine hydrolases were puri...

  9. Hypolipidemic and antioxidant activity of the novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor KY-455 in rabbits and hamsters.

    PubMed

    Nakamura, Shohei; Kamiya, Shoji; Shirahase, Hiroaki; Kanda, Mamoru; Yoshimi, Akihisa; Tarumi, Tadatsugu; Kurahashi, Kazuyoshi

    2004-01-01

    The hypolipidemic and antioxidant effects of N-(4,6-dimethyl-1-pentylindolin-7-yl)-2,2-dimethylpropanamide (CAS 178469-71-1, KY-455), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, were examined in hyperlipidemic rabbits and normolipidemic hamsters. KY-455 inhibited rabbit intestinal, hepatic, macrophage and adrenal ACAT with IC50 values of 0.4, 0.9, 2.9 and 4.1 micromol/l, respectively. KY-455 also inhibited rabbit plasma and LDL-peroxidation (IC50: 0.4 and 1.7 micromol/l, respectively). In rabbits fed a high-cholesterol diet, treatment with KY-455 (30 mg/kg/day) for 8 days markedly lowered serum esterified, free, low-density lipoprotein (LDL)-cholesterol, and hepatic esterified cholesterol levels. KY-455 tended to inhibit ex vivo hepatic ACAT activity 5 h after the final administration. KY-455 also inhibited ex vivo peroxidation of plasma lipids 1 and 5 h after the final administration in rabbits. In normolipidemic hamsters fed a regular diet, treatment with KY-455 (30 mg/kg, twice a day) for 4 days significantly reduced serum esterified, free and LDL-cholesterol, and hepatic esterified and free cholesterol levels. A single administration of KY-455 (30 mg/kg) significantly inhibited ex vivo hepatic ACAT activity in hamsters. In conclusion, KY-455 showed in vitro inhibitory effects on LDL-peroxidation and macrophage ACAT activity at similar concentrations, and in vivo hypolipidemic and ex vivo antioxidative effects at the same dose. Long-term administration of KY-455 is expected to prevent the progress of atherosclerosis by lowering plasma lipid levels, inhibiting both LDL-oxidation and accumulation of cholesterol in macrophages.

  10. Binding and Inactivation Mechanism of a Humanized Fatty Acid Amide Hydrolase by [alpha]-Ketoheterocycle Inhibitors Revealed from Cocrystal Structures

    SciTech Connect

    Mileni, Mauro; Garfunkle, Joie; DeMartino, Jessica K.; Cravatt, Benjamin F.; Boger, Dale L.; Stevens, Raymond C.

    2010-08-17

    The cocrystal X-ray structures of two isomeric {alpha}-ketooxazole inhibitors (1 (OL-135) and 2) bound to fatty acid amide hydrolase (FAAH), a key enzymatic regulator of endocannabinoid signaling, are disclosed. The active site catalytic Ser241 is covalently bound to the inhibitors electrophilic carbonyl groups, providing the first structures of FAAH bound to an inhibitor as a deprotonated hemiketal mimicking the enzymatic tetrahedral intermediate. The work also offers a detailed view of the oxyanion hole and an exceptional 'in-action' depiction of the unusual Ser-Ser-Lys catalytic triad. These structures capture the first picture of inhibitors that span the active site into the cytosolic port providing new insights that help to explain FAAH's interaction with substrate leaving groups and their role in modulating inhibitor potency and selectivity. The role for the activating central heterocycle is clearly defined and distinguished from that observed in prior applications with serine proteases, reconciling the large electronic effect of attached substituents found unique to this class of inhibitors with FAAH. Additional striking active site flexibility is seen upon binding of the inhibitors, providing insights into the existence of a now well-defined membrane access channel with the disappearance of a spatially independent portion of the acyl chain-binding pocket. Finally, comparison of the structures of OL-135 (1) and its isomer 2 indicates that they bind identically to FAAH, albeit with reversed orientations of the central activating heterocycle, revealing that the terminal 2-pyridyl substituent and the acyl chain phenyl group provide key anchoring interactions and confirming the distinguishing role of the activating oxazole.

  11. The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic acid, a side-chain precursor for cephem antibiotics.

    PubMed

    Koetsier, Martijn J; Gombert, Andreas K; Fekken, Susan; Bovenberg, Roel A L; van den Berg, Marco A; Kiel, Jan A K W; Jekel, Peter A; Janssen, Dick B; Pronk, Jack T; van der Klei, Ida J; Daran, Jean-Marc

    2010-01-01

    Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the beta-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-aminopenicillanic acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins.

  12. Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids.

    PubMed Central

    Wadum, Majken C T; Villadsen, Jens K; Feddersen, Søren; Møller, Rikke S; Neergaard, Thomas B F; Kragelund, Birthe B; Højrup, Peter; Faergeman, Nils J; Knudsen, Jens

    2002-01-01

    Long-chain acyl-CoA esters are key metabolites in lipid synthesis and beta-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression. Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl-CoA-binding protein were replaced by cysteine residues, which were covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make the two fluorescent acyl-CoA indicators (FACIs) FACI-24 and FACI-53. FACI-24 and FACI-53 showed fluorescence emission maximum at 510 and 525 nm respectively, in the absence of ligand (excitation 387 nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495 nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460 nm upon binding of C14-C20 saturated and unsaturated acyl-CoA esters. Both indicators bind long-chain (>C14) acyl-CoA esters with high specificity and affinity (K(d)=0.6-1.7 nM). FACI-53 showed a high fluorescence yield for C8-C12 acyl chains. It is shown that FACI-24 acts as a sensitive acyl-CoA sensor for measuring the concentration of free acyl-CoA, acyl-CoA synthetase activity and the concentrations of free fatty acids after conversion of the fatty acid into their respective acyl-CoA esters. PMID:12071849

  13. The Level of Circulating Octanoate Does Not Predict Ghrelin O-Acyl Transferase (GOAT)-Mediated Acylation of Ghrelin During Fasting

    PubMed Central

    Nikolayev, Alexander; Liu, Jianhua; Pezzoli, Suzan S.; Farhy, Leon S.; Patrie, James; Gaylinn, Bruce D.; Heiman, Mark; Thorner, Michael O.

    2015-01-01

    Background: Acyl-ghrelin is a 28-amino acid peptide released from the stomach. Ghrelin O-acyl transferase (GOAT) attaches an 8-carbon medium-chain fatty acid (MCFA) (octanoate) to serine 3 of ghrelin. This acylation is necessary for the activity of ghrelin. Animal data suggest that MCFAs provide substrate for GOAT and an increase in nutritional octanoate increases acyl-ghrelin. Objectives: To address the question of the source of substrate for acylation, we studied whether the decline in ghrelin acylation during fasting is associated with a decline in circulating MCFAs. Methods: Eight healthy young men (aged 18–28 years, body mass index range, 20.6–26.2 kg/m2) had blood drawn every 10 minutes for acyl- and desacyl-ghrelin and every hour for free fatty acids (FFAs) during the last 24 hours of a 61.5-hour fast and during a fed day. FFAs were measured by a highly sensitive liquid chromatography-mass spectroscopy method. Acyl- and desacyl-ghrelin were measured in an in-house assay; the results were published previously. Ghrelin acylation was assessed by the ratio of acyl-ghrelin to total ghrelin. Results: With the exception of MCFAs C8 and C10, all other FFAs, the MCFAs (C6 and C12), and the long-chain fatty acids (C14–C18) significantly increased with fasting (P < .05). There was no significant association between the fold change in ghrelin acylation and circulating FFAs. Conclusions: These results suggest that changes in circulating MCFAs are not linked to the decline in ghrelin acylation during fasting and support the hypothesis that acylation of ghrelin depends at least partially on the availability of gastroluminal MCFAs or the regulation of GOAT activity. PMID:25337923

  14. Probing the active sites of butyrylcholinesterase and cholesterol esterase with isomalathion: conserved stereoselective inactivation of serine hydrolases structurally related to acetylcholinesterase.

    PubMed

    Doorn, J A; Talley, T T; Thompson, C M; Richardson, R J

    2001-07-01

    Previous work has shown that acetylcholinesterase (AChE), a member of the alpha/beta-hydrolase superfamily, is stereoselectively inhibited by the four stereoisomers of isomalathion. Recent kinetic and mass spectral data demonstrated that a difference in mechanism of inactivation exists for AChE treated with (1R)- versus (1S,3S)-stereoisomers. This study sought to determine whether other alpha/beta-hydrolases are stereoselectively inhibited by isomalathion and if the difference in mechanism of AChE inactivation between (1R)- and (1S,3S)-isomers is conserved for other alpha/beta-hydrolases. Bimolecular rate constants of inhibition (k(i)) were measured for human and equine butyrylcholinesterase (HBChE and EBChE, respectively) and bovine cholesterol esterase (BCholE) with all four isomers. Isomalathion isomers inhibited these enzymes with the following order of potency: (1R,3R) > (1R,3S) > (1S,3R) > or = (1S,3S). Ratios of k(i) values for the most potent to the least potent isomer were 10.5 (HBChE), 11.9 (EBChE), and 68.6 (BCholE). Rate constants of reactivation (k(3)) were measured for enzyme inhibited by isomalathion isomers. HBChE, EBChE, and BCholE inactivated by the (1R)-isomers readily reactivated. However, enzymes modified by (1S)-isomalathions were refractory toward reactivation, and k(3) values were not significantly different from zero for HBChE and BCholE treated with the (1S,3S)-isomer. Computer-based docking experiments were performed for BCholE with (1R,3R)- and (1S,3S)-enantiomers. Calculated structures predicted a difference in primary leaving group: diethyl thiosuccinate for (1R,3R)-isomalathion and thiomethyl for the (1S,3S)-isomer. The data demonstrate that the alpha/beta-hydrolases used in this study are stereoselectively inhibited by isomalathion. Furthermore, the results suggest that the mechanistic shift demonstrated to occur for inhibition of AChE by (1R)- versus (1S,3S)-isomers is conserved for butyrylcholinesterase and cholesterol esterase.

  15. Synthetic Modification of 9α- and 9β-Hydroxyparthenolide by Heck or Acylation Reactions and Evaluation of Cytotoxic Activities.

    PubMed

    El Bouakher, Abderrahman; Jismy, Badr; Allouchi, Hassan; Duverger, Eric; Barkaoui, Latifa; El Hakmaoui, Ahmed; Daniellou, Richard; Guillaumet, Gérald; Akssira, Mohamed

    2016-12-05

    Motivated by the widely reported anticancer activity of parthenolides and their derivatives, a series of new substituted parthenolides was efficiently synthesized. Structural modifications were performed at the C-9 and C-13 positions of 9α- and 9β-hydroxyparthenolide, which were isolated from the aerial parts of Anvillea radiata. Twenty-one derivatives were synthesized and evaluated for their in vitro cytotoxic activity against HS-683, SK-MEL-28, A549, and MCF-7 human cancer cell lines using the MTT colorimetric assay. Among the derivatives, seven exhibited excellent activity compared to 5-fluorouracil and etoposide against the four cell lines tested, with IC50 values ranging from 1.1 to 9.4 µM.

  16. The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its membrane topology.

    PubMed Central

    Friedberg, T; Holler, R; Löllmann, B; Arand, M; Oesch, F

    1996-01-01

    Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786-5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been disputed. Here we demonstrate that, in contrast with the theoretical prediction, the rat mEH has exclusively a type I membrane topology. Moreover we show that this topology can be inverted without affecting the catalytic activity of mEH. Our conclusions are supported by the observation that two mEH constructs (mEHg1 and mEHg2), containing engineered potential glycosylation sites at two separate locations after the C-terminal site of the membrane anchor, were not glycosylated in fibroblasts. However, changing the net charge at the N-terminus of these engineered mEH proteins by +3 resulted in proteins (++mEHg1 and ++mEHg2) that became glycosylated and consequently had a type II topology. The sensitivity of these glycosylated proteins to endoglycosidase H indicated that, like the native mEH, they are still retained in the ER. The engineered mEH proteins were integrated into membranes as they were resistant to alkaline extraction. Interestingly, an insect mEH with a charge distribution in its N-terminus similar to ++mEHg1 has recently been isolated. This enzyme might well display a type II topology instead of the type I topology of the rat mEH. Importantly, mEHg1, having the natural cytosolic orientation, as well as ++mEHg1, having an artificial huminal orientation, displayed rather

  17. Labrenzia sp. BM1: A Quorum Quenching Bacterium That Degrades N-acyl Homoserine Lactones via Lactonase Activity

    PubMed Central

    Ghani, Norshazliza Ab; Norizan, Siti Nur Maisarah; Chan, Xin Yue; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    We report the degradation of quorum sensing N-acylhomoserine lactone molecules by a bacterium isolated from a Malaysian marine water sample. MALDI-TOF and phylogenetic analysis indicated this isolate BM1 clustered closely to Labrenzia sp. The quorum quenching activity of this isolate was confirmed by using a series of bioassays and rapid resolution liquid chromatography analysis. Labrenzia sp. degraded a wide range of N-acylhomoserine lactones namely N-(3-hexanoyl)-l-homoserine lactone (C6-HSL), N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL) and N-(3-hydroxyhexanoyl)-l-homoserine lactone (3-hydroxy-C6-HSL). Re-lactonisation bioassays confirmed Labrenzia sp. BM1 degraded these signalling molecules efficiently via lactonase activity. To the best of our knowledge, this is the first documentation of a Labrenzia sp. capable of degrading N-acylhomoserine lactones and confirmation of its lactonase-based mechanism of action. PMID:24995373

  18. Fish protein hydrolysate reduces plasma total cholesterol, increases the proportion of HDL cholesterol, and lowers acyl-CoA:cholesterol acyltransferase activity in liver of Zucker rats.

    PubMed

    Wergedahl, Hege; Liaset, Bjørn; Gudbrandsen, Oddrun Anita; Lied, Einar; Espe, Marit; Muna, Ziad; Mørk, Sverre; Berge, Rolf K

    2004-06-01

    There is growing evidence that soy protein improves the blood lipid profiles of animals and humans. We compared the effects of fish protein hydrolysate (FPH), soy protein, and casein (control) on lipid metabolism in Wistar rats and genetically obese Zucker (fa/fa) rats. In Zucker rats, FPH treatment affected the fatty acid composition in liver, plasma, and triacylglycerol-rich lipoproteins. The mRNA levels of Delta 5 and Delta 6 desaturases were reduced by FPH and soy protein feeding compared with casein feeding. In Zucker rats both FPH and soy protein treatment reduced the plasma cholesterol level. Furthermore, the HDL cholesterol:total cholesterol ratio was greater in these rats and in the Wistar rats fed FPH and soy protein compared with those fed casein. Although fecal total bile acids were greater in soy protein-fed Zucker rats than in casein-fed controls, those fed FPH did not differ from the controls. However, the acyl-CoA:cholesterol acyltransferase activity was reduced in Zucker rats fed FPH and tended to be lower (P = 0.13) in those fed soy protein compared with those fed casein. Low ratios of methionine to glycine and lysine to arginine in the FPH and soy protein diets, compared with the casein diet, may be involved in lowering the plasma cholesterol concentration. Our results indicate that the effects of FPH and soy protein on fatty acid metabolism are similar in many respects, but the hypocholesterolemic effects of FPH and soy protein appear to be due to different mechanisms. FPH may have a role as a cardioprotective nutrient.

  19. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein.

    PubMed Central

    Mulbry, W W; Karns, J S

    1989-01-01

    The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid. Images PMID:2556372

  20. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein.

    PubMed

    Mulbry, W W; Karns, J S

    1989-12-01

    The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid.

  1. Isolation and characterization of Chinese hamster ovary (CHO) cells deficient in acyl coenzyme A: cholesterol acyltransferase (ACAT) activity

    SciTech Connect

    Cadigan, K.M.; Heider, J.G.; Chang, T.Y.

    1986-05-01

    The specific ACAT inhibitor compound 58-035 has been used to mimic the phenotype of an ACAT deficient mutant in 25-RA cells. 25-RA is a CHO cell line resistant to 25-hydroxycholesterol and contains five times more cholesterol ester than wild-type (WT) cells. 25-RA cells preincubated with 58-035 are 100 to 500 times more resistant to amphotericin B killing than untreated 25-RA. 100 x 10/sup 6/ mutagenized 25-RA cells underwent three rounds of amphotericin B killing and two rounds of 25-hydroxycholesterol killing (to remove WT revertants which are amphotericin B resistant). Thus far, three biochemically distinct mutants have been isolated containing 33% (AC27), 25% (AC90), and 10% (AC232) of the parental ACAT activity as measured by an /sup 3/H-oleate pulse in intact cells. When parental and mutant cell extracts are reconstituted into cholesterol containing liposomes the differences in ACAT activity remain. They have also found that 25-RA cells can survive in cholesterol free medium containing TMD, an inhibitor of cholesterol biosynthesis, presumably because of adequate supply of endogenous cholesterol from hydrolysis of its stored cholesterol ester. In contrast, under the same conditions, mutant AC232 is effectively killed ( greater than or equal to 99%) by cholesterol starvation, thus providing a potential selection procedure for isolating revertants of ACAT mutants.

  2. The macamide N-3-methoxybenzyl-linoleamide is a time-dependent fatty acid amide hydrolase (FAAH) inhibitor.

    PubMed

    Almukadi, Haifa; Wu, Hui; Böhlke, Mark; Kelley, Charles J; Maher, Timothy J; Pino-Figueroa, Alejandro

    2013-10-01

    The Peruvian plant Lepidium meyenii (Maca) has been shown to possess neuroprotective activity both in vitro and in vivo. Previous studies have also demonstrated the activity of the pentane extract and its macamides, the most representative lipophilic constituents of Maca, in the endocannabinoid system as fatty acid amide hydrolase (FAAH) inhibitors. One of the most active macamides, N-3-methoxybenzyl-linoleamide, was studied to determine its mechanism of interaction with FAAH and whether it has inhibitory activity on mono-acyl glycerol lipase (MAGL), the second enzyme responsible for endocannabinoid degradation. Macamide concentrations from 1 to 100 μM were tested using FAAH and MAGL inhibitor assay methods and showed no effect on MAGL. Tests with other conditions were performed in order to characterize the inhibitory mechanism of FAAH inhibition. N-3-methoxybenzyl-linoleamide displayed significant time-dependent and dose-dependent FAAH inhibitory activity. The mechanism of inhibition was most likely irreversible or slowly reversible. These results suggest the potential application of macamides isolated from Maca as FAAH inhibitors, as they might act on the central nervous system to provide analgesic, anti-inflammatory, or neuroprotective effects, by modulating the release of neurotransmitters.

  3. Sequence of the bphD gene encoding 2-hydroxy-6-oxo-(phenyl/chlorophenyl)hexa-2,4-dienoic acid (HOP/cPDA) hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway in Comamonas testosteroni: evidence suggesting involvement of Ser112 in catalytic activity.

    PubMed

    Ahmad, D; Fraser, J; Sylvestre, M; Larose, A; Khan, A; Bergeron, J; Juteau, J M; Sondossi, M

    1995-04-14

    The nucleotide sequence of bphD, encoding 2-hydroxy-6-oxo-(phenyl/chlorophenyl)hexa-2,4-dienoic acid hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway of Comamonas testosteroni strain B-356, was determined. Comparison of the deduced amino-acid sequence with published sequences led to the identification of a 'lipase box', containing a consensus pentapeptide sequence GlyXaaSerXaaGly. This suggested that the mechanism of action of this enzyme may involve an Asp-Ser-His catalytic triad similar to that of classical lipases and serine hydrolases. Further biochemical and genetic evidence for the active-site involvement of Ser112 was obtained by showing that a semipurified enzyme was inhibited by PMSF, a classic inhibitor of serine hydrolases, and by site-directed Ser112-->Ala mutagenesis.

  4. Kinetic and Structural Insight into the Mechanism of BphD, a C-C Bond Hydrolase from the Biphenyl Degradation Pathway†

    PubMed Central

    Horsman, Geoff P.; Ke, Jiyuan; Dai, Shaodong; Seah, Stephen Y. K.; Bolin, Jeffrey T.; Eltis, Lindsay D.

    2008-01-01

    Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphDLB400) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 °C showed that the enzyme rapidly (1/τ1 ∼ 500 s−1) transforms HOPDA (λmax = 434 nm) to a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/τ2 = 54 s−1, 1/τ3 = 6 s−1 ∼ kcat) with simultaneous biphasic appearance (48 and 8 s−1) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/τ1 and 1/τ2, but affected neither 1/τ3 nor kcat, suggesting that 1/τ2 may reflect diffusive HPD dissociation, while 1/τ3 represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and therefore postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (λmax = 492 nm). Crystal structures of wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 Å resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogen-bonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate. PMID:16964968

  5. Photo-inducible cytotoxic and clastogenic activities of 3,6-di-substituted acridines obtained by acylation of proflavine.

    PubMed

    Benchabane, Yohann; Di Giorgio, Carole; Boyer, Gérard; Sabatier, Anne-Sophie; Allegro, Diane; Peyrot, Vincent; De Méo, Michel

    2009-06-01

    The cytotoxicity and photo-enhanced cytotoxicity of a series of 18 3,6-di-substituted acridines were evaluated on both tumour CHO cells and human normal keratinocytes, and compared to their corresponding clastogenicity as assessed by the micronucleus assay. Compounds 2f tert-butyl N-[(6-tert-butoxycarbonylamino)acridin-3-yl]carbamate and 2d N-[6-(pivalamino)acridin-3-yl]pivalamide displayed a specific cytotoxicity on CHO cells. These results suggested that the two derivatives could be considered as interesting candidates for anticancer chemotherapy and hypothesized that the presence of 1,1-dimethylethyl substituents was responsible for a strong nonclastogenic cytotoxicity. Compounds 2b and 2c, on the contrary, displayed a strong clastogenicity. They indicated that the presence of nonbranched aliphatic chains on positions 3 and 6 of the acridine rings tended to induce a significant clastogenic effect. Finally, they established that most of the acridine compounds could be photo-activated by UVA-visible rays and focussed on the significant role of light irradiation on their biological properties.

  6. Acylation of Ferrocene: A Greener Approach

    ERIC Educational Resources Information Center

    Birdwhistell, Kurt R.; Nguyen, Andy; Ramos, Eric J.; Kobelja, Robert

    2008-01-01

    The acylation of ferrocene is a common reaction used in organic laboratories to demonstrate Friedel-Crafts acylation and the purification of compounds using column chromatography. This article describes an acylation of ferrocene experiment that is more eco-friendly than the conventional acylation experiment. The traditional experiment was modified…

  7. Curation of characterized glycoside hydrolases of Fungal origin

    PubMed Central

    Murphy, Caitlin; Powlowski, Justin; Wu, Min; Butler, Greg; Tsang, Adrian

    2011-01-01

    Fungi produce a wide range of extracellular enzymes to break down plant cell walls, which are composed mainly of cellulose, lignin and hemicellulose. Among them are the glycoside hydrolases (GH), the largest and most diverse family of enzymes active on these substrates. To facilitate research and development of enzymes for the conversion of cell-wall polysaccharides into fermentable sugars, we have manually curated a comprehensive set of characterized fungal glycoside hydrolases. Characterized glycoside hydrolases were retrieved from protein and enzyme databases, as well as literature repositories. A total of 453 characterized glycoside hydrolases have been cataloged. They come from 131 different fungal species, most of which belong to the phylum Ascomycota. These enzymes represent 46 different GH activities and cover 44 of the 115 CAZy GH families. In addition to enzyme source and enzyme family, available biochemical properties such as temperature and pH optima, specific activity, kinetic parameters and substrate specificities were recorded. To simplify comparative studies, enzyme and species abbreviations have been standardized, Gene Ontology terms assigned and reference to supporting evidence provided. The annotated genes have been organized in a searchable, online database called mycoCLAP (Characterized Lignocellulose-Active Proteins of fungal origin). It is anticipated that this manually curated collection of biochemically characterized fungal proteins will be used to enhance functional annotation of novel GH genes. Database URL: http://mycoCLAP.fungalgenomics.ca/ PMID:21622642

  8. Effect of alpha lipoic acid on leukotriene A4 hydrolase.

    PubMed

    Torres, María José; Fierro, Angélica; Pessoa-Mahana, C David; Romero-Parra, Javier; Cabrera, Gonzalo; Faúndez, Mario

    2017-03-15

    Leukotriene A4 hydrolase is a soluble enzyme with epoxide hydrolase and aminopeptidase activities catalysing the conversion of leukotriene A4 to leukotriene B4 and the hydrolysis of the peptide proline-glycine-proline. Imbalances in leukotriene B4 synthesis are related to several pathologic conditions. Currently there are no available drugs capable to modulate the synthesis of leukotriene B4 or to block its receptors. Here we show the inhibitory profile of alpha lipoic acid on the activity of leukotriene A4 Hydrolase. Alpha lipoic acid inhibited both activities of the enzyme at concentrations lower than 10μM. The 5-lipoxygenase inhibitor zileuton, or the 5-lipoxygenase activating protein inhibitor MK-886, were unable to inhibit the activity of the enzyme. Acute promyelocytic leukaemia HL-60 cells were differentiated to leukotriene A4 hydrolase expressing neutrophil-like cells. Alpha lipoic acid inhibited the aminopeptidase activity of the cytosolic fraction from neutrophil-like cells but had no effect on the cytosolic fraction from undifferentiated cells. Docking and molecular dynamic approximations revealed that alpha lipoic acid participates in electrostatic interactions with K-565 and R-563, which are key residues for the carboxylate group recognition of endogenous substrates by the enzyme. Alpha lipoic acid is a compound widely used in clinical practice, most of its therapeutic effects are associated with its antioxidants properties, however, antioxidant effect alone is unable to explain all clinical effects observed with alpha lipoic acid. Our results invite to evaluate the significance of the inhibitory effect of alpha lipoic acid on the catalytic activity of leukotriene A4 hydrolase using in vivo models.

  9. Triacylglycerol synthesis by PDAT1 in the absence of DGAT1 activity is dependent on re-acylation of LPC by LPCAT2

    PubMed Central

    2012-01-01

    Background The Arabidopsis thaliana dgat1 mutant, AS11, has an oil content which is decreased by 30%, and a strongly increased ratio of 18:3/20:1, compared to wild type. Despite lacking a functional DGAT1, AS11 still manages to make 70% of WT seed oil levels. Recently, it was demonstrated that in the absence of DGAT1, PDAT1 was essential for normal seed development, and is a dominant determinant in Arabidopsis TAG biosynthesis. Methods Biochemical, metabolic and gene expression studies combined with genetic crossing of selected Arabidopsis mutants have been carried out to demonstrate the contribution of Arabidopsis PDAT1 and LPCAT2 in the absence of DGAT1 activity. Results Through microarray and RT-PCR gene expression analyses of AS11 vs. WT mid-developing siliques, we observed consistent trends between the two methods. FAD2 and FAD3 were up-regulated and FAE1 down-regulated, consistent with the AS11 acyl phenotype. PDAT1 expression was up-regulated by ca 65% while PDAT2 expression was up-regulated only 15%, reinforcing the dominant role of PDAT1 in AS11 TAG biosynthesis. The expression of LPCAT2 was up-regulated by 50-75%, while LPCAT1 expression was not significantly affected. In vitro LPCAT activity was enhanced by 75-125% in microsomal protein preparations from mid-developing AS11 seed vs WT. Co-incident homozygous knockout lines of dgat1/lpcat2 exhibited severe penalties on TAG biosynthesis, delayed plant development and seed set, even with a functional PDAT1; the double mutant dgat1/lpcat1 showed only marginally lower oil content than AS11. Conclusions Collectively, the data strongly support that in AS11 it is LPCAT2 up-regulation which is primarily responsible for assisting in PDAT1-catalyzed TAG biosynthesis, maintaining a supply of PC as co-substrate to transfer sn-2 moieties to the sn-3 position of the enlarged AS11 DAG pool. PMID:22233193

  10. SREBP2 Activation Induces Hepatic Long-chain Acyl-CoA Synthetase 1 (ACSL1) Expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter.

    PubMed

    Singh, Amar Bahadur; Kan, Chin Fung Kelvin; Dong, Bin; Liu, Jingwen

    2016-03-04

    Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism. To identify novel transcriptional modulators of ACSL1, we examined ACSL1 expression in liver tissues of hamsters fed a normal diet, a high fat diet, or a high cholesterol and high fat diet (HCHFD). Feeding hamsters HCHFD markedly reduced hepatic Acsl1 mRNA and protein levels as well as acyl-CoA synthetase activity. Decreases in Acsl1 expression strongly correlated with reductions in hepatic Srebp2 mRNA level and mature Srebp2 protein abundance. Conversely, administration of rosuvastatin (RSV) to hamsters increased hepatic Acsl1 expression. These new findings were reproduced in mice treated with RSV or fed the HCHFD. Furthermore, the RSV induction of acyl-CoA activity in mouse liver resulted in increases in plasma and hepatic cholesterol ester concentrations and reductions in free cholesterol amounts. Investigations on different ACSL1 transcript variants in HepG2 cells revealed that the mRNA expression of C-ACSL1 was specifically regulated by the sterol regulatory element (SRE)-binding protein (SREBP) pathway, and RSV treatment increased the C-ACSL1 abundance from a minor mRNA species to an abundant transcript. We analyzed 5'-flanking sequence of exon 1C of the human ACSL1 gene and identified one putative SRE site. By performing a promoter activity assay and DNA binding assays, we firmly demonstrated the key role of this SRE motif in SREBP2-mediated activation of C-ACSL1 gene transcription. Finally, we demonstrated that knockdown of endogenous SREBP2 in HepG2 cells lowered ACSL1 mRNA and protein levels. Altogether, this work discovered an unprecedented link between ACSL1 and SREBP2 via the specific regulation of the C-ACSL1 transcript.

  11. SREBP2 Activation Induces Hepatic Long-chain Acyl-CoA Synthetase 1 (ACSL1) Expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter*

    PubMed Central

    Singh, Amar Bahadur; Kan, Chin Fung Kelvin; Dong, Bin; Liu, Jingwen

    2016-01-01

    Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism. To identify novel transcriptional modulators of ACSL1, we examined ACSL1 expression in liver tissues of hamsters fed a normal diet, a high fat diet, or a high cholesterol and high fat diet (HCHFD). Feeding hamsters HCHFD markedly reduced hepatic Acsl1 mRNA and protein levels as well as acyl-CoA synthetase activity. Decreases in Acsl1 expression strongly correlated with reductions in hepatic Srebp2 mRNA level and mature Srebp2 protein abundance. Conversely, administration of rosuvastatin (RSV) to hamsters increased hepatic Acsl1 expression. These new findings were reproduced in mice treated with RSV or fed the HCHFD. Furthermore, the RSV induction of acyl-CoA activity in mouse liver resulted in increases in plasma and hepatic cholesterol ester concentrations and reductions in free cholesterol amounts. Investigations on different ACSL1 transcript variants in HepG2 cells revealed that the mRNA expression of C-ACSL1 was specifically regulated by the sterol regulatory element (SRE)-binding protein (SREBP) pathway, and RSV treatment increased the C-ACSL1 abundance from a minor mRNA species to an abundant transcript. We analyzed 5′-flanking sequence of exon 1C of the human ACSL1 gene and identified one putative SRE site. By performing a promoter activity assay and DNA binding assays, we firmly demonstrated the key role of this SRE motif in SREBP2-mediated activation of C-ACSL1 gene transcription. Finally, we demonstrated that knockdown of endogenous SREBP2 in HepG2 cells lowered ACSL1 mRNA and protein levels. Altogether, this work discovered an unprecedented link between ACSL1 and SREBP2 via the specific regulation of the C-ACSL1 transcript. PMID:26728456

  12. Fatty Acyl Chains of Mycobacterium marinum Lipooligosaccharides

    PubMed Central

    Rombouts, Yoann; Alibaud, Laeticia; Carrère-Kremer, Séverine; Maes, Emmanuel; Tokarski, Caroline; Elass, Elisabeth; Kremer, Laurent; Guérardel, Yann

    2011-01-01

    We have recently established the fine structure of the glycan backbone of lipooligosaccharides (LOS-I to LOS-IV) isolated from Mycobacterium marinum, a close relative of Mycobacterium tuberculosis. These studies culminated with the description of an unusual terminal N-acylated monosaccharide that confers important biological functions to LOS-IV, such as macrophage activation, that may be relevant to granuloma formation. It was, however, also suggested that the lipid moiety was required for LOSs to exert their immunomodulatory activity. Herein, using highly purified LOSs from M. marinum, we have determined through a combination of mass spectrometric and NMR techniques, the structure and localization of the fatty acids composing the lipid moiety. The occurrence of two distinct polymethyl-branched fatty acids presenting specific localizations is consistent with the presence of two highly related polyketide synthases (Pks5 and Pks5.1) in M. marinum and presumably involved in the synthesis of these fatty acyl chains. In addition, a bioinformatic search permitted us to identify a set of enzymes potentially involved in the biosynthesis or transfer of these lipids to the LOS trehalose unit. These include MMAR_2343, a member of the Pap (polyketide-associated protein) family, that acylates trehalose-based glycolipids in M. marinum. The participation of MMAR_2343 to LOS assembly was demonstrated using a M. marinum mutant carrying a transposon insertion in the MMAR_2343 gene. Disruption of MMAR_2343 resulted in a severe LOS breakdown, indicating that MMAR_2343, hereafter designated PapA4, fulfills the requirements for LOS acylation and assembly. PMID:21803773

  13. Characterization of a structurally and functionally diverged acyl-acyl carrier protein desaturase from milkweed seed.

    PubMed

    Cahoon, E B; Coughlan, S J; Shanklin, J

    1997-04-01

    A cDNA for a structurally variant acyl-acyl carrier protein (ACP) desaturase was isolated from milkweed (Asclepias syriaca) seed, a tissue enriched in palmitoleic (16:1delta9)* and cis-vaccenic (18:1delta11) acids. Extracts of Escherichia coli that express the milkweed cDNA catalyzed delta9 desaturation of acyl-ACP substrates, and the recombinant enzyme exhibited seven- to ten-fold greater specificity for palmitoyl (16:0)-ACP and 30-fold greater specificity for myristoyl (14:0)-ACP than did known delta9-stearoyl (18:0)-ACP desaturases. Like other variant acyl-ACP desaturases reported to date, the milkweed enzyme contains fewer amino acids near its N-terminus compared to previously characterized delta9-18:0-ACP desaturases. Based on the activity of an N-terminal deletion mutant of a delta9-18:0-ACP desaturase, this structural feature likely does not account for differences in substrate specificities.

  14. Rosiglitazone Inhibits Acyl-CoA Synthetase Activity and Fatty Acid Partitioning to Diacylglycerol and Triacylglycerol via a Peroxisome Proliferator–Activated Receptor-γ–Independent Mechanism in Human Arterial Smooth Muscle Cells and Macrophages

    PubMed Central

    Askari, Bardia; Kanter, Jenny E.; Sherrid, Ashley M.; Golej, Deidre L.; Bender, Andrew T.; Liu, Joey; Hsueh, Willa A.; Beavo, Joseph A.; Coleman, Rosalind A.; Bornfeldt, Karin E.

    2010-01-01

    Rosiglitazone is an insulin-sensitizing agent that has recently been shown to exert beneficial effects on atherosclerosis. In addition to peroxisome proliferator–activated receptor (PPAR)-γ, rosiglitazone can affect other targets, such as directly inhibiting recombinant long-chain acyl-CoA synthetase (ACSL)-4 activity. Because it is unknown if ACSL4 is expressed in vascular cells involved in atherosclerosis, we investigated the ability of rosiglitazone to inhibit ACSL activity and fatty acid partitioning in human and murine arterial smooth muscle cells (SMCs) and macrophages. Human and murine SMCs and human macrophages expressed Acsl4, and rosiglitazone inhibited Acsl activity in these cells. Furthermore, rosiglitazone acutely inhibited partitioning of fatty acids into phospholipids in human SMCs and inhibited fatty acid partitioning into diacylglycerol and triacylglycerol in human SMCs and macrophages through a PPAR-γ–independent mechanism. Conversely, murine macrophages did not express ACSL4, and rosiglitazone did not inhibit ACSL activity in these cells, nor did it affect acute fatty acid partitioning into cellular lipids. Thus, rosiglitazone inhibits ACSL activity and fatty acid partitioning in human and murine SMCs and in human macrophages through a PPAR-γ–independent mechanism likely to be mediated by ACSL4 inhibition. Therefore, rosiglitazone might alter the biological effects of fatty acids in these cells and in atherosclerosis. PMID:17259370

  15. Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

    PubMed Central

    Dodge, Anthony G.; Preiner, Chelsea S.

    2013-01-01

    The known enzymes that open the s-triazine ring, the cyanuric acid hydrolases, have been confined almost exclusively to the kingdom Bacteria and are all homologous members of the rare cyanuric acid hydrolase/barbiturase protein family. In the present study, a filamentous fungus, Sarocladium sp. strain CA, was isolated from soil by enrichment culturing using cyanuric acid as the sole source of nitrogen. A reverse-genetic approach identified a fungal cyanuric acid hydrolase gene composed of two exons and one intron. The translated spliced sequence was 39 to 53% identical to previously characterized bacterial cyanuric acid hydrolases. The sequence was used to generate a gene optimized for expression in Escherichia coli and encoding an N-terminally histidine-tagged protein. The protein was purified by nickel affinity and anion-exchange chromatography. The purified protein was shown by 13C nuclear magnetic resonance (13C-NMR) to produce carboxybiuret as the product, which spontaneously decarboxylated to yield biuret and carbon dioxide. The protein was very narrow in substrate specificity, showing activity only with cyanuric acid and N-methyl cyanuric acid. Barbituric acid was an inhibitor of enzyme activity. Sequence analysis identified genes with introns in other fungi from the Ascomycota that, if spliced, are predicted to encode proteins with cyanuric acid hydrolase activity. The Ascomycota cyanuric acid hydrolase homologs are most closely related to cyanuric acid hydrolases from Actinobacteria. PMID:24039269

  16. Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives.

    PubMed

    Kotik, Michael; Brichac, Jiri; Kyslík, Pavel

    2005-12-06

    Microbial isolates from biofilters and petroleum-polluted bioremediation sites were screened for the presence of enantioselective epoxide hydrolases active towards tert-butyl glycidyl ether, benzyl glycidyl ether, and allyl glycidyl ether. Out of 270 isolated strains, which comprised bacteria, yeasts, and filamentous fungi, four were selected based on the enantioselectivities of their epoxide hydrolases determined in biotransformation reactions. The enzyme of Aspergillus niger M200 preferentially hydrolyses (S)-tert-butyl glycidyl ether to (S)-3-tert-butoxy-1,2-propanediol with a relatively high enantioselectivity (the enantiomeric ratio E is about 30 at a reaction temperature of 28 degrees C). Epoxide hydrolases of Rhodotorula mucilaginosa M002 and Rhodococcus fascians M022 hydrolyse benzyl glycidyl ether with relatively low enantioselectivities, the former reacting predominantly with the (S)-enantiomer, the latter preferring the (R)-enantiomer. Enzymatic hydrolysis of allyl glycidyl ether by Cryptococcus laurentii M001 proceeds with low enantioselectivity (E=3). (R)-tert-Butyl glycidyl ether with an enantiomeric excess (ee) of over 99%, and (S)-3-tert-butoxy-1,2-propanediol with an ee-value of 86% have been prepared on a gram-scale using whole cells of A. niger M200. An enantiomeric ratio of approximately 100 has been determined under optimised biotransformation conditions with the partially purified epoxide hydrolase from A. niger M200. The regioselectivity of this enzyme was determined to be total for both (S)-tert-butyl glycidyl ether and (R)-tert-butyl glycidyl ether.

  17. Acylated flavonol glycoside from Platanus orientalis.

    PubMed

    Tantry, Mudasir A; Akbar, Seema; Dar, Javid A; Irtiza, Syed; Galal, Ahmed; Khuroo, Mohammad A; Ghazanfar, Khalid

    2012-03-01

    The ethylacetate and n-butanol fractions of ethanolic extract of Platanus orientalis leaves led to the isolation of new acylated flavonol glycoside as 3',5,7-trihydroxy-4'-methoxyflavonol 3-[O-2-O-(2,4-Dihydroxy)-E-cinnamoyl-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl (1→2)]-β-D-glucopyranoside, along with seven known compounds. All the compounds were characterized by NMR including 2D NMR techniques. The isolates were evaluated for NF-κB, nitric oxide (NO), aromatase and QR2 chemoprevention activities and some of them appeared to be modestly active.

  18. Isolation and characterization of Xenopus soluble epoxide hydrolase.

    PubMed

    Purba, Endang R; Oguro, Ami; Imaoka, Susumu

    2014-07-01

    Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.

  19. Identification and characterization of a new epoxide hydrolase from mouse liver microsomes.

    PubMed

    Guenthner, T M; Oesch, F

    1983-12-25

    A new microsomal epoxide hydrolase (mEH2) has been identified and characterized. This enzyme has properties which distinguish it from previously described cytosolic (cEH) or membrane-bound (mEH1) epoxide hydrolases. The enzyme is an integral microsomal protein which is not dissociated from the membrane by repeated washing, high ionic strength salt, or chaotropic agent solutions, or by sonication. It is very different from the normally described microsomal epoxide hydrolase (mEH1) as shown by its different substrate specificity and kinetic properties and by immunological criteria. In contrast to the hitherto described microsomal epoxide hydrolase, mEH1, the new enzyme effectively catalyzes the hydration of transdisubstituted oxiranes such as trans-stilbene oxide and trans-beta-ethyl styrene oxide and has no appreciable activity toward benzo(a)pyrene 4,5-oxide. It is also structurally distinct, in that it does not cross-react with antibodies raised against the normally described microsomal epoxide hydrolase mEH1. This newly described microsomal epoxide hydrolase probably represents an important factor in the control of reactive epoxides; its location in the membrane ensures access to lipophilic epoxides generated by membrane-bound monooxygenases, and its substrate specificity is such that it can hydrolyze epoxides poorly metabolized by the previously described microsomal epoxide hydrolase.

  20. Bitter gourd (Momordica charantia) extract activates peroxisome proliferator-activated receptors and upregulates the expression of the acyl CoA oxidase gene in H4IIEC3 hepatoma cells.

    PubMed

    Chao, Che-Yi; Huang, Ching-jang

    2003-01-01

    Peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-dependent transcription factor that regulates the expression of genes involved in lipid metabolism and transport. Ligands/activators of PPARalpha, like fibrate-type drugs, may have hypolipidemic effects. To identify food that contains activators of PPARalpha, a transactivation assay employing a clone of CHO-K1 cells stably transfected with a (UAS)(4)-tk-alkaline phosphatase reporter and a chimeric receptor of Gal4-rPPARalpha LBD was used to screen ethyl acetate (EA) extracts of a large variety of food materials. It was found that the EA extract of bitter gourd (Momordica charantia), a common oriental vegetable, activated PPARalpha to an extent that was equivalent to or even higher than 10 microM Wy-14643, a known ligand of PPARalpha. This extract also activated PPARgamma to a significant extent which was comparable to 0.5 microM BRL-49653. The activity toward PPARalpha was mainly in the soluble fraction of the organic solvent. The EA extract prepared from the whole fruit showed significantly higher activity than that from seeds or flesh alone. The bitter gourd EA extract was then incorporated into the medium for treatment of a peroxisome proliferator-responsive murine hepatoma cell line, H4IIEC3, for 72 h. Treated cells showed significantly higher activity of acyl CoA oxidase and higher expressions of mRNA of this enzyme and fatty acid-binding protein, indicating that the bitter gourd EA extract was able to act on a natural PPARalpha signaling pathway in this cell line. It is thus worth further investigating the PPAR-associated health benefits of bitter gourd.

  1. Asymmetric Allylboration of Acyl Imines Catalyzed by Chiral Diols

    PubMed Central

    Lou, Sha; Moquist, Philip N.; Schaus, Scott E.

    2008-01-01

    Chiral BINOL-derived diols catalyze the enantioselective asymmetric allylboration of acyl imines. The reaction requires 15 mol% of (S)-3,3′-Ph2-BINOL as the catalyst and allyldiisopropoxyborane as the nucleophile. The reaction products are obtained in good yields (75 – 94%) and high enantiomeric ratios (95:5 – 99.5:0.5) for aromatic and aliphatic imines. High diastereoselectivities (dr > 98:2) and enantioselectivities (er > 98:2) are obtained in the reactions of acyl imines with crotyldiisopropoxyboranes. This asymmetric transformation is directly applied to the synthesis of maraviroc, the selective CCR5 antagonist with potent activity against HIV-1 infection. Mechanistic investigations of the allylboration reaction including IR, NMR, and mass spectrometry study indicate that acyclic boronates are activated by chiral diols via exchange of one of the boronate alkoxy groups with activation of the acyl imine via hydrogen bonding. PMID:18020334

  2. A simplified electrostatic model for hydrolase catalysis.

    PubMed

    Pessoa Filho, Pedro de Alcantara; Prausnitz, John M

    2015-07-01

    Toward the development of an electrostatic model for enzyme catalysis, the active site of the enzyme is represented by a cavity whose surface (and beyond) is populated by electric charges as determined by pH and the enzyme's structure. The electric field in the cavity is obtained from electrostatics and a suitable computer program. The key chemical bond in the substrate, at its ends, has partial charges with opposite signs determined from published force-field parameters. The electric field attracts one end of the bond and repels the other, causing bond tension. If that tension exceeds the attractive force between the atoms, the bond breaks; the enzyme is then a successful catalyst. To illustrate this very simple model, based on numerous assumptions, some results are presented for three hydrolases: hen-egg white lysozyme, bovine trypsin and bovine ribonuclease. Attention is given to the effect of pH.

  3. Polyglycine hydrolases: fungal b-lactamase-like endoproteases that cleave polyglycine regions within plant class IV chitinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyglycine hydrolases are secreted fungal proteases that cleave glycine-glycine peptide bonds in the inter-domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es-cmp) and Cochli...

  4. Central acylated ghrelin improves memory function and hippocampal AMPK activation and partly reverses the impairment of energy and glucose metabolism in rats infused with β-amyloid.

    PubMed

    Kang, Suna; Moon, Na Rang; Kim, Da Sol; Kim, Sung Hoon; Park, Sunmin

    2015-09-01

    Ghrelin is a gastric hormone released during the fasting state that targets the hypothalamus where it induces hunger; however, emerging evidence suggests it may also affect memory function. We examined the effect of central acylated-ghrelin and DES-acetylated ghrelin (native ghrelin) on memory function and glucose metabolism in an experimentally induced Alzheimer's disease (AD) rat model. AD rats were divided into 3 groups and Non-AD rats were used as a normal-control group. Each rat in the AD groups had intracerebroventricular (ICV) infusion of β-amyloid (25-35; 16.8nmol/day) into the lateral ventricle for 3 days, and then the pumps were changed to infuse either acylated-ghrelin (0.2nmol/h; AD-G), DES-acylated ghrelin (0.2nmol/h; AD-DES-G), or saline (control; AD-C) for 3 weeks. The Non-AD group had ICV infusion of β-amyloid (35-25) which does not deposit in the hippocampus. During the next 3 weeks memory function, food intake, body weight gain, body fat composition, and glucose metabolism were measured. AD-C exhibited greater β-amyloid deposition compared to Non-AD-C, and AD-G suppressed the increased β-amyloid deposition and potentiated the phosphorylation AMPK. In addition, AD-G increased the phosphorylation GSK and decreased the phosphorylation of Tau in comparison to AD-C and AD-DES-G. Cognitive function, measured by passive avoidance and water maze tests, was much lower in AD-C than Non-AD-C whereas AD-G but not AD-DES-G prevented the decrease (p<0.021). Body weight gain was lower in AD-C group than Non-AD-C group without changing epididymal fat mass. AD-G reversed the decrease in body weight which was due to increased energy intake and decreased energy expenditure. The AD-G group exhibited a decrease in the second part of serum glucose levels during an oral glucose tolerance test (OGTT) compared to the AD-C and AD-DES-G group (p<0.009). However, area under the curve of insulin during the first part of OGTT was higher in AD-DES-G than other groups

  5. Theoretical and structural analysis of the active site of the transcriptional regulators LasR and TraR, using molecular docking methodology for identifying potential analogues of acyl homoserine lactones (AHLs) with anti-quorum sensing activity.

    PubMed

    Ahumedo, Maicol; Díaz, Antonio; Vivas-Reyes, Ricardo

    2010-02-01

    In the present study the homology of transcriptional receptors LuxR type were evaluated using as point of reference the receptors TraR and LasR of the bacterial types Agrobacterium tumefaciens and Pseudomonas aureginosa respectively. A series of alignments were performed in order to demonstrate that the active site of the protein is conserved in wide range of gram negative bacteria. Moreover, some docking calculations were carried out for analogs of the acyl homoserin lactones (AHLs) and regulatory proteins LasR and TraR, to understand the complex microenvironment in which the ligands are exposed. The molecular alignments show clearly that there are preserved motifs in the residues (Y53, Y61, W57, D70, W85 to TraR, Y56, Y64, W60, D73, W88 to LasR) analyzed, which may serve as site-specific targets for the development of potential antagonists. In this study was found that the anti-quorum sensing activity of the AHLs molecular analogs appears to depend on; the structure of the lactone ring and on appropriate combination of absolute and relative stereochemistry of the carbonyl (C=O) and amide (NH(2)) groups of the side chain of these AHLs molecular analogs, in combination with the interactions with the conserved amino acids (D73, W60, Y56, S129 to LasR and D70, W57, Y53 to TraR) of the LuxR type protein family.

  6. A novel plasmid for detection of N-acyl homoserine lactones.

    PubMed

    Ling, Elizabeth A; Ellison, Matthew L; Pesci, Everett C

    2009-07-01

    Many bacteria utilize acyl-homoserine lactones as cell to cell signals that can regulate the expression of numerous genes. Structural differences in acyl-homoserine lactones produced by different bacteria, such as acyl side chain length and the presence or absence of an oxy group, make many of the commonly used detection bioassays impractical for broad range detection. Here we present a simple, broad range acyl-homoserine lactone detection bioassay that can be used to detect a wide range of these chemical signals. A plasmid (pEAL01) was constructed and transformed into Pseudomonas aeruginosa strain QSC105 to allow for detection of a broad range of acyl-homoserine lactones through induction of a lasB'-lacZ transcriptional fusion. Monitoring beta-galactosidase activity from this bioassay showed that P. aeruginosa strain QSC105 (pEAL01) could detect the presence of eight acyl-homoserine lactones tested at physiological concentrations. This novel strain could also detect acyl-homoserine lactones from the extracts of four different bacteria that produce different acyl-homoserine lactones signals. These data indicate that strain QSC105 (pEAL01) can be used to detect a wide variety of acyl-homoserine lactones by a simple beta-galactosidase assay and this bioassay could be a useful and inexpensive tool to quickly identify the presence of these signal molecules.

  7. Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions*

    PubMed Central

    Lager, Ida; Yilmaz, Jenny Lindberg; Zhou, Xue-Rong; Jasieniecka, Katarzyna; Kazachkov, Michael; Wang, Peng; Zou, Jitao; Weselake, Randall; Smith, Mark A.; Bayon, Shen; Dyer, John M.; Shockey, Jay M.; Heinz, Ernst; Green, Allan; Banas, Antoni; Stymne, Sten

    2013-01-01

    Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C18-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism. PMID:24189065

  8. Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1.

    PubMed

    Sio, Charles F; Otten, Linda G; Cool, Robbert H; Diggle, Stephen P; Braun, Peter G; Bos, Rein; Daykin, Mavis; Cámara, Miguel; Williams, Paul; Quax, Wim J

    2006-03-01

    The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3' position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.

  9. Quorum Quenching by an N-Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1

    PubMed Central

    Sio, Charles F.; Otten, Linda G.; Cool, Robbert H.; Diggle, Stephen P.; Braun, Peter G.; Bos, Rein; Daykin, Mavis; Cámara, Miguel; Williams, Paul; Quax, Wim J.

    2006-01-01

    The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3′ position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies. PMID:16495538

  10. Two fatty acyl reductases involved in moth pheromone biosynthesis

    PubMed Central

    Antony, Binu; Ding, Bao-Jian; Moto, Ken’Ichi; Aldosari, Saleh A.; Aldawood, Abdulrahman S.

    2016-01-01

    Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective ‘single pgFARs’ produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a ‘single reductase’ can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

  11. Potent Urea and Carbamate Inhibitors of Soluble Epoxide Hydrolases

    NASA Astrophysics Data System (ADS)

    Morisseau, Christophe; Goodrow, Marvin H.; Dowdy, Deanna; Zheng, Jiang; Greene, Jessica F.; Sanborn, James R.; Hammock, Bruce D.

    1999-08-01

    The soluble epoxide hydrolase (sEH) plays a significant role in the biosynthesis of inflammation mediators as well as xenobiotic transformations. Herein, we report the discovery of substituted ureas and carbamates as potent inhibitors of sEH. Some of these selective, competitive tightbinding inhibitors with nanomolar Ki values interacted stoichiometrically with the homogenous recombinant murine and human sEHs. These inhibitors enhance cytotoxicity of trans-stilbene oxide, which is active as the epoxide, but reduce cytotoxicity of leukotoxin, which is activated by epoxide hydrolase to its toxic diol. They also reduce toxicity of leukotoxin in vivo in mice and prevent symptoms suggestive of acute respiratory distress syndrome. These potent inhibitors may be valuable tools for testing hypotheses of involvement of diol and epoxide lipids in chemical mediation in vitro or in vivo systems.

  12. Identification of the C-Terminal GH5 Domain from CbCel9B/Man5A as the First Glycoside Hydrolase with Thermal Activation Property from a Multimodular Bifunctional Enzyme

    PubMed Central

    Wang, Rong; Gong, Li; Xue, Xianli; Qin, Xing; Ma, Rui; Luo, Huiying; Zhang, Yongjie; Yao, Bin; Su, Xiaoyun

    2016-01-01

    Caldicellulosiruptor bescii encodes at least six unique multimodular glycoside hydrolases crucial for plant cell wall polysaccharides degradation, with each having two catalytic domains separated by two to three carbohydrate binding modules. Among the six enzymes, three have one N- or C-terminal GH5 domain with identical amino acid sequences. Despite a few reports on some of these multimodular enzymes, little is known about how the conserved GH5 domains behave, which are believed to be important due to the gene duplication. We thus cloned a representative GH5 domain from the C-terminus of a multimodular protein, i.e. the bifunctional cellulase/mannanase CbCel9B/Man5A which has been reported, and expressed it in Escherichia coli. Without any appending CBMs, the recombinant CbMan5A was still able to hydrolyze a variety of mannan substrates with different backbone linkages or side-chain decorations. While CbMan5A displayed the same pH optimum as CbCel9B/Man5A, it had an increased optimal temperature (90°C) and moreover, was activated by heating at 70°C and 80°C, a property not ever reported for the full-length protein. The turnover numbers of CbMan5A on mannan substrates were, however, lower than those of CbCel9B/Man5A. These data suggested that evolution of CbMan5A and the other domains into a single polypeptide is not a simple assembly; rather, the behavior of one module may be affected by the other ones in the full-length enzyme. The differential scanning calorimetry analysis further indicated that heating CbMan5A was not a simple transition state process. To the best knowledge of the authors, CbMan5A is the first glycoside hydrolase with thermal activation property identified from a multimodular bifunctional enzyme. PMID:27258548

  13. Human Valacyclovir Hydrolase/Biphenyl Hydrolase-Like Protein Is a Highly Efficient Homocysteine Thiolactonase

    PubMed Central

    McDonald, Matthew G.; Rademacher, Peter M.; MacCoss, Michael J.; Hsieh, Edward J.; Rettie, Allan E.; Furlong, Clement E.

    2014-01-01

    Homocysteinylation of lysine residues by homocysteine thiolactone (HCTL), a reactive homocysteine metabolite, results in protein aggregation and malfunction, and is a well-known risk factor for cardiovascular, autoimmune and neurological diseases. Human plasma paraoxonase-1 (PON1) and bleomycin hydrolase (Blmh) have been reported as the physiological HCTL detoxifying enzymes. However, the catalytic efficiency of HCTL hydrolysis by Blmh is low and not saturated at 20 mM HCTL. The catalytic efficiency of PON1 for HCTL hydrolysis is 100-fold lower than that of Blmh. A homocysteine thiolactonase (HCTLase) was purified from human liver and identified by mass spectrometry (MS) as the previously described human biphenyl hydrolase-like protein (BPHL). To further characterize this newly described HCTLase activity, BPHL was expressed in Escherichia coli and purified. The sequence of the recombinant BPHL (rBPHL) and hydrolytic products of the substrates HCTL and valacyclovir were verified by MS. We found that the catalytic efficiency (kcat/Km) of rBPHL for HCTL hydrolysis was 7.7 × 104 M−1s−1, orders of magnitude higher than that of PON1 or Blmh, indicating a more significant physiological role for BPHL in detoxifying HCTL. PMID:25333274

  14. Differential effects of laparoscopic sleeve gastrectomy and laparoscopic gastric bypass on appetite, circulating acyl-ghrelin, peptide YY3-36 and active GLP-1 levels in non-diabetic humans.

    PubMed

    Yousseif, Ahmed; Emmanuel, Julian; Karra, Efthimia; Millet, Queensta; Elkalaawy, Mohamed; Jenkinson, Andrew D; Hashemi, Majid; Adamo, Marco; Finer, Nicholas; Fiennes, Alberic G; Withers, Dominic J; Batterham, Rachel L

    2014-02-01

    Laparoscopic Roux-en-Y gastric bypass (LRYGBP) reduces appetite and induces significant and sustainable weight loss. Circulating gut hormones changes engendered by LRYGBP are implicated in mediating these beneficial effects. Laparoscopic sleeve gastrectomy (LSG) is advocated as an alternative to LRYGBP, with comparable short-term weight loss and metabolic outcomes. LRYGBP and LSG are anatomically distinct procedures causing differential entero-endocrine cell nutrient exposure and thus potentially different gut hormone changes. Studies reporting the comparative effects of LRYGBP and LSG on appetite and circulating gut hormones are controversial, with no data to date on the effects of LSG on circulating peptide YY3-36 (PYY3-36) levels, the specific PYY anorectic isoform. In this study, we prospectively investigated appetite and gut hormone changes in response to LRYGBP and LSG in adiposity-matched non-diabetic patients. Anthropometric indices, leptin, fasted and nutrient-stimulated acyl-ghrelin, active glucagon-like peptide-1 (GLP-1), PYY3-36 levels and appetite were determined pre-operatively and at 6 and 12 weeks post-operatively in obese, non-diabetic females, with ten undergoing LRYGBP and eight adiposity-matched females undergoing LSG. LRYGBP and LSG comparably reduced adiposity. LSG decreased fasting and post-prandial plasma acyl-ghrelin compared to pre-surgery and to LRYGBP. Nutrient-stimulated PYY3-36 and active GLP-1 concentrations increased post-operatively in both groups. However, LRYGBP induced greater, more sustained PYY3-36 and active GLP-1 increments compared to LSG. LRYGBP suppressed fasting hunger compared to LSG. A similar increase in post-prandial fullness was observed post-surgery following both procedures. LRYGBP and LSG produced comparable enhanced satiety and weight loss. However, LSG and LRYGBP differentially altered gut hormone profiles.

  15. Fatty acid acylation of rat brain myelin proteolipid protein in vitro: identification of the lipid donor.

    PubMed

    Bizzozero, O A; Lees, M B

    1986-02-01

    The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chloroform/methanol, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl-CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl-CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl-CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.

  16. Isolation, characterization, and localization of AgaSGNH cDNA: a new SGNH-motif plant hydrolase specific to Agave americana L. leaf epidermis.

    PubMed

    Reina, José J; Guerrero, Consuelo; Heredia, Antonio

    2007-01-01

    GDSL and SGNH hydrolases are lipases involved in a wide range of functions, behaving in many cases as bifunctional enzymes. In this work, the isolation and characterization of AgaSGNH, a cDNA encoding a member of the SGNH-hydrolase superfamily from young leaf epidermis of the monocot Agave americana L., is reported. The protein possesses a typical signal peptide at its N-terminus that allows its secretion to the epidermis cell wall, as verified by immunolocalization experiments. In addition, the AgaSGNH sequence contains a His-Leu-Gly-Ala-Glu (HLGAE) motif which is similar to that observed in other plant acyltransferases. Expression levels by northern blot and in situ localization of the corresponding mRNA, as well as the immunolocalization of the protein in Agave young leaves indicate that the protein is specifically present in the epidermal cells. The detailed study performed in different parts of the Agave leaf confirms two aspects: first, the expression of AgaSGNH is limited to the epidermis, and second, the maximum mRNA levels are found in the epidermis of the youngest zones of the leaf which are especially active in cutin biosynthesis. These levels dramatically decrease in the oldest zone of the leaf, where the presence of AgaSGNH mRNA is undetectable, and the biosynthesis of different cuticle components is severely reduced. These data could be compatible with the hypothesis that AgaSGNH could carry out both the hydrolysis and the transfer, from an activated acyl-CoA to a crescent cutin in Agave americana leaves and, therefore, be involved in the still unknown mechanism of plant cutin biosynthesis.

  17. Modulating the synthetase activity of penicillin G acylase in organic media by addition of N-methylimidazole: using vinyl acetate as activated acyl donor.

    PubMed

    Liu, Bokai; Wu, Qi; Lv, Deshui; Lin, Xianfu

    2011-05-20

    This paper reported the modulation of enzyme activity by organic small molecule. The esterification activity of Penicillin G acylase (PGA) was improved more than 70-fold by the addition of 10% N-methylimidazole. Some control experiments have been designed to demonstrate the catalytic specificity of PGA. The structure and the amount of additive were optimized to improve the product yield. The influence of N-methylimidazole on the PGA conformation was investigated by FTIR and autodock simulation. Seven substrates were used to evaluate the effect of structure on the PGA-catalyzed transesterification. A series of products were successfully synthesized with the yield ranged from 56% to 84% and PGA showed specific recognition on the substrate with phenyl group in the presence of 10% N-methylimidazole.

  18. S-Mercuration of ubiquitin carboxyl-terminal hydrolase L1 through Cys152 by methylmercury causes inhibition of its catalytic activity and reduction of monoubiquitin levels in SH-SY5Y cells.

    PubMed

    Toyama, Takashi; Abiko, Yumi; Katayama, Yuko; Kaji, Toshiyuki; Kumagai, Yoshito

    2015-12-01

    Methylmercury (MeHg) is an environmental electrophile that covalently modifies cellular proteins. In this study, we identified proteins that undergo S-mercuration by MeHg. By combining two-dimensional SDS-PAGE, atomic absorption spectrometry and ultra performance liquid chromatography mass spectrometry (UPLC/MS/MS), we revealed that ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a target for S-mercuration in human neuroblastoma SH-SY5Y cells exposed to MeHg (1 µM, 9 hr). The modification site of UCH-L1 by MeHg was Cys152, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. MeHg was shown to inhibit the catalytic activity of recombinant human UCH-L1 in a concentration-dependent manner. Knockdown of UCH-L1 indicated that this enzyme plays a critical role in regulating mono-ubiquitin (monoUb) levels in SH-SY5Y cells and exposure of SH-SY5Y cells to MeHg caused a reduction in the level of monoUb in these cells. These observations suggest that UCH-L1 readily undergoes S-mercuration by MeHg through Cys152 and this covalent modification inhibits UCH-L1, leading to the potential disruption of the maintenance of cellular monoUb levels.

  19. Contribution of hydrolase and phosphatase domains in soluble epoxide hydrolase to vascular endothelial growth factor expression and cell growth.

    PubMed

    Oguro, Ami; Sakamoto, Koichi; Suzuki, Sachiko; Imaoka, Susumu

    2009-12-01

    Soluble epoxide hydrolase (sEH) is an important pharmacological target because it metabolizes potent bioactive substrates, epoxyeicosatrienoinc acids (EETs) and other lipid epoxide. EETs have a variety of biological functions including angiogenesis and cancer metastasis. However, the regulation and physiological function of sEH is not well understood. In this study, we found that hypoxia significantly suppressed the expression of sEH in mouse liver and a human hepatoma cell line, Hep3B. Hypoxia promotes the proliferation of vascular endothelial cells or carcinoma cells. Knockdown of sEH in Hep3B cells induced vascular endothelial growth factor (VEGF) mRNA and cell growth, both of which were suppressed by overexpression of sEH. sEH has phosphatase activity as well as epoxide hydrolase (EH) activity. We prepared mutant clones which lacking EH or phosphatase activity using the amino acid change Asp335Ser or Asp9Ala, respectively. The effects of WT sEH on cell growth were lost by mutation of either the EH domain or phosphatase domain. However, mutation of the phosphatase domain but not EH domain did not influence the expression of VEGF. These results suggest that sEH plays an important role in the physiology of cells including proliferation and that the epoxide hydrolase and phosphatase domains of sEH have different biological functions.

  20. A Phylogenetically Informed Comparison of GH1 Hydrolases between Arabidopsis and Rice Response to Stressors

    PubMed Central

    Cao, Yun-Ying; Yang, Jing-Fang; Liu, Tie-Yuan; Su, Zhen-Feng; Zhu, Fu-Yuan; Chen, Mo-Xian; Fan, Tao; Ye, Neng-Hui; Feng, Zhen; Wang, Ling-Juan; Hao, Ge-Fei; Zhang, Jianhua; Liu, Ying-Gao

    2017-01-01

    Glycoside hydrolases Family 1 (GH1) comprises enzymes that can hydrolyze β-O-glycosidic bond from a carbohydrate moiety. The plant GH1 hydrolases participate in a number of developmental processes and stress responses, including cell wall modification, plant hormone activation or deactivation and herbivore resistance. A large number of members has been observed in this family, suggesting their potential redundant functions in various biological processes. In this study, we have used 304 sequences of plant GH1 hydrolases to study the evolution of this gene family in plant lineage. Gene duplication was found to be a common phenomenon in this gene family. Although many members of GH1 hydrolases showed a high degree of similarity in Arabidopsis and rice, they showed substantial tissue specificity and differential responses to various stress treatments. This differential regulation implies each enzyme may play a distinct role in plants. Furthermore, some of salt-responsive Arabidopsis GH1 hydrolases were selected to test their genetic involvement in salt responses. The knockout mutants of AtBGLU1 and AtBGLU19 were observed to be less-sensitive during NaCl treatment in comparison to the wild type seedlings, indicating their participation in salt stress response. In summary, Arabidopsis and rice GH1 glycoside hydrolases showed distinct features in their evolutionary path, transcriptional regulation and genetic functions. PMID:28392792

  1. Purification and characterization of three parathion hydrolases from gram-negative bacterial strains.

    PubMed

    Mulbry, W W; Karns, J S

    1989-02-01

    Three unique parathion hydrolases were purified from gram-negative bacterial isolates and characterized. All three purified enzymes had roughly comparable affinities for ethyl parathion and had broad temperature optima at ca. 40 degrees C. The membrane-bound hydrolase of Flavobacterium sp. strain ATCC 27551 was composed of a single subunit of approximately 35,000 daltons (Da) and was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as CuCl2. The cytosolic hydrolase of strain B-1 was composed of a single subunit of approximately 43,000 Da and was stimulated by DTT and inhibited by CuCl2. The membrane-bound hydrolase of strain SC was composed of four identical subunits of 67,000 Da and was inhibited by DTT and stimulated by CuCl2. The substrate ranges of the three enzymes also differed, as evidenced by their relative affinities for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothioate (EPN). The B-1 hydrolase displayed equal affinity for both compounds, the Flavobacterium enzyme showed twofold-lower affinity for EPN than for parathion, and the SC hydrolase displayed no activity toward EPN. The range in characteristics of these three enzymes can be exploited in different waste disposal strategies.

  2. Purification and characterization of three parathion hydrolases from gram-negative bacterial strains.

    PubMed Central

    Mulbry, W W; Karns, J S

    1989-01-01

    Three unique parathion hydrolases were purified from gram-negative bacterial isolates and characterized. All three purified enzymes had roughly comparable affinities for ethyl parathion and had broad temperature optima at ca. 40 degrees C. The membrane-bound hydrolase of Flavobacterium sp. strain ATCC 27551 was composed of a single subunit of approximately 35,000 daltons (Da) and was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as CuCl2. The cytosolic hydrolase of strain B-1 was composed of a single subunit of approximately 43,000 Da and was stimulated by DTT and inhibited by CuCl2. The membrane-bound hydrolase of strain SC was composed of four identical subunits of 67,000 Da and was inhibited by DTT and stimulated by CuCl2. The substrate ranges of the three enzymes also differed, as evidenced by their relative affinities for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothioate (EPN). The B-1 hydrolase displayed equal affinity for both compounds, the Flavobacterium enzyme showed twofold-lower affinity for EPN than for parathion, and the SC hydrolase displayed no activity toward EPN. The range in characteristics of these three enzymes can be exploited in different waste disposal strategies. Images PMID:2541658

  3. Reaction Pathway for Cocaine Hydrolase-Catalyzed Hydrolysis of (+)-Cocaine

    PubMed Central

    Yao, Yuan; Liu, Junjun; Zheng, Fang; Zhan, Chang-Guo

    2017-01-01

    A recently designed and discovered cocaine hydrolase (CocH), engineered from human butyrylcholinesterase (BChE), has been proven promising as a novel enzyme therapy for treatment of cocaine overdose and addiction because it is highly efficient in catalyzing hydrolysis of naturally occurring (−)-cocaine. It has been known that the CocH also has a high catalytic efficiency against (+)-cocaine, a synthetic enantiomer of cocaine. Reaction pathway and the corresponding free energy profile for the CocH-catalyzed hydrolysis of (+)-cocaine have been determined, in the present study, by performing first-principles pseudobond quantum mechanical/molecular mechanical (QM/MM)-free energy (FE) calculations. Acordingt to the QM/MM-FE results, the catalytic hydrolysis process is initiated by the nucleophilic attack on carbonyl carbon of (−)-cocaine benzoyl ester via hydroxyl oxygen of S198 side chain, and the second reaction step (i.e. dissociation of benzoyl ester) is rate-determining. This finding for CocH-catalyzed hydrolysis of (+)-cocaine is remarkably different from that for the (+)-cocaine hydrolysis catalyzed by bacterial cocaine esterase in which the first reaction step of the deacylation is associated with the highest free energy barrier (~17.9 kcal/mol). The overall free energy barrier (~16.0 kcal/mol) calculated for the acylation stage of CocH-catalyzed hydrolysis of (+)-cocaine is in good agreement with the experimental free energy barrier of ~14.5 kcal/mol derivated from the experimental kinetic data.

  4. ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT.

    PubMed

    Chao, Hsu; Zhou, Minglong; McIntosh, Avery; Schroeder, Friedhelm; Kier, Ann B

    2003-01-01

    Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.

  5. The Role of Mitochondrial Non-Enzymatic Protein Acylation in Ageing

    PubMed Central

    Hong, Shin Yee; Ng, Li Theng; Ng, Li Fang; Inoue, Takao; Tolwinski, Nicholas S.; Hagen, Thilo; Gruber, Jan

    2016-01-01

    In recent years, various large-scale proteomic studies have demonstrated that mitochondrial proteins are highly acylated, most commonly by addition of acetyl and succinyl groups. These acyl modifications may be enzyme catalysed but can also be driven non-enzymatically. The latter mechanism is promoted in mitochondria due to the nature of the mitochondrial microenvironment, which is alkaline and contains high concentrations of acyl-CoA species. Protein acylation may modify enzyme activity, typically inhibiting it. We posited that organismal ageing might be accompanied by an accumulation of acylated proteins, especially in mitochondria, and that this might compromise mitochondrial function and contribute to ageing. In this study, we used R. norvegicus, C. elegans and D. melanogaster to compare the acylation status of mitochondrial proteins between young and old animals. We observed a specific age-dependent increase in protein succinylation in worms and flies but not in rat. Rats have two substrate-specific mitochondrial deacylases, SIRT3 and SIRT5 while both flies and worms lack these enzymes. We propose that accumulation of mitochondrial protein acylation contributes to age-dependent mitochondrial functional decline and that SIRT3 and SIRT5 enzymes may promote longevity through regulation of mitochondrial protein acylation during ageing. PMID:28033361

  6. Multi-organ abnormalities and mTORC1 activation in zebrafish model of multiple acyl-CoA dehydrogenase deficiency.

    PubMed

    Kim, Seok-Hyung; Scott, Sarah A; Bennett, Michael J; Carson, Robert P; Fessel, Joshua; Brown, H Alex; Ess, Kevin C

    2013-06-01

    Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxa(vu463) ) that has an inactivating mutation in the etfa gene. dxa(vu463) recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxa(vu463) zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxa(vu463) zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.

  7. Prediction of drug-drug interactions with carbamazepine-10,11-epoxide using a new in vitro assay for epoxide hydrolase inhibition.

    PubMed

    Rosa, Maria; Bonnaillie, Pierre; Chanteux, Hugues

    2016-12-01

    1. Carbamazepine is an antiepileptic drug which is metabolized by CYP3A4 into carbamazepine-10,11-epoxide. This metabolite is then detoxified by epoxide hydrolase. As carbamazepine-10,11-epoxide has been associated with neurotoxicity, it is critical to identify whether a new antiepileptic drug has the potential to inhibit epoxide hydrolase and therefore increase carbamazepine-10,11-epoxide plasma levels. 2. In this study, an in vitro assay was developed to evaluate epoxide hydrolase activity by using carbamazepine-10,11-epoxide as probe substrate. The ability of this assay to predict drug-drug interactions (DDI) at the epoxide hydrolase level was also investigated. 3. To this aim, known inhibitors of epoxide hydrolase for which in vivo data are available were used. Firstly, carbamazepine-10,11-epoxide hydrolase activity was determined in liver microsomes, cytosol and hepatocytes. Thereafter, the IC50 of epoxide hydrolase inhibitors (progabide, valproic acid, valpromide and valnoctamide) was determined in liver microsomes and hepatocytes. Finally, prediction of AUC increase was performed using the in vitro data generated. 4. Interestingly, epoxide hydrolase activity was found to be much higher in human hepatocytes compared to liver microsomes/cytosol. Even though assessed on a limited number of compounds, this study demonstrated that the use of hepatocytes seems to be a more relevant model to assess and predict DDI at the epoxide hydrolase level.

  8. Discovery of Triterpenoids as Reversible Inhibitors of α/β-hydrolase Domain Containing 12 (ABHD12)

    PubMed Central

    Parkkari, Teija; Haavikko, Raisa; Laitinen, Tuomo; Navia-Paldanius, Dina; Rytilahti, Roosa; Vaara, Miia; Lehtonen, Marko; Alakurtti, Sami; Yli-Kauhaluoma, Jari; Nevalainen, Tapio; Savinainen, Juha R.; Laitinen, Jarmo T.

    2014-01-01

    Background α/β-hydrolase domain containing (ABHD)12 is a recently discovered serine hydrolase that acts in vivo as a lysophospholipase for lysophosphatidylserine. Dysfunctional ABHD12 has been linked to the rare neurodegenerative disorder called PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, cataract). In vitro, ABHD12 has been implicated in the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG). Further studies on ABHD12 function are hampered as no selective inhibitor have been identified to date. In contrast to the situation with the other endocannabinoid hydrolases, ABHD12 has remained a challenging target for inhibitor development as no crystal structures are available to facilitate drug design. Methodology/Principal Findings Here we report the unexpected discovery that certain triterpene-based structures inhibit human ABHD12 hydrolase activity in a reversible manner, the best compounds showing submicromolar potency. Based on structure activity relationship (SAR) data collected for 68 natural and synthetic triterpenoid structures, a pharmacophore model has been constructed. A pentacyclic triterpene backbone with carboxyl group at position 17, small hydrophobic substituent at the position 4, hydrogen bond donor or acceptor at position 3 accompanied with four axial methyl substituents was found crucial for ABHD12 inhibitor activity. Although the triterpenoids typically may have multiple protein targets, we witnessed unprecedented selectivity for ABHD12 among the metabolic serine hydrolases, as activity-based protein profiling of mouse brain membrane proteome indicated that the representative ABHD12 inhibitors did not inhibit other serine hydrolases, nor did they target cannabinoid receptors. Conclusions/Significance We have identified reversibly-acting triterpene-based inhibitors that show remarkable selectivity for ABHD12 over other metabolic serine hydrolases. Based on SAR data, we have constructed the first pharmacophore

  9. Co-purification of microsomal epoxide hydrolase with the warfarin-sensitive vitamin K1 oxide reductase of the vitamin K cycle.

    PubMed

    Guenthner, T M; Cai, D; Wallin, R

    1998-01-15

    Vitamin K1 oxide reductase activity has been partially purified from rat liver microsomes. A three-step procedure produced a preparation in which warfarin-sensitive vitamin K1 oxide reductase activity was 118-fold enriched over the activity in intact rat liver microsomes. A major component of the multi-protein mixture was identified as a 50 kDa protein that strongly cross-reacts with antiserum prepared against homogeneous rat liver microsomal epoxide hydrolase. The reductase preparation also had a high level or epoxide hydrolase activity against two xenobiotic epoxide substrates. The K(m) values for hydrolysis by the reductase preparation were similar to those for homogeneous microsomal epoxide hydrolase itself, and the specific hydrolase activities of the reductase preparation were 25-35% of the specific activities measured for the homogeneous hydrolase preparation. Antibodies prepared against homogeneous microsomal epoxide hydrolase inhibited up to 80% of reductase activity of the reductase preparation. Homogeneous microsomal epoxide hydrolase had no vitamin K1 oxide reductase activity. This evidence suggests that microsomal epoxide hydrolase, or a protein that is very similar to it, is a major functional component of a multi-protein complex that is responsible for vitamin K1 oxide reduction in rat liver microsomes.

  10. Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria

    PubMed Central

    Garland, P. B.; Yates, D. W.; Haddock, B. A.

    1970-01-01

    1. Deca-2,4,6,8-tetraenoic acid is a substrate for both ATP-specific (EC 6.2.1.2 or 3) and GTP-specific (EC 6.2.1.–) acyl-CoA synthetases of rat liver mitochondria. The enzymic synthesis of decatetraenoyl-CoA results in new spectral characteristics. The difference spectrum for the acyl-CoA minus free acid has a maximum at 376nm with εmM 34. Isosbestic points are at 345nm and 440nm. 2. The acylation of CoA by decatetraenoate in mitochondrial suspensions can be continuously measured with a dual-wavelength spectrophotometer. 3. By using this technique, three distinct types of acyl-CoA synthetase activity were demonstrated in rat liver mitochondria. One of these utilized added CoA and ATP, required added Mg2+ and corresponded to a previously described `external' acyl-CoA synthetase. The other two acyl-CoA synthetase activities utilized intramitochondrial CoA and did not require added Mg2+. Of these two `internal' acyl-CoA synthetases, one was insensitive to uncoupling agents, was inhibited by phosphate or arsenate, and corresponded to the GTP-specific enzyme. The other corresponded to the ATP-specific enzyme. 4. Atractylate inhibited the activity of the two internal acyl-CoA synthetases only when the energy source was added ATP. 5. The amount of intramitochondrial CoA acylated by decatetraenoate was independent of whether the internal ATP-specific or GTP-specific acyl-CoA synthetase was active. It is concluded that these two internal acyl-CoA synthetases have access to the same intramitochondrial pool of CoA. 6. The amount of intramitochondrial CoA that could be acylated with decatetraenoate was decreased by the addition of palmitoyl-dl-carnitine, 2-oxoglutarate, or pyruvate. These observations indicated that pyruvate dehydrogenase (EC 1.2.4.1), oxoglutarate dehydrogenase (EC 1.2.4.2), carnitine palmitoyltransferase (EC 2.3.1.–), citrate synthase (EC 4.1.3.7), and succinyl-CoA synthetase (EC 6.2.1.4) all have access to the same intramitochondrial pool of CoA as do

  11. Structural Relationship of the Lipid A Acyl Groups to Activation of Murine Toll-Like Receptor 4 by Lipopolysaccharides from Pathogenic Strains of Burkholderia mallei, Acinetobacter baumannii, and Pseudomonas aeruginosa

    PubMed Central

    Korneev, Kirill V.; Arbatsky, Nikolay P.; Molinaro, Antonio; Palmigiano, Angelo; Shaikhutdinova, Rima Z.; Shneider, Mikhail M.; Pier, Gerald B.; Kondakova, Anna N.; Sviriaeva, Ekaterina N.; Sturiale, Luisa; Garozzo, Domenico; Kruglov, Andrey A.; Nedospasov, Sergei A.; Drutskaya, Marina S.; Knirel, Yuriy A.; Kuprash, Dmitry V.

    2015-01-01

    Toll-like receptor 4 (TLR4) is required for activation of innate immunity upon recognition of lipopolysaccharide (LPS) of Gram-negative bacteria. The ability of TLR4 to respond to a particular LPS species is important since insufficient activation may not prevent bacterial growth while excessive immune reaction may lead to immunopathology associated with sepsis. Here, we investigated the biological activity of LPS from Burkholderia mallei that causes glanders, and from the two well-known opportunistic pathogens Acinetobacter baumannii and Pseudomonas aeruginosa (causative agents of nosocomial infections). For each bacterial strain, R-form LPS preparations were purified by hydrophobic chromatography and the chemical structure of lipid A, an LPS structural component, was elucidated by HR-MALDI-TOF mass spectrometry. The biological activity of LPS samples was evaluated by their ability to induce production of proinflammatory cytokines, such as IL-6 and TNF, by bone marrow-derived macrophages. Our results demonstrate direct correlation between the biological activity of LPS from these pathogenic bacteria and the extent of their lipid A acylation. PMID:26635809

  12. A high-throughput screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases.

    PubMed

    Christensen, Quin H; Grove, Tyler L; Booker, Squire J; Greenberg, E Peter

    2013-08-20

    Many Proteobacteria use N-acyl-homoserine lactone (acyl-HSL) quorum sensing to control specific genes. Acyl-HSL synthesis requires unique enzymes that use S-adenosyl methionine as an acyl acceptor and amino acid donor. We developed and executed an enzyme-coupled high-throughput cell-free screen to discover acyl-HSL synthase inhibitors. The three strongest inhibitors were equally active against two different acyl-HSL synthases: Burkholderia mallei BmaI1 and Yersinia pestis YspI. Two of these inhibitors showed activity in whole cells. The most potent compound behaves as a noncompetitive inhibitor with a Ki of 0.7 µM and showed activity in a cell-based assay. Quorum-sensing signal synthesis inhibitors will be useful in attempts to understand acyl-HSL synthase catalysis and as a tool in studies of quorum-sensing control of gene expression. Because acyl-HSL quorum-sensing controls virulence of some bacterial pathogens, anti-quorum-sensing chemicals have been sought as potential therapeutic agents. Our screen and identification of acyl-HSL synthase inhibitors serve as a basis for efforts to target quorum-sensing signal synthesis as an antivirulence approach.

  13. Preparation and characterization of O-acylated fucosylated chondroitin sulfate from sea cucumber.

    PubMed

    Gao, Na; Wu, Mingyi; Liu, Shao; Lian, Wu; Li, Zi; Zhao, Jinhua

    2012-08-01

    Fucosylated chondroitin sulfate (FuCS), a kind of complex glycosaminoglycan from sea cucumber, has potent anticoagulant activity. In order to understand the relationship between structures and activity, the depolymerized FuCS (dFuCS) was chosen to prepare its derivates by selective substitution at OH groups. Its O-acylation was carried out in a homogeneous way using carboxylic acid anhydrides. The structures of O-acylated derivatives were characterized by NMR. The results indicated that the 4-O-sulfated fucose residues may be easier to be acylated than the other ones in the sulfated fucose branches. But the O-acylation was always accompanied by the β-elimination, and the degree of elimination was higher as that of acylation was higher. The results of clotting assay indicated that the effect of partial O-acylation of the dFuCS on their anticoagulant potency was not significant and the O-acylation of 2-OH groups of 4-O-sulfated fucose units did not affect the anticoagulant activity.

  14. The Physiology of Protein S-acylation

    PubMed Central

    Chamberlain, Luke H.; Shipston, Michael J.

    2015-01-01

    Protein S-acylation, the only fully reversible posttranslational lipid modification of proteins, is emerging as a ubiquitous mechanism to control the properties and function of a diverse array of proteins and consequently physiological processes. S-acylation results from the enzymatic addition of long-chain lipids, most typically palmitate, onto intracellular cysteine residues of soluble and transmembrane proteins via a labile thioester linkage. Addition of lipid results in increases in protein hydrophobicity that can impact on protein structure, assembly, maturation, trafficking, and function. The recent explosion in global S-acylation (palmitoyl) proteomic profiling as a result of improved biochemical tools to assay S-acylation, in conjunction with the recent identification of enzymes that control protein S-acylation and de-acylation, has opened a new vista into the physiological function of S-acylation. This review introduces key features of S-acylation and tools to interrogate this process, and highlights the eclectic array of proteins regulated including membrane receptors, ion channels and transporters, enzymes and kinases, signaling adapters and chaperones, cell adhesion, and structural proteins. We highlight recent findings correlating disruption of S-acylation to pathophysiology and disease and discuss some of the major challenges and opportunities in this rapidly expanding field. PMID:25834228

  15. Des-acyl ghrelin prevents heatstroke-like symptoms in rats exposed to high temperature and high humidity.

    PubMed

    Inoue, Yoshiyuki; Hayashi, Yujiro; Kangawa, Kenji; Suzuki, Yoshihiro; Murakami, Noboru; Nakahara, Keiko

    2016-02-26

    We have shown previously that des-acyl ghrelin decreases body temperature in rats through activation of the parasympathetic nervous system. Here we investigated whether des-acyl ghrelin ameliorates heatstroke in rats exposed to high temperature. Peripheral administration of des-acyl ghrelin significantly attenuated hyperthermia induced by exposure to high-temperature (35°C) together with high humidity (70-80%). Although biochemical analysis revealed that exposure to high temperature significantly increased hematocrit and the serum levels of aspartate amino transferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine and electrolytes (Na(+), K(+), Cl(-)), most of these heatstroke-associated reactions were significantly reduced by treatment with des-acyl ghrelin. The level of des-acyl ghrelin in plasma was also found to be significantly increased under high-temperature conditions. These results suggest that des-acyl ghrelin could be useful for preventing heatstroke under high temperature condition.

  16. Classification of the adenylation and acyl-transferase activity of NRPS and PKS systems using ensembles of substrate specific hidden Markov models.

    PubMed

    Khayatt, Barzan I; Overmars, Lex; Siezen, Roland J; Francke, Christof

    2013-01-01

    There is a growing interest in the Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) of microbes, fungi and plants because they can produce bioactive peptides such as antibiotics. The ability to identify the substrate specificity of the enzyme's adenylation (A) and acyl-transferase (AT) domains is essential to rationally deduce or engineer new products. We here report on a Hidden Markov Model (HMM)-based ensemble method to predict the substrate specificity at high quality. We collected a new reference set of experimentally validated sequences. An initial classification based on alignment and Neighbor Joining was performed in line with most of the previously published prediction methods. We then created and tested single substrate specific HMMs and found that their use improved the correct identification significantly for A as well as for AT domains. A major advantage of the use of HMMs is that it abolishes the dependency on multiple sequence alignment and residue selection that is hampering the alignment-based clustering methods. Using our models we obtained a high prediction quality for the substrate specificity of the A domains similar to two recently published tools that make use of HMMs or Support Vector Machines (NRPSsp and NRPS predictor2, respectively). Moreover, replacement of the single substrate specific HMMs by ensembles of models caused a clear increase in prediction quality. We argue that the superiority of the ensemble over the single model is caused by the way substrate specificity evolves for the studied systems. It is likely that this also holds true for other protein domains. The ensemble predictor has been implemented in a simple web-based tool that is available at http://www.cmbi.ru.nl/NRPS-PKS-substrate-predictor/.

  17. Investigation of the mechanism of phosphonoacetaldehyde hydrolase

    SciTech Connect

    Hepburn, T.W.; Olsen, D.B.; Dunaway-Mariano, D.; Mariano, P.S.

    1986-05-01

    The authors are presently studying enzymes which catalyze the formation and cleavage of carbon phosphorous bonds. In 1970 LaNauze et. al. reported the isolation of one enzyme of interest - phosphonoacetaldehyde hydrolase from a mutant of Bacillus cereus. This enzyme catalyzes the hydrolysis of phosphonoaldehyde to acetaldehyde and inorganic phosphate. They have isolated phosphonatase from wild type B. cereus (grown on 2-aminoethylphosphonate as the P/sub i/ source) and have used /sup 1/H-NMR and /sup 31/P-NMR techniques to determine the products of the enzyme reaction as phosphate and acetaldehyde. The mechanism of the enzyme could involve the formation of a Schiff base between phosphonoacetaldehyde and lysine or it might only require Mg/sup + +/, an essential cofactor for activity. To distinguish between these possibilities they have begun to look at the Schiff base formation in more detail. NaBH/sub 4/ was found to inactivate the enzyme in the presence of substrate but not in its absence. This is consistent with results obtained for the enzyme isolated from the mutant bacteria. In addition treatment of the wild type enzyme with tritiated NaBH/sub 4/ resulted in significant incorporation of radiolabel into the protein as compared to the control. These results tentatively suggest that hydrolysis proceeds via a covalent imine intermediate.

  18. Acyl-CoA Synthetase Is Located in the Outer Membrane and Acyl-CoA Thioesterase in the Inner Membrane of Pea Chloroplast Envelopes 1

    PubMed Central

    Andrews, Jaen; Keegstra, Kenneth

    1983-01-01

    Both acyl-CoA synthetase and acyl-CoA thioesterase activities are present in chloroplast envelope membranes. The functions of these enzymes in lipid metabolism remains unresolved, although the synthetase has been proposed to be involved in either plastid galactolipid synthesis or the export of plastid-synthesized fatty acids to the cytoplasm. We have examined the locations of both enzymes within the two envelope membranes of pea (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Inner and outer envelope membranes were purified from unfractionated envelope preparations by linear density sucrose gradient centrifugation. Acyl-CoA synthetase was located in the outer envelope membrane while acyl-CoA thioesterase was located in the inner envelope membrane. Thus, it seems unlikely that the synthetase is directly involved in galactolipid assembly. Instead, its localization supports the hypothesis that it functions in the transport of plastid-synthesized fatty acids to the endoplasmic reticulum. PMID:16663076

  19. Rates of thrombin acylation and deacylation upon reaction with low molecular weight acylating agents, carbamylating agents and carbonylating agents.

    PubMed

    Brown, A D; Powers, J C

    1995-08-01

    Acylated derivatives of thrombin have been made using low molecular weight acylating agents, carbamylating agents and carbonylating agents. The compounds used to acylate the active site serine include isatoic anhydrides, benzoxazinones, benzylisocyanate, N-(benzylcarbonyloxy)succinimide and p-(dimethylamino)benzoylimidazolide. The rates of acylation and deacylation were determined. The best overall inhibitors of thrombin are 2-ethoxy-4H-3,1-benzoxazin-4-one, isatoic anhydride and tert-butyl-2,4-dioxo-2H-3,1-benzoxazine-1(4H)-acetate, which have k2/Ki values of 52,700 M-1s-1, 48,900 M-1s-1 and 5400 M-1s-1, respectively. The carbamyl derivative of thrombin formed with benzylisocyanate had the slowest rate of deacylation (2.3 x 10(-7) s-1), while the ester derivative formed with 2-(N,N-dimethylamino)methylimino-4H-3,1-benzoxazin-4-one had the fastest rate of deacylation (1.9 x 10(-4) s-1).

  20. Effect of Dimer Dissociation on Activity and Thermostability of the α-Glucuronidase from Geobacillus stearothermophilus: Dissecting the Different Oligomeric Forms of Family 67 Glycoside Hydrolases

    PubMed Central

    Shallom, Dalia; Golan, Gali; Shoham, Gil; Shoham, Yuval

    2004-01-01

    The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. α-Glucuronidases are family 67 glycosidases that cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of α-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the α-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial α-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in α-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35°C, compared to 65°C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9°C, was almost identical to that of the wild-type, 73.4°C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region

  1. Effect of dimer dissociation on activity and thermostability of the alpha-glucuronidase from Geobacillus stearothermophilus: dissecting the different oligomeric forms of family 67 glycoside hydrolases.

    PubMed

    Shallom, Dalia; Golan, Gali; Shoham, Gil; Shoham, Yuval

    2004-10-01

    The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. alpha-Glucuronidases are family 67 glycosidases that cleave the alpha-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of alpha-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the alpha-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial alpha-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in alpha-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35 degrees C, compared to 65 degrees C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9 degrees C, was almost identical to that of the wild-type, 73.4 degrees C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure

  2. The Salmonella Effector SpvD Is a Cysteine Hydrolase with a Serovar-specific Polymorphism Influencing Catalytic Activity, Suppression of Immune Responses, and Bacterial Virulence*

    PubMed Central

    Grabe, Grzegorz J.; Zhang, Yue; Przydacz, Michal; Rolhion, Nathalie; Yang, Yi; Pruneda, Jonathan N.; Komander, David; Holden, David W.; Hare, Stephen A.

    2016-01-01

    Many bacterial pathogens secrete virulence (effector) proteins that interfere with immune signaling in their host. SpvD is a Salmonella enterica effector protein that we previously demonstrated to negatively regulate the NF-κB signaling pathway and promote virulence of S. enterica serovar Typhimurium in mice. To shed light on the mechanistic basis for these observations, we determined the crystal structure of SpvD and show that it adopts a papain-like fold with a characteristic cysteine-histidine-aspartate catalytic triad comprising Cys-73, His-162, and Asp-182. SpvD possessed an in vitro deconjugative activity on aminoluciferin-linked peptide and protein substrates in vitro. A C73A mutation abolished SpvD activity, demonstrating that an intact catalytic triad is required for its function. Taken together, these results strongly suggest that SpvD is a cysteine protease. The amino acid sequence of SpvD is highly conserved across different S. enterica serovars, but residue 161, located close to the catalytic triad, is variable, with serovar Typhimurium SpvD having an arginine and serovar Enteritidis a glycine at this position. This variation affected hydrolytic activity of the enzyme on artificial substrates and can be explained by substrate accessibility to the active site. Interestingly, the SpvDG161 variant more potently inhibited NF-κB-mediated immune responses in cells in vitro and increased virulence of serovar Typhimurium in mice. In summary, our results explain the biochemical basis for the effect of virulence protein SpvD and demonstrate that a single amino acid polymorphism can affect the overall virulence of a bacterial pathogen in its host. PMID:27789710

  3. Effects of farmyard manure and nitrogen fertilizers on mobility of phosphorus and sulphur in wheat and activity of selected hydrolases in soil

    NASA Astrophysics Data System (ADS)

    Lemanowicz, Joanna; Siwik-Ziomek, Anetta; Koper, Jan

    2014-03-01

    The paper demonstrates the results of research on the mobility of phosphorus and sulphur in winter wheat fertilized with several rates (0, 20, 40, 60, 80 t ha-1) of farmyard manure and nitrogen (0, 40, 80, 120 kg N ha-1). The content of these nutrients was related to the activity of acid phosphatase and arylsulphatase in a Haplic Luvisol. The highest content of available phosphorus (91.58 mg P kg-1) was reported in the soil amended with farmyard manure at the rate of 60 t ha-1. The content of sulphates (VI) in the Haplic Luvisol was high and, on average, equal to 25.22 mg kg-1. The activity of acid phosphatase in the soil increased with increasing mineral nitrogen rates. The highest content of sulphates (VI) and the lowest activity of arylsulphatase were identified at the nitrogen rate of 40 kg N ha-1. The mobility of phosphorus in winter wheat was the highest when farmyard manure at the rate of 60 t ha-1 and mineral nitrogen at the rate of 120 kg N ha-1 were incorporated into the soil. The greatest translocation of sulphur was reported at the high farmyard manure rates (40, 60 and 80 t ha-1) and the nitrogen rate of 80 kg N ha-1.

  4. The Glycoside Hydrolase Family 8 Reducing-End Xylose-Releasing Exo-oligoxylanase Rex8A from Paenibacillus barcinonensis BP-23 Is Active on Branched Xylooligosaccharides

    PubMed Central

    Lopez, Sergi; Biely, Peter; Sanz-Aparicio, Julia; Pastor, F. I. Javier

    2016-01-01

    ABSTRACT A GH8 family enzyme involved in xylan depolymerization has been characterized. The enzyme, Rex8A, is a reducing-end xylose-releasing exo-oligoxylanase (Rex) that efficiently hydrolyzes xylooligosaccharides and shows minor activity on polymeric xylan. Rex8A hydrolyzes xylooligomers of 3 to 6 xylose units to xylose and xylobiose in long-term incubations. Kinetic constants of Rex8A were determined on xylotriose, showing a Km of 1.64 ± 0.03 mM and a kcat value of 118.8 s−1. Besides linear xylooligosaccharides, the enzyme hydrolyzed decorated xylooligomers. The catalytic activity on branched xylooligosaccharides, i.e., the release of xylose from the reducing end, is a newly described trait of xylose-releasing exo-oligoxylanases, as the exo-activity on these substrates has not been reported for the few of these enzymes characterized to date. Modeling of the three-dimensional (3D) structure of Rex8A shows an (α/α)6 barrel fold where the loops connecting the α-helices contour the active site. These loops, which show high sequence diversity among GH8 enzymes, shape a catalytic cleft with a −2 subsite that can accommodate methyl-glucuronic acid decorations. The hydrolytic ability of Rex8A on branched oligomers can be crucial for the complete depolymerization of highly substituted xylans, which is indispensable to accomplish biomass deconstruction and to generate efficient catalysts. IMPORTANCE A GH8 family enzyme involved in xylan depolymerization has been characterized. The Rex8A enzyme from Paenibacillus barcinonensis is involved in depolymerization of glucuronoxylan, a major component of the lignocellulosic substrates. The study shows that Rex8A is a reducing-end xylose-releasing exo-oligoxylanase that efficiently hydrolyzes xylose from neutral and acidic xylooligosaccharides generated by the action of other xylanases also secreted by the strain. The activity of a Rex enzyme on branched xylooligosaccharides has not been described to date. This report

  5. A New Insight into the Physiological Role of Bile Salt Hydrolase among Intestinal Bacteria from the Genus Bifidobacterium

    PubMed Central

    Jarocki, Piotr; Podleśny, Marcin; Glibowski, Paweł; Targoński, Zdzisław

    2014-01-01

    This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche. PMID:25470405

  6. The TIP GROWTH DEFECTIVE1 S-acyl transferase regulates plant cell growth in Arabidopsis.

    PubMed

    Hemsley, Piers A; Kemp, Alison C; Grierson, Claire S

    2005-09-01

    TIP GROWTH DEFECTIVE1 (TIP1) of Arabidopsis thaliana affects cell growth throughout the plant and has a particularly strong effect on root hair growth. We have identified TIP1 by map-based cloning and complementation of the mutant phenotype. TIP1 encodes an ankyrin repeat protein with a DHHC Cys-rich domain that is expressed in roots, leaves, inflorescence stems, and floral tissue. Two homologues of TIP1 in yeast (Saccharomyces cerevisiae) and human (Homo sapiens) have been shown to have S-acyl transferase (also known as palmitoyl transferase) activity. S-acylation is a reversible hydrophobic protein modification that offers swift, flexible control of protein hydrophobicity and affects protein association with membranes, signal transduction, and vesicle trafficking within cells. We show that TIP1 binds the acyl group palmitate, that it can rescue the morphological, temperature sensitivity, and yeast casein kinase2 localization defects of the yeast S-acyl transferase mutant akr1Delta, and that inhibition of acylation in wild-type Arabidopsis roots reproduces the Tip1- mutant phenotype. Our results demonstrate that S-acylation is essential for normal plant cell growth and identify a plant S-acyl transferase, an essential research tool if we are to understand how this important, reversible lipid modification operates in plant cells.

  7. N-Acylation During Glidobactin Biosynthesis by the Tridomain Nonribosomal Peptide Synthetase Module GlbF

    PubMed Central

    Imker, Heidi J.; Krahn, Daniel; Clerc, Jérôme; Kaiser, Markus; Walsh, Christopher T.

    2011-01-01

    Summary Glidobactins are hybrid NRPS-PKS natural products that function as irreversible proteasome inhibitors. A variety of medium chain 2(E),4(E)-diene fatty acids N-acylate the peptidolactam core and contribute significantly to the potency of proteasome inhibition. We have expressed the initiation NRPS module GlbF (C-A-T) in Escherichia coli and observe soluble active protein only on co-expression with the 8 kDa MbtH-like protein, GlbE. Following adenylation and installation of Thr as a T-domain thioester, the starter condensation domain utilizes fatty acyl-CoA donors to acylate the Thr1 amino group and generate the fatty acyl-Thr1-S-pantetheinyl-GlbF intermediate to be used in subsequent chain elongation. Previously proposed to be mediated via acyl carrier protein fatty acid donors, direct utilization of fatty acyl-CoA donors for N-acylation of T-domain tethered amino acids is likely a common strategy for chain initiation in NRPS-mediated lipopeptide biosynthesis. PMID:21035730

  8. Synthesis of acyl arbutin by an immobilized lipase and its suppressive ability against lipid oxidation in a bulk system and O/W emulsion.

    PubMed

    Nagai, Mizuka; Watanabe, Yoshiyuki; Nomura, Masato

    2009-11-01

    Acyl arbutin was synthesized through the condensation of arbutin with a saturated fatty acid (C6-18) by the immobilized lipase in a batch reaction. The conversion at 10 and 20 g/l-solvent of immobilized lipase reached 45% over 2 d, but the initial reaction rate per amount of immobilized lipase decreased at 20 g/l-solvent. The radical scavenging activity of acyl arbutin in an ethanol solution was independent of the acyl chain length, although the rate constant, k, estimated for the oxidation of methyl linoleate in a bulk system with acyl arbutin by using the Weibull equation, decreased as the acyl chain length increased. This indicates the antioxidative ability of acyl arbutin with a long acyl chain to be due to its lipophilicity. Furthermore, it is suggested that dodecanoyl arbutin mainly acted on the interface between the oil and water phases in an O/W emulsion, and effectively suppressed the oxidation induced at the interface.

  9. 2-O-Acyl-3-O-(1-acyloxyalkyl) Prodrugs of 5,6-Isopropylidene-l-Ascorbic Acid and l-Ascorbic Acid: Antioxidant Activity and Ability to Permeate Silicone Membranes

    PubMed Central

    Thiele, Nikki A.; McGowan, Jennifer; Sloan, Kenneth B.

    2016-01-01

    2-O-Acyl-3-O-(1-acyloxyalkyl) prodrug derivatives, 15, of 5,6-isopropylidene-l-ascorbic acid, VCA, and l-ascorbic acid, VC, have been characterized by measuring (1) their solubilities in water (SAQ) and in 1-octanol (SOCT); (2) the ability of one member of the homologous series, 15a, to diffuse through a silicone membrane from its application in propylene glycol:water (PG:AQ), 30:70; (3) the ability of another member of the series, 15e, to express cellular antioxidant activity (CAA) in HaCaT cells; and (4) the ability of 15e to support cell viability in HaCaT cells. All of the prodrugs were more soluble in 1-octanol than VC or VCA were. 15a, which exhibited a good balance between SOCT and SAQ, was found to deliver approximately 15 times more 15a than VCA delivered VCA through a silicone membrane from PG:AQ, 30:70. Under those conditions, no VC permeated the membrane. 15e, which hydrolyzed to release acetaldehyde as a byproduct instead of the toxin formaldehyde, exhibited approximately 30 times the antioxidant activity of VC in CaHaT cells and supported cell viability up to 900 μM in HaCaT cells. PMID:27438850

  10. Molecular characterization of the acyl-coenzyme A:isopenicillin N acyltransferase gene (penDE) from Penicillium chrysogenum and Aspergillus nidulans and activity of recombinant enzyme in Escherichia coli.

    PubMed Central

    Tobin, M B; Fleming, M D; Skatrud, P L; Miller, J R

    1990-01-01

    The final step in the biosynthesis of beta-lactam antibiotics in Penicillium chrysogenum and Aspergillus nidulans involves removal of the L-alpha-aminoadipyl side chain from isopenicillin N (IPN) and exchange with a nonpolar side chain. The enzyme catalyzing this reaction, acyl-coenzyme A:isopenicillin N acyltransferase (acyltransferase), was purified from P. chrysogenum and A. nidulans. Based on NH2-terminal amino acid sequence information, the acyltransferase gene (penDE) from P. chrysogenum and A. nidulans were cloned. In both organisms, penDE was located immediately downstream from the isopenicillin N synthetase gene (pcbC) and consisted of four exons encoding an enzyme of 357 amino acids (approximately 40 kilodaltons [kDa]). The DNA coding sequences showed approximately 73% identity, while the amino acid sequences were approximately 76% identical. Noncoding DNA regions (including the region between pcbC and penDE) were not conserved. Acyltransferase activity from Escherichia coli producing the 40-kDa protein accepted either 6-aminopenicillanic acid or IPN as the substrate and made a penicillinase-sensitive antibiotic in the presence of phenylacetyl coenzyme A. Therefore, a single gene is responsible for converting IPN to penicillin G. The active form of the enzyme may result from processing of the 40-kDa monomeric precursor to a heterodimer containing subunits of 11 and 29 kDa. Images PMID:2120195

  11. Ethanol Metabolism Modifies Hepatic Protein Acylation in Mice

    PubMed Central

    Fritz, Kristofer S.; Green, Michelle F.; Petersen, Dennis R.; Hirschey, Matthew D.

    2013-01-01

    Mitochondrial protein acetylation increases in response to chronic ethanol ingestion in mice, and is thought to reduce mitochondrial function and contribute to the pathogenesis of alcoholic liver disease. The mitochondrial deacetylase SIRT3 regulates the acetylation status of several mitochondrial proteins, including those involved in ethanol metabolism. The newly discovered desuccinylase activity of the mitochondrial sirtuin SIRT5 suggests that protein succinylation could be an important post-translational modification regulating mitochondrial metabolism. To assess the possible role of protein succinylation in ethanol metabolism, we surveyed hepatic sub-cellular protein fractions from mice fed a control or ethanol-supplemented diet for succinyl-lysine, as well as acetyl-, propionyl-, and butyryl-lysine post-translational modifications. We found mitochondrial protein propionylation increases, similar to mitochondrial protein acetylation. In contrast, mitochondrial protein succinylation is reduced. These mitochondrial protein modifications appear to be primarily driven by ethanol metabolism, and not by changes in mitochondrial sirtuin levels. Similar trends in acyl modifications were observed in the nucleus. However, comparatively fewer acyl modifications were observed in the cytoplasmic or the microsomal compartments, and were generally unchanged by ethanol metabolism. Using a mass spectrometry proteomics approach, we identified several candidate acetylated, propionylated, and succinylated proteins, which were enriched using antibodies against each modification. Additionally, we identified several acetyl and propionyl lysine residues on the same sites for a number of proteins and supports the idea of the overlapping nature of lysine-specific acylation. Thus, we show that novel post-translational modifications are present in hepatic mitochondrial, nuclear, cytoplasmic, and microsomal compartments and ethanol ingestion, and its associated metabolism, induce specific

  12. Strategies to reduce end-product inhibition in family 48 glycoside hydrolases.

    PubMed

    Chen, Mo; Bu, Lintao; Alahuhta, Markus; Brunecky, Roman; Xu, Qi; Lunin, Vladimir V; Brady, John W; Crowley, Michael F; Himmel, Michael E; Bomble, Yannick J

    2016-03-01

    Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the product inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. The theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.

  13. Structure-Guided Engineering of Molinate Hydrolase for the Degradation of Thiocarbamate Pesticides

    PubMed Central

    Paiva, Ana M.; Ferreira-da-Silva, Frederico; Matias, Pedro M.; Nunes, Olga C.; Gales, Luís

    2015-01-01

    Molinate is a recalcitrant thiocarbamate used to control grass weeds in rice fields. The recently described molinate hydrolase, from Gulosibacter molinativorax ON4T, plays a key role in the only known molinate degradation pathway ending in the formation of innocuous compounds. Here we report the crystal structure of recombinant molinate hydrolase at 2.27 Å. The structure reveals a homotetramer with a single mononuclear metal-dependent active site per monomer. The active site architecture shows similarities with other amidohydrolases and enables us to propose a general acid-base catalysis mechanism for molinate hydrolysis. Molinate hydrolase is unable to degrade bulkier thiocarbamate pesticides such as thiobencarb which is used mostly in rice crops. Using a structural-based approach, we were able to generate a mutant (Arg187Ala) that efficiently degrades thiobencarb. The engineered enzyme is suitable for the development of a broader thiocarbamate bioremediation system. PMID:25905461

  14. Strategies to reduce end-product inhibition in family 48 glycoside hydrolases

    SciTech Connect

    Chen, Mo; Bu, Lintao; Alahuhta, Markus; Brunecky, Roman; Xu, Qi; Lunin, Vladimir V.; Brady, John W.; Crowley, Michael F.; Himmel, Michael E.; Bomble, Yannick J.

    2016-02-01

    Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the product inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. As a result, the theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.

  15. Structure-guided engineering of molinate hydrolase for the degradation of thiocarbamate pesticides.

    PubMed

    Leite, José P; Duarte, Márcia; Paiva, Ana M; Ferreira-da-Silva, Frederico; Matias, Pedro M; Nunes, Olga C; Gales, Luís

    2015-01-01

    Molinate is a recalcitrant thiocarbamate used to control grass weeds in rice fields. The recently described molinate hydrolase, from Gulosibacter molinativorax ON4T, plays a key role in the only known molinate degradation pathway ending in the formation of innocuous compounds. Here we report the crystal structure of recombinant molinate hydrolase at 2.27 Å. The structure reveals a homotetramer with a single mononuclear metal-dependent active site per monomer. The active site architecture shows similarities with other amidohydrolases and enables us to propose a general acid-base catalysis mechanism for molinate hydrolysis. Molinate hydrolase is unable to degrade bulkier thiocarbamate pesticides such as thiobencarb which is used mostly in rice crops. Using a structural-based approach, we were able to generate a mutant (Arg187Ala) that efficiently degrades thiobencarb. The engineered enzyme is suitable for the development of a broader thiocarbamate bioremediation system.

  16. Phosphonoacetic acid utilization by fungal isolates: occurrence and properties of a phosphonoacetate hydrolase in some penicillia.

    PubMed

    Forlani, Giuseppe; Klimek-Ochab, Magdalena; Jaworski, Jakub; Lejczak, Barbara; Picco, Anna M

    2006-12-01

    Among a collection of 18 fungal strains representing eight genera, only two strains (Penicillium oxalicum and P. minioluteum) were capable of growth on phosphonoacetic acid as sole phosphorous source. Enrichment liquid cultures in minimal medium with the compound as the only P-source selected four isolates, that were also identified as Penicillium spp. Phosphonoacetate metabolism did not lead to extracellular release of inorganic phosphate. In all cases phosphonoacetate hydrolase activity was detected in partially purified extracts, and a protein of the expected molecular mass reacted with polyclonal antibodies raised against the enzyme from P. oxalicum. There was no relation between phosphonoacetate hydrolase specific activity and growth rate or yield. Phosphonoacetic acid was the inducer of the hydrolase, independently of the concurrent availability of inorganic phosphate. Notwithstanding this, the utilization of the phosphonate was significantly inhibited in the presence of phosphate, suggesting an interference of the latter with phosphonoacetic acid uptake.

  17. Discovery of a Bacterial Glycoside Hydrolase Family 3 (GH3) β-Glucosidase with Myrosinase Activity from a Citrobacter Strain Isolated from Soil.

    PubMed

    Albaser, Abdulhadi; Kazana, Eleanna; Bennett, Mark H; Cebeci, Fatma; Luang-In, Vijitra; Spanu, Pietro D; Rossiter, John T

    2016-02-24

    A Citrobacter strain (WYE1) was isolated from a UK soil by enrichment using the glucosinolate sinigrin as sole carbon source. The enzyme myrosinase was purified using a combination of ion exchange and gel filtration to give a pure protein of approximately 66 kDa. The N-terminal amino acid and internal peptide sequence of the purified protein were determined and used to identify the gene, which, based on InterPro sequence analysis, belongs to the family GH3, contains a signal peptide, and is a periplasmic protein with a predicted molecular mass of 71.8 kDa. A preliminary characterization was carried out using protein extracts from cell-free preparations. The apparent KM and Vmax were 0.46 mM and 4.91 mmol dm(-3) min(-1) mg(-1), respectively, with sinigrin as substrate. The optimum temperature and pH for enzyme activity were 25 °C and 6.0, respectively. The enzyme was marginally activated with ascorbate by a factor of 1.67.

  18. A genome wide shRNA screen identifies α/β hydrolase domain containing 4 (ABHD4) as a novel regulator of anoikis resistance.

    PubMed

    Simpson, Craig D; Hurren, Rose; Kasimer, Dahlia; MacLean, Neil; Eberhard, Yanina; Ketela, Troy; Moffat, Jason; Schimmer, Aaron D

    2012-07-01

    Acquisition of resistance to anchorage dependant cell death, a process termed anoikis, is a requirement for cancer cell metastasis. However, the molecular determinants of anoikis resistance and sensitivity are poorly understood. To better understand resistance to anoikis we conducted a genome wide lentiviral shRNA screen to identify genes whose knockdown render anoikis-sensitive RWPE-1 prostate cells resistant to anoikis. RWPE-1 cells were infected with a pooled lentiviral shRNA library with 54,021 shRNA targeting 11,255 genes. After infection, an anoikis-resistant cell population was selected and shRNA sequences were amplified and sequenced. Thirty-four shRNA sequences reproducibly protected RWPE-1 cells from anoikis after culture under suspension conditions including the top validated hit, α/β hydrolase domain containing 4 (ABHD4). In validation studies, ABHD4 knockdown inhibited anoikis in RWPE-1 cells as well as anoikis sensitive NP69 nasopharyngeal and OVCAR3 ovarian cancer cells, while over-expression of the gene increased sensitivity. Induction of anoikis after ABHD4 knockdown was associated with cleavage of PARP and activation of caspases-3, but was independent in changes of FLIP, FAK and Src expression. Interestingly, induction of anoikis after ABHD4 knockdown was independent of the known role of ABHD4 in the anandamide synthesis pathway and the generation of glycerophospho-N-acyl ethanolamines. Thus, ABHD4 is a novel genetic regulator of anoikis sensitivity.

  19. High-Throughput In Vitro Glycoside Hydrolase (HIGH) Screening for Enzyme Discovery

    SciTech Connect

    Kim, Tae-Wan; Chokhawala, Harshal A.; Hess, Matthias; Dana, Craig M.; Baer, Zachary; Sczyrba, Alexander; Rubin, Edward M.; Blanch, Harvey W.; Clark, Douglas S.

    2011-09-16

    A high-throughput protein-expression and screening method (HIGH method, see picture) provides a rapid approach to the discovery of active glycoside hydrolases in environmental samples. Finally, HIGH screening combines cloning, protein expression, and enzyme hydrolysis in one pot; thus, the entire process from gene expression to activity detection requires only three hours.

  20. Critical active-site residues identified by site-directed mutagenesis in Pseudomonas aeruginosa phosphorylcholine phosphatase, a new member of the haloacid dehalogenases hydrolase superfamily.

    PubMed

    Beassoni, Paola R; Otero, Lisandro H; Massimelli, Maria J; Lisa, Angela T; Domenech, Carlos E

    2006-12-01

    Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP), the product of the PA5292 gene, is synthesized when the bacteria are grown with choline, betaine, dimethylglycine, or carnitine. In the presence of Mg(2+), PChP catalyzes the hydrolysis of both phosphorylcholine (PCh) and p-nitrophenylphosphate (p-NPP). PCh saturation curve analysis of the enzyme with or without the signal peptide indicated that the peptide was the fundamental factor responsible for decreasing the affinity of the second site of PChP for PCh, either at pH 5.0 or pH 7.4. PChP contained three conserved motifs characteristic of the haloacid dehalogenases superfamily. In the PChP without the signal peptide, motifs I, II, and III correspond to the residues (31)DMDNT(35), (166)SAA(168), and K(242)/(261)GDTPDSD(267), respectively. To determine the catalytic importance of the D31, D33, T35, S166, K242, D262, D265, and D267 on the enzyme activity, site-directed mutagenesis was performed. D31, D33, D262, and D267 were identified as the more important residues for catalysis. D265 and D267 may be involved in the stabilization of motif III, or might contribute to substrate specificity. The substitution of T35 by S35 resulted in an enzyme with a low PChP activity, but conserves the catalytic sites involved in the hydrolysis of PCh (K(m1) 0.03 mM: , K(m2) 0.5 mM: ) or p-NPP (K(m) 2.1 mM: ). Mutating either S166 or K242 revealed that these residues are also important to catalyze the hydrolysis of both substrates. The substitution of lysine by arginine or by glutamine revealed the importance of the positive charged group, either from the amino or guanidinium groups, because K242Q was inactive, whereas K242R was a functional enzyme.

  1. A pre-steady state and steady state kinetic analysis of the N-ribosyl hydrolase activity of hCD157.

    PubMed

    Preugschat, Frank; Carter, Luke H; Boros, Eric E; Porter, David J T; Stewart, Eugene L; Shewchuk, Lisa M

    2014-12-15

    hCD157 catalyzes the hydrolysis of nicotinamide riboside (NR) and nicotinic acid riboside (NAR). The release of nicotinamide or nicotinic acid from NR or NAR was confirmed by spectrophotometric, HPLC and NMR analyses. hCD157 is inactivated by a mechanism-based inhibitor, 2'-deoxy-2'-fluoro-nicotinamide arabinoside (fNR). Modification of the enzyme during the catalytic cycle by NR, NAR, or fNR increased the intrinsic protein fluorescence by approximately 50%. Pre-steady state and steady state data were used to derive a minimal kinetic scheme for the hydrolysis of NR. After initial complex formation a reversible step (360 and 30s(-1)) is followed by a slow irreversible step (0.1s(-1)) that defined the rate limiting step, or kcat. The calculated KMapp value for NR in the hydrolytic reaction is 6nM. The values of the kinetic constants suggest that one biological function of cell-surface hCD157 is to bind and slowly hydrolyze NR, possibly converting it to a ligand-activated receptor. Differences in substrate preference between hCD157 and hCD38 were rationalized through a comparison of the crystal structures of the two proteins. This comparison identified several residues in hCD157 (F108 and F173) that can potentially hinder the binding of dinucleotide substrates (NAD+).

  2. Effect of PCB's on plasma vitamin A (retinol) and hepatic retinyl palmitate hydrolase (RPH) activity in female Sprague-Dawley rats

    SciTech Connect

    Powers, R.H.; Gilbert, L.C.; Aust, S.D.

    1987-05-01

    A single i.p. dose of 15, 5 or 1 mg/kg 3,4,3',4'-tetrachlorobiphenyl (TCB) caused a dose-dependent depression of plasma retinol levels 24 hours after treatment of female Sprague-Dawley rats. The loss of plasma retinol appeared to be a function of depressed levels of the retinol-retinol binding protein (RBP)-transthyretin ternary complex. No free retinol-RBP was observed in plasma from treated animals. Hepatic RPH activity was also depressed, and highly and positively correlated to the plasma retinol levels. TCB was determined to be a non-competitive inhibitor of partially purified RPH with a KI of 91 uM. Metabolism of TCB by microsomes in vitro decreased the inhibition of RPH. Equimolar doses of either 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) or 3,4,5,3',4',5'-HCB failed to cause a similar depression of plasma retinol of treated female rats. They conclude that, unlike other PCB congeners, TCB causes a depression of plasma retinol by inhibition of hepatic RPH.

  3. Lipopolysaccharides with acylation defects potentiate TLR4 signaling and shape T cell responses.

    PubMed

    Martirosyan, Anna; Ohne, Yoichiro; Degos, Clara; Gorvel, Laurent; Moriyón, Ignacio; Oh, Sangkon; Gorvel, Jean-Pierre

    2013-01-01

    Lipopolysaccharides or endotoxins are components of Gram-negative enterobacteria that cause septic shock in mammals. However, a LPS carrying hexa-acyl lipid A moieties is highly endotoxic compared to a tetra-acyl LPS and the latter has been considered as an antagonist of hexa-acyl LPS-mediated TLR4 signaling. We investigated the relationship between the structure and the function of bacterial LPS in the context of human and mouse dendritic cell activation. Strikingly, LPS with acylation defects were capable of triggering a strong and early TLR4-dependent DC activation, which in turn led to the activation of the proteasome machinery dampening the pro-inflammatory cytokine secretion. Upon activation with tetra-acyl LPS both mouse and human dendritic cells triggered CD4(+) T and CD8(+) T cell responses and, importantly, human myeloid dendritic cells favored the induction of regulatory T cells. Altogether, our data suggest that LPS acylation controlled by pathogenic bacteria might be an important strategy to subvert adaptive immunity.

  4. Purification and characterization of allophanate hydrolase (AtzF) from Pseudomonas sp. strain ADP.

    PubMed

    Shapir, Nir; Sadowsky, Michael J; Wackett, Lawrence P

    2005-06-01

    AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a k(cat)/K(m) of 1.1 x 10(4) s(-1) M(-1), and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced k(cat)/K(m) of 21 s(-1) M(-1). Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of

  5. Purification and Characterization of Allophanate Hydrolase (AtzF) from Pseudomonas sp. Strain ADP

    PubMed Central

    Shapir, Nir; Sadowsky, Michael J.; Wackett, Lawrence P.

    2005-01-01

    AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a kcat/Km of 1.1 × 104 s−1 M−1, and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced kcat/Km of 21 s−1 M−1. Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes

  6. Long-term consequences of perinatal fatty acid amino hydrolase inhibition

    PubMed Central

    Wu, Chia-Shan; Morgan, Daniel; Jew, Chris P; Haskins, Chris; Andrews, Mary-Jeanette; Leishman, Emma; Spencer, Corinne M; Czyzyk, Traci; Bradshaw, Heather; Mackie, Ken; Lu, Hui-Chen

    2014-01-01

    Background and PurposeFatty acid amide hydrolase inhibitors show promise as a treatment for anxiety, depression and pain. Here we investigated whether perinatal exposure to URB597, a fatty acid amide hydrolase inhibitor, alters brain development and affects behaviour in adult mice. Experimental ApproachMouse dams were treated daily from gestational day 10.5 to 16.5 with 1, 3 or 10 mg kg−1 URB597. MS was used to measure a panel of endocannabinoids and related lipid compounds and brain development was assessed at embryonic day 16.5. Separate cohorts of mouse dams were treated with 10 mg kg−1 URB597, from gestational day 10.5 to postnatal day 7, and the adult offspring were assessed with a battery of behavioural tests. Key ResultsPerinatal URB597 exposure elevated anandamide and related N-acyl amides. URB597 did not induce signs of toxicity or affect dam weight gain, neurogenesis or axonal development at embryonic day 16.5. It did lead to subtle behavioural deficits in adult offspring, manifested by reduced cocaine-conditioned preference, increased depressive behaviours and impaired working memory. Anxiety levels, motor function and sensory-motor gating were not significantly altered. Conclusions and ImplicationsTaken together, the present results highlight how exposure to elevated levels of anandamide and related N-acyl amides during brain development can lead to subtle alterations in behaviour in adulthood. Linked ArticlesThis article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6 PMID:24730060

  7. Acyl-ACP thioesterases from macadamia (Macadamia tetraphylla) nuts: cloning, characterization and their impact on oil composition.

    PubMed

    Moreno-Pérez, Antonio J; Sánchez-García, Alicia; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2011-01-01

    The mechanisms by which macadamia nuts accumulate the unusual palmitoleic and asclepic acyl moieties, which constitute up to 20% of the fatty acids in some varieties, are still unknown. Acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) are intraplastidial enzymes that terminate the synthesis of fatty acids in plants and that facilitate the export of the acyl moieties to the endoplasmic reticulum where they can be used in the production of glycerolipids. Here, we have investigated the possible role of acyl-ACP thioesterase activity in the composition of macadamia kernel oil. Accordingly, two acyl-ACP thioesterases were cloned from developing macadamia kernels, one of the FatA type and the other of the FatB type. These enzymes were heterologously expressed in Escherichia coli, and the recombinant thioesterases were purified, characterized kinetically and assayed with a variety of substrates, demonstrating the high specificity of macadamia FatA towards 16:1-ACP. Acyl-ACP thioesterase activity was also characterized in crude extracts from two different varieties of macadamia, Cate and Beaumont, which accumulate different amounts of n-7 fatty acids. The impact of acyl-ACP thioesterase activities on the oil composition of these kernels is discussed in the light of these results.

  8. Assessment of rat liver microsomal epoxide hydrolase as a marker of hepatocarcinogenesis.

    PubMed

    Kizer, D E; Clouse, J A; Ringer, D P; Hanson-Painton, O; Vaz, A D; Palakodety, R B; Griffin, M J

    1985-05-15

    The influence of eleven xenobiotics on the activity and amount of hepatic microsomal epoxide hydrolase was determined. Activity was assayed using three different substrates after rats were fed, throughout 3 weeks, diets containing one of six hepatocarcinogens, viz. 2-acetylaminofluorene, 3'-methyl-4-dimethylaminoazobenzene, 4'-fluoro-4-dimethylaminoazobenzene, thioacetamide, aflatoxin B1 and ethionine. Five hepatocarcinogens induced activity 4- to 10-fold; ethionine was relatively ineffective as an inducer. Two non-carcinogenic analogues of hepatocarcinogens, viz. fluorene and p-aminoazobenzene, caused no appreciable increase in enzyme activity, but phenobarbital, barbital and 1-naphthylisothiocyanate induced activity 2- to 3-fold. All eleven xenobiotics increased the amount of microsomal epoxide hydrolase 2- to 9-fold when examined immunochemically using either a radial diffusion assay or an enzyme-linked immunosorbent assay (ELISA). Serum glutamic oxaloacetic acid transaminase activity was not appreciably elevated by feeding ten of the xenobiotics, suggesting that inductions were not owing to toxicity. Using ELISA, microsomal epoxide hydrolase was detected in post-microsomal (PM) supernatant fractions from control rat liver, thus confirming an earlier report by Gill et al. [Carcinogenesis 3, 1307 (1982)]. The eleven xenobiotics induced the amount of ELISA-detectable antigen in PM supernatant fractions by 3- to 34-fold. Longer centrifugation of PM supernatant fractions yielded a pellet fraction that contained 92 +/- 1.2% of the ELISA-detectable antigen irrespective of the xenobiotic regimen. Relationships between xenobiotic induction of microsomal epoxide hydrolase activity and amount and hepatocarcinogenesis are discussed.

  9. Distinct rat hepatic microsomal epoxide hydrolases catalyze the hydration of cholesterol 5,6 alpha-oxide and certain xenobiotic alkene and arene oxides.

    PubMed

    Levin, W; Michaud, D P; Thomas, P E; Jerina, D M

    1983-02-01

    Metabolism of cholesterol 5,6 alpha-oxide to the 5,6-glycol is catalyzed by a rat liver microsomal epoxide hydrolase that is distinct from the microsomal epoxide hydrolase that metabolizes a wide range of xenobiotic alkene and arene oxides. The two enzymes are antigenically distinct, and the purified microsomal epoxide hydrolase that metabolizes xenobiotic oxides does not catalyze the hydration of cholesterol 5,6 alpha-oxide. In vivo treatment of rats with inducers of microsomal epoxide hydrolase does not enhance the activity of cholesterol 5,6 alpha-oxide hydrolase and, in some cases, actually depresses enzyme activity in the resultant microsomal preparations. Octene 1,2-oxide and benz[a]anthracene 5,6-oxide, both good substrates for xenobiotic epoxide hydrolase, are not competitive inhibitors of cholesterol oxide hydration by rat liver microsomes. The above results establish the existence of a liver microsomal epoxide hydrolase that is under different regulatory control and that appears to have a different substrate specificity than the well-characterized microsomal epoxide hydrolase involved in the metabolism of a widely diverse group of alkene and arene oxides.

  10. Twisting of glycosidic bonds by hydrolases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Patterns of scissile bond twisting have been found in crystal structures of glycoside hydrolases (GHs) that are complexed with substrates and inhibitors. To estimate the increased potential energy in the substrates that results from this twisting, we have plotted torsion angles for the scissile bond...

  11. Structure and function of polyglycine hydrolases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave polyglycine linkers of targeted plant defense chitinases. Unlike typical endoproteases that cleave a specific peptide bond, these 640 amino acid glycoproteins selectively cleave one of multiple peptide bonds within polyglyci...

  12. Acylation type determines ghrelin's effects on energy homeostasis in rodents.

    PubMed

    Heppner, Kristy M; Chaudhary, Nilika; Müller, Timo D; Kirchner, Henriette; Habegger, Kirk M; Ottaway, Nickki; Smiley, David L; Dimarchi, Richard; Hofmann, Susanna M; Woods, Stephen C; Sivertsen, Bjørn; Holst, Birgitte; Pfluger, Paul T; Perez-Tilve, Diego; Tschöp, Matthias H

    2012-10-01

    Ghrelin is a gastrointestinal polypeptide that acts through the ghrelin receptor (GHSR) to promote food intake and increase adiposity. Activation of GHSR requires the presence of a fatty-acid (FA) side chain on amino acid residue serine 3 of the ghrelin molecule. However, little is known about the role that the type of FA used for acylation plays in the biological action of ghrelin. We therefore evaluated a series of differentially acylated peptides to determine whether alterations in length or stability of the FA side chain have an impact on the ability of ghrelin to activate GHSR in vitro or to differentially alter food intake, body weight, and body composition in vivo. Fatty acids principally available in the diet (such as palmitate C16) and therefore representing potential substrates for the ghrelin-activating enzyme ghrelin O-acyltransferase (GOAT) were used for dose-, time-, and administration/route-dependent effects of ghrelin on food intake, body weight, and body composition in rats and mice. Our data demonstrate that altering the length of the FA side chain of ghrelin results in the differential activation of GHSR. Additionally, we found that acylation of ghrelin with a long-chain FA (C16) delays the acute central stimulation of food intake. Lastly, we found that, depending on acylation length, systemic and central chronic actions of ghrelin on adiposity can be enhanced or reduced. Together our data suggest that modification of the FA side-chain length can be a novel approach to modulate the efficacy of pharmacologically administered ghrelin.

  13. Evaluation of NHS Carbamates as a Potent and Selective Class of Endocannabinoid Hydrolase Inhibitors

    PubMed Central

    2013-01-01

    Monoacylglycerol lipase (MAGL) is a principal metabolic enzyme responsible for hydrolyzing the endogenous cannabinoid (endocannabinoid) 2-arachidonoylglycerol (2-AG). Selective inhibitors of MAGL offer valuable probes to further understand the enzyme’s function in biological systems and may lead to drugs for treating a variety of diseases, including psychiatric disorders, neuroinflammation, and pain. N-Hydroxysuccinimidyl (NHS) carbamates have recently been identified as a promising class of serine hydrolase inhibitors that shows minimal cross-reactivity with other proteins in the proteome. Here, we explore NHS carbamates more broadly and demonstrate their potential as inhibitors of endocannabinoid hydrolases and additional enzymes from the serine hydrolase class. We extensively characterize an NHS carbamate 1a (MJN110) as a potent, selective, and in-vivo-active MAGL inhibitor. Finally, we demonstrate that MJN110 alleviates mechanical allodynia in a rat model of diabetic neuropathy, marking NHS carbamates as a promising class of MAGL inhibitors. PMID:23731016

  14. Structural basis for acyl-group discrimination by human Gcn5L2

    PubMed Central

    Ringel, Alison E.; Wolberger, Cynthia

    2016-01-01

    Gcn5 is a conserved acetyltransferase that regulates transcription by acetylating the N-terminal tails of histones. Motivated by recent studies identifying a chemically diverse array of lysine acyl modifications in vivo, the acyl-chain specificity of the acetyltransferase human Gcn5 (Gcn5L2) was examined. Whereas Gcn5L2 robustly catalyzes lysine acetylation, the acyltransferase activity of Gcn5L2 becomes progressively weaker with increasing acyl-chain length. To understand how Gcn5 discriminates between different acyl-CoA molecules, structures of the catalytic domain of human Gcn5L2 bound to propionyl-CoA and butyryl-CoA were determined. Although the active site of Gcn5L2 can accommodate propionyl-CoA and butyryl-CoA without major structural rearrangements, butyryl-CoA adopts a conformation incompatible with catalysis that obstructs the path of the incoming lysine residue and acts as a competitive inhibitor of Gcn5L2 versus acetyl-CoA. These structures demonstrate how Gcn5L2 discriminates between acyl-chain donors and explain why Gcn5L2 has weak activity for acyl moieties that are larger than an acetyl group. PMID:27377381

  15. Structure Determination and Characterization of the Vitamin B[superscript 6] Degradative Enzyme (E)-2-(Acetamidomethylene)succinate Hydrolase

    SciTech Connect

    McCulloch, Kathryn M.; Mukherjee, Tathagata; Begley, Tadhg P.; Ealick, Steven E.

    2010-06-22

    The gene identification and kinetic characterization of (E)-2-(acetamidomethylene)succinate (E-2AMS) hydrolase has recently been described. This enzyme catalyzes the final reaction in the degradation of vitamin B{sub 6} and produces succinic semialdehyde, acetate, ammonia, and carbon dioxide from E-2AMS. The structure of E-2AMS hydrolase was determined to 2.3 {angstrom} using SAD phasing. E-2AMS hydrolase is a member of the {alpha}/{beta} hydrolase superfamily and utilizes a serine/histidine/aspartic acid catalytic triad. Mutation of either the nucleophilic serine or the aspartate resulted in inactive enzyme. Mutation of an additional serine residue in the active site causes the enzyme to be unstable and is likely structurally important. The structure also provides insight into the mechanism of hydrolysis of E-2AMS and identifies several potential catalytically important residues.

  16. A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides.

    PubMed

    Nemoto, Takayuki K; Ohara-Nemoto, Yuko; Bezerra, Gustavo Arruda; Shimoyama, Yu; Kimura, Shigenobu

    2016-03-11

    Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser(615) and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9 family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 μm(-1) s(-1), optimal pH was 7-8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met(16)-Glu(101)). Three-dimensional modeling revealed the three domain structures (residues Met(16)-Ala(126), which has no similar homologue with known structure; residues Leu(127)-Met(495) (β-propeller domain); and residues Ala(496)-Phe(736) (α/β-hydrolase domain)) and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides.

  17. A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides*

    PubMed Central

    Nemoto, Takayuki K.; Ohara-Nemoto, Yuko; Bezerra, Gustavo Arruda; Shimoyama, Yu; Kimura, Shigenobu

    2016-01-01

    Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser615 and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9 family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 μm−1 s−1, optimal pH was 7–8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met16–Glu101). Three-dimensional modeling revealed the three domain structures (residues Met16–Ala126, which has no similar homologue with known structure; residues Leu127–Met495 (β-propeller domain); and residues Ala496–Phe736 (α/β-hydrolase domain)) and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides. PMID:26733202

  18. Antinociceptive property of new 4-acyl-arylhydrazone pyrazole compounds.

    PubMed

    Matheus, M E; Oliveira, L F; Freitas, A C; Carvalho, A M; Barreiro, E J

    1991-01-01

    A series of new 4-acyl-arylhydrazone pyrazole compounds were tested for antinociceptive activity using the inhibition of abdominal contortions induced by acetylcholine (4 mg/kg, ip) in the mouse. Dipyrone was used for comparison of the antinociceptive potency of the compounds being tested. All drugs were administered po in saline (dipyrone) or in propylene glycol (4-acyl-arylhydrazones). The maximum response induced by dipyrone (86% inhibition) was assigned an efficacy index of 1.0. Although none of the compounds had an efficacy index greater than 1.0, all three reached 1.0. The two most potent compounds, W1d and W1g, which also had an efficacy similar to that of dipyrone, contain a p-N(CH3)2 and m-OH,p-OCH3 group in the aromatic ring of the acyl-hydrazone, respectively. W1d presented the lowest antinociceptive ED50 in the series (1.41 mg/kg) and was eleven times more potent than dipyrone (ED50 = 15.80 mg/kg). Other substitutions at the para position had lower potency than W1d. The present results indicate that the introduction of a group at the para position of the acyl-arylhydrazone ring increases the antinociceptive activity of these compounds to provide compounds of the same efficacy but greater potency than dipyrone to which these new compounds are structurally related. Other assays of nociceptive activity are being used to characterize the mechanism of action of the potential new drugs.

  19. Proteins with an alpha/beta hydrolase fold: Relationships between subfamilies in an ever-growing superfamily.

    PubMed

    Lenfant, Nicolas; Hotelier, Thierry; Bourne, Yves; Marchot, Pascale; Chatonnet, Arnaud

    2013-03-25

    Alpha/beta hydrolases function as hydrolases, lyases, transferases, hormone precursors or transporters, chaperones or routers of other proteins. The amount of structural and functional available data related to this protein superfamily expands exponentially, as does the number of proteins classified as alpha/beta hydrolases despite poor sequence similarity and lack of experimental data. However the superfamily can be rationally divided according to sequence or structural homologies, leading to subfamilies of proteins with potentially similar functions. Since the discovery of proteins homologous to cholinesterases but devoid of enzymatic activity (e.g., the neuroligins), divergent functions have been ascribed to members of other subfamilies (e.g., lipases, dipeptidylaminopeptidase IV, etc.). To study the potentially moonlighting properties of alpha/beta hydrolases, the ESTHER database (for ESTerase and alpha/beta Hydrolase Enzymes and Relatives; http://bioweb.ensam.inra.fr/esther), which collects, organizes and disseminates structural and functional information related to alpha/beta hydrolases, has been updated with new tools and the web server interface has been upgraded. A new Overall Table along with a new Tree based on HMM models has been included to tentatively group subfamilies. These tools provide starting points for phylogenetic studies aimed at pinpointing the origin of duplications leading to paralogous genes (e.g., acetylcholinesterase versus butyrylcholinesterase, or neuroligin versus carboxylesterase). Another of our goals is to implement new tools to distinguish catalytically active enzymes from non-catalytic proteins in poorly studied or annotated subfamilies.

  20. γ-Glutamyl Hydrolase: Kinetic Characterization of Isopeptide Hydrolysis Using Fluorogenic Substrates†

    PubMed Central

    Alexander, Jessica P.; Ryan, Thomas J.; Ballou, David P.; Coward, James K.

    2008-01-01

    γ-Glutamyl hydrolase, a cysteine peptidase, catalyzes the hydrolysis of poly-γ-glutamate derivatives of folate co-factors and many antifolate drugs. We have used internally quenched fluorogenic derivatives of glutamyl-γ-glutamate and (4,4-difluoro)glutamyl-γ-glutamate to examine the effect of fluorine substitution adjacent to the scissile isopeptide bond. Using a newly developed continuous fluorescence assay, the hydrolysis of both substrates could be described by Michaelis-Menten kinetics. Fluorine substitution resulted in a significant decrease in observed rates of hydrolysis under steady-state conditions due primarily to a ~ 15-fold increase in Km. Using stopped-flow techniques, hydrolysis of the non-fluorinated isopeptide was characterized by a burst phase followed by a steady-state rate, indicating that formation of the acyl enzyme is not rate-limiting for hydrolysis of this isopeptide. This conclusion was confirmed by analysis of the progress curves over a wide range of substrate concentration, which demonstrated that the acylation rate (k2) is ~ 10-fold higher than the deacylation rate (k3). The increased value of Km associated with the difluoro derivative limited the ability to obtain comparable pre-steady-state kinetics data at saturating concentration of substrate due to inner filter effects. However, even under non-saturating conditions, a modest burst was observed for the difluoro derivative. These data indicate that either deacylation or rearrangement of the enzyme-product complex is rate-limiting in this isopeptide hydrolysis reaction. PMID:18171026

  1. Peripheral FAAH and soluble epoxide hydrolase inhibitors are synergistically antinociceptive.

    PubMed

    Sasso, Oscar; Wagner, Karen; Morisseau, Christophe; Inceoglu, Bora; Hammock, Bruce D; Piomelli, Daniele

    2015-07-01

    We need better medicines to control acute and chronic pain. Fatty acid amide hydrolase (FAAH) and soluble epoxide hydrolase (sEH) catalyze the deactivating hydrolysis of two classes of bioactive lipid mediators--fatty acid ethanolamides (FAEs) and epoxidized fatty acids (EpFAs), respectively--which are biogenetically distinct but share the ability to attenuate pain responses and inflammation. In these experiments, we evaluated the antihyperalgesic activity of small-molecule inhibitors of FAAH and sEH, administered alone or in combination, in two pain models: carrageenan-induced hyperalgesia in mice and streptozocin-induced allodynia in rats. When administered separately, the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea (TPPU) and the peripherally restricted FAAH inhibitor URB937 were highly active in the two models. The combination TPPU plus URB937 was markedly synergistic, as assessed using isobolographic analyses. The results of these experiments reveal the existence of a possible functional crosstalk between FAEs and EpFAs in regulating pain responses. Additionally, the results suggest that combinations of sEH and FAAH inhibitors might be exploited therapeutically to achieve greater analgesic efficacy.

  2. Inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) as hypocholesterolemic agents: synthesis and structure-activity relationships of novel series of sulfonamides, acylphosphonamides and acylphosphoramidates.

    PubMed

    Lee, H T; Roark, W H; Picard, J A; Sliskovic, D R; Roth, B D; Stanfield, R L; Hamelehle, K L; Bousley, R F; Krause, B R

    1998-02-03

    Sulfoacetic acid, phosphoramidate, and phosphoramide analogs of the ACAT inhibitors, CI-999 and CI-1011 were synthesized. The structure-activity relationships of these compounds as ACAT inhibitors are described.

  3. Fatty acyl-CoA reductase

    SciTech Connect

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

  4. Discovery of novel leukotriene A4 hydrolase inhibitors based on piperidine and piperazine scaffolds.

    PubMed

    Sandanayaka, Vincent; Mamat, Bjorn; Bhagat, Nikhil; Bedell, Louis; Halldorsdottir, Gudrun; Sigthorsdottir, Heida; Andrésson, Thorkell; Kiselyov, Alex; Gurney, Mark; Singh, Jasbir

    2010-05-01

    Novel piperidine and piperazine derivatives have been designed and tested as inhibitors of LTA(4) hydrolase (LTA(4)H). Most potent compounds showed good potency in both enzymatic and functional human whole blood assay. Crystallography studies further confirmed observed structure-activity relationship and LTA(4)H binding mode for analogs from the piperidine series.

  5. Functional characterization and structural modeling of synthetic polyester-degrading hydrolases from Thermomonospora curvata

    PubMed Central

    2014-01-01

    Thermomonospora curvata is a thermophilic actinomycete phylogenetically related to Thermobifida fusca that produces extracellular hydrolases capable of degrading synthetic polyesters. Analysis of the genome of T. curvata DSM43183 revealed two genes coding for putative polyester hydrolases Tcur1278 and Tcur0390 sharing 61% sequence identity with the T. fusca enzymes. Mature proteins of Tcur1278 and Tcur0390 were cloned and expressed in Escherichia coli TOP10. Tcur1278 and Tcur0390 exhibited an optimal reaction temperature against p-nitrophenyl butyrate at 60°C and 55°C, respectively. The optimal pH for both enzymes was determined at pH 8.5. Tcur1278 retained more than 80% and Tcur0390 less than 10% of their initial activity following incubation for 60 min at 55°C. Tcur0390 showed a higher hydrolytic activity against poly(ε-caprolactone) and polyethylene terephthalate (PET) nanoparticles compared to Tcur1278 at reaction temperatures up to 50°C. At 55°C and 60°C, hydrolytic activity against PET nanoparticles was only detected with Tcur1278. In silico modeling of the polyester hydrolases and docking with a model substrate composed of two repeating units of PET revealed the typical fold of α/β serine hydrolases with an exposed catalytic triad. Molecular dynamics simulations confirmed the superior thermal stability of Tcur1278 considered as the main reason for its higher hydrolytic activity on PET. PMID:25405080

  6. Proteomic Analysis of a Novel Bacillus Jumbo Phage Revealing Glycoside Hydrolase As Structural Component

    PubMed Central

    Yuan, Yihui; Gao, Meiying

    2016-01-01

    Tailed phages with genomes of larger than 200 kbp are classified as Jumbo phages and exhibited extremely high uncharted diversity. The genomic annotation of Jumbo phage is often disappointing because most of the predicted proteins, including structural proteins, failed to make good hits to the sequences in the databases. In this study, 23 proteins of a novel Bacillus Jumbo phage, vB_BpuM_BpSp, were identified as phage structural proteins by the structural proteome analysis, including 14 proteins of unknown function, 5 proteins with predicted function as structural proteins, a glycoside hydrolase, a Holliday junction resolvase, a RNA-polymerase β-subunit, and a host-coding portal protein, which might be hijacked from the host strain during phage virion assembly. The glycoside hydrolase (Gp255) was identified as phage virion component and was found to interact with the phage baseplate protein. Gp255 shows specific lytic activity against the phage host strain GR8 and has high temperature tolerance. In situ peptidoglycan-hydrolyzing activities analysis revealed that the expressed Gp255 and phage structural proteome exhibited glycoside hydrolysis activity against the tested GR8 cell extracts. This study identified the first functional individual structural glycoside hydrolase in phage virion. The presence of activated glycoside hydrolase in phage virions might facilitate the injection of the phage genome during infection by forming pores on the bacterial cell wall. PMID:27242758

  7. Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme, Sphingobium fuliginis ATCC 27551.

    PubMed

    Nakayama, Kosuke; Ohmori, Takeshi; Ishikawa, Satoshi; Iwata, Natsumi; Seto, Yasuo; Kawahara, Kazuyoshi

    2016-05-01

    The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.

  8. Synthesis and antileishmanial activity of 6-mono-substituted and 3,6-di-substituted acridines obtained by acylation of proflavine.

    PubMed

    Di Giorgio, Carole; Shimi, Kamal; Boyer, Gérard; Delmas, Florence; Galy, Jean-Pierre

    2007-10-01

    Two new series of diaminoacridinic derivatives obtained from proflavine and N-(6-amino-3-acridinyl)acetamide were synthesised and assessed for their cytotoxic and antileishmanial activities. Two compounds, N-[6-(acetylamino)-3-acridinyl]acetamide and N-[6-(benzoylamino)-3-acridinyl]benzamide demonstrated highly specific antileishmanial properties against the intracellular amastigote form of the parasite. Structure-activity relationships established that the antiproliferative activity against human cells was greatly enhanced by the presence of a benzoylamino group in 6-mono-substituted acridines, while the presence of two acetylamino or benzoylamino groups in 3,6-di-substituted acridines strongly increased the specificity of the molecules for Leishmania parasite, suggesting that symmetric conformations could preferentially interfere with Leishmania metabolism.

  9. Acylated pregnane glycosides from Caralluma quadrangula.

    PubMed

    Abdallah, Hossam M; Osman, Abdel-Moneim M; Almehdar, Hussein; Abdel-Sattar, Essam

    2013-04-01

    In a previous study, the methanolic extract as well as the chloroform fraction of the aerial parts of Caralluma quadrangula (Forssk.) N.E.Br. indigenous to Saudi Arabia showed significant in vitro cytotoxic activity against breast cancer (MCF7) cell line. In a biologically-guided fractionation approach, four acylated pregnane glycosides were isolated from the chloroform fraction of C. quadrangula. The structures of the isolated compounds were elucidated by the analysis of their MS and NMR data. The compounds were identified as 12,20-di-O-benzoylboucerin 3-O-β-D-digitoxopyranosyl-(1→4)-β-D-canaropyranosyl-(1→4)-β-D-cymaropyranoside (1), 12,20-di-O-benzoylboucerin 3-O-β-D-cymaropyranosyl-(1→4)-β-D-canaropyranosyl-(1→4)-β-D-cymaropyranoside (2), 12,20-di-O-benzoylboucerin 3-O-β-D-glucopyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)-β-D-canaropyranosyl-(1→4)-β-D-cymaropyranoside (3) and 12,20-di-O-benzoyl-3β,5α,12β,14β,20-pentahydroxy-(20R)-pregn-6-ene 3-O-β-D-glucopyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)-β-D-canaropyranosyl-(1→4)-β-D-cymaropyranoside (4). The isolated compounds were tested for their cytotoxic activity against breast cancer (MCF7) cell line.

  10. Unusual long-chain N-acyl homoserine lactone production by and presence of quorum quenching activity in bacterial isolates from diseased tilapia fish.

    PubMed

    Chang, Chien-Yi; Koh, Chong-Lek; Sam, Choon-Kook; Chan, Xin-Yue; Yin, Wai Fong; Chan, Kok Gan

    2012-01-01

    Growth-dependent cell-cell communication termed quorum sensing is a key regulatory system in bacteria for controlling gene expression including virulence factors. In this study five potential bacterial pathogens including Bacillus sp. W2.2, Klebsiella sp. W4.2, Pseudomonas sp. W3 and W3.1 and Serratia sp. W2.3 were isolated from diseased Tilapia fish in Malaysia, supplied by the leading global fish supplier. Proteolytic activity assays confirmed that with the exception of Klebsiella sp. W4.2, all isolates showed distinct proteolytic activity. Furthermore Bacillus sp. W2.2 and Pseudomonas sp. strains W3 and W3.1 also displayed haemolytic activity. By using high resolution liquid chromatography mass spectrometry, we revealed the presence of unusually long-chain N-(3-oxohexadecanoyl)-homoserine lactone (3-oxo-C16-HSL) from Pseudomonas sp. W3.1 and N-dodecanoyl-homoserine lactone (C12-HSL) from Serratia sp. W2.3, respectively. Interestingly, Pseudomonas sp. W3.1 also produced a wide range of Pseudomonas quinolone signalling (PQS) molecules. Pseudomonas sp. W3 did not show any quorum sensing properties but possessed quorum quenching activity that inactivated AHLs. This study is the first documentation that shows unusual long-chain AHLs production in Serratia sp. and Pseudomonas sp. isolated from diseased fish and the latter also produce a wide range of PQS molecules.

  11. Unusual Long-Chain N-Acyl Homoserine Lactone Production by and Presence of Quorum Quenching Activity in Bacterial Isolates from Diseased Tilapia Fish

    PubMed Central

    Chang, Chien-Yi; Koh, Chong-Lek; Sam, Choon-Kook; Chan, Xin-Yue; Yin, Wai Fong; Chan, Kok Gan

    2012-01-01

    Growth-dependent cell-cell communication termed quorum sensing is a key regulatory system in bacteria for controlling gene expression including virulence factors. In this study five potential bacterial pathogens including Bacillus sp. W2.2, Klebsiella sp. W4.2, Pseudomonas sp. W3 and W3.1 and Serratia sp. W2.3 were isolated from diseased Tilapia fish in Malaysia, supplied by the leading global fish supplier. Proteolytic activity assays confirmed that with the exception of Klebsiella sp. W4.2, all isolates showed distinct proteolytic activity. Furthermore Bacillus sp. W2.2 and Pseudomonas sp. strains W3 and W3.1 also displayed haemolytic activity. By using high resolution liquid chromatography mass spectrometry, we revealed the presence of unusually long-chain N-(3-oxohexadecanoyl)-homoserine lactone (3-oxo-C16-HSL) from Pseudomonas sp. W3.1 and N-dodecanoyl-homoserine lactone (C12-HSL) from Serratia sp. W2.3, respectively. Interestingly, Pseudomonas sp. W3.1 also produced a wide range of Pseudomonas quinolone signalling (PQS) molecules. Pseudomonas sp. W3 did not show any quorum sensing properties but possessed quorum quenching activity that inactivated AHLs. This study is the first documentation that shows unusual long-chain AHLs production in Serratia sp. and Pseudomonas sp. isolated from diseased fish and the latter also produce a wide range of PQS molecules. PMID:22952864

  12. Trapping of the Enoyl-Acyl Carrier Protein Reductase–Acyl Carrier Protein Interaction

    PubMed Central

    Tallorin, Lorillee; Finzel, Kara; Nguyen, Quynh G.; Beld, Joris; La Clair, James J.; Burkart, Michael D.

    2016-01-01

    An ideal target for metabolic engineering, fatty acid biosynthesis remains poorly understood on a molecular level. These carrier protein-dependent pathways require fundamental protein–protein interactions to guide reactivity and processivity, and their control has become one of the major hurdles in successfully adapting these biological machines. Our laboratory has developed methods to prepare acyl carrier proteins (ACPs) loaded with substrate mimetics and cross-linkers to visualize and trap interactions with partner enzymes, and we continue to expand the tools for studying these pathways. We now describe application of the slow-onset, tight-binding inhibitor triclosan to explore the interactions between the type II fatty acid ACP from Escherichia coli, AcpP, and its corresponding enoyl-ACP reductase, FabI. We show that the AcpP–triclosan complex demonstrates nM binding, inhibits in vitro activity, and can be used to isolate FabI in complex proteomes. PMID:26938266

  13. Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens*

    PubMed Central

    Jongkees, Seino A. K.; Yoo, Hayoung; Withers, Stephen G.

    2014-01-01

    Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct 1H NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2. PMID:24573682

  14. Glycosyltransferases from oat (Avena) implicated in the acylation of avenacins.

    PubMed

    Owatworakit, Amorn; Townsend, Belinda; Louveau, Thomas; Jenner, Helen; Rejzek, Martin; Hughes, Richard K; Saalbach, Gerhard; Qi, Xiaoquan; Bakht, Saleha; Roy, Abhijeet Deb; Mugford, Sam T; Goss, Rebecca J M; Field, Robert A; Osbourn, Anne

    2013-02-08

    Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid.

  15. Synthesis and structure-activity relationship studies on a novel series of naphthylidinoylureas as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT).

    PubMed

    Ohnuma, Satoshi; Muraoka, Masami; Ioriya, Katsuhisa; Ohashi, Naohito

    2004-03-08

    The synthesis and structure-activity relationships of N-phenyl-N'-[3-(4-phenylnaphthylidinoyl)]urea derivatives 3 as a novel structural class of potent ACAT inhibitors is described. A 3-methoxy group substituted on the naphthylidinone 4-phenyl ring, together with a 1-N-(n)butyl substitution, SM-32504 (3m), gave a potent ACAT inhibitor, in vitro, respectively. The most potent compound, SM-32504 (3m), decreased the serum cholesterol level significantly in a high fat and high cholesterol-fed mouse model.

  16. Activity-Based Protein Profiling Reveals Broad Reactivity of the Nerve Agent Sarin.

    PubMed

    Tuin, Adriaan W; Mol, Marijke A E; van den Berg, Roland M; Fidder, A; van der Marel, Gijs A; Overkleeft, Herman S; Noort, Daan

    2009-04-01

    Elucidation of noncholinesterase protein targets of organophosphates, and nerve agents in particular, may reveal additional mechanisms for their high toxicity as well as clues for novel therapeutic approaches toward intoxications with these agents. Within this framework, we here describe the synthesis of the activity-based probe 3, which contains a phosphonofluoridate moiety, a P-Me moiety, and a biotinylated O-alkyl group, and its use in activity-based protein profiling with two relevant biological samples, that is, rhesus monkey liver and cultured human A549 lung cells. In this way, we have unearthed eight serine hydrolases (fatty acid synthase, acylpeptide hydrolase, dipeptidyl peptidase 9, prolyl oligopeptidase, carboxylesterase, long-chain acyl coenzyme A thioesterase, PAF acetylhydrolase 1b, and esterase D/S-formyl glutathione hydrolase) as targets that are modified by the nerve agent sarin. It is also shown that the newly developed probe 3 might find its way into the development of alternative, less laborious purification protocols for human butyrylcholinesterase, a potent bioscavenger currently under clinical investigation as a prophylactic/therapeutic for nerve agent intoxications.

  17. Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast.

    PubMed

    Pogorelko, Gennady; Fursova, Oksana; Lin, Ming; Pyle, Eric; Jass, Johanna; Zabotina, Olga A

    2011-11-01

    The systematic creation of defined cell wall modifications in the model plant Arabidopsis thaliana by expression of microbial hydrolases with known specific activities is a promising approach to examine the impacts of cell wall composition and structure on both plant fitness and cell wall recalcitrance. Moreover, this approach allows the direct evaluation in living plants of hydrolase specificity, which can differ from in vitro specificity. To express genes encoding microbial hydrolases in A. thaliana, and target the hydrolases to the apoplast compartment, we constructed an expression cassette composed of the Cauliflower Mosaic Virus 35S RNA promoter, the A. thaliana β-expansin signal peptide, and the fluorescent marker protein YFP. Using this construct we successfully introduced into Colombia-0 plants three Aspergillus nidulans hydrolases, β-xylosidase/α-arabinosidase, feruloyl esterase, acetylxylan esterase, and a Xanthomonas oryzae putative a-L: -arabinofuranosidase. Fusion with YFP permitted quick and easy screening of transformants, detection of apoplastic localization, and protein size confirmation. Compared to wild-type Col-0, all transgenic lines showed a significant increase in the corresponding hydrolytic activity in the apoplast and changes in cell wall composition. Examination of hydrolytic activity in the transgenic plants also showed, for the first time, that the X. oryzae gene indeed encoded an enzyme with α-L: -arabinofuranosidase activity. None of the transgenic plants showed a visible phenotype; however, the induced compositional changes increased the degradability of biomass from plants expressing feruloyl esterase and β-xylosidase/α-arabinosidase. Our results demonstrate the viability of creating a set of transgenic A. thaliana plants with modified cell walls to use as a toolset for investigation of how cell wall composition contributes to recalcitrance and affects plant fitness.

  18. Reversible Nε-Lysine Acetylation Regulates the Activity of Acyl-CoA Synthetases Involved in Anaerobic Benzoate Catabolism in Rhodopseudomonas palustris

    PubMed Central

    Crosby, Heidi A.; Heiniger, Erin K.; Harwood, Caroline S.; Escalante-Semerena, Jorge C.

    2010-01-01

    Rhodopseudomonas palustris grows photoheterotrophically on aromatic compounds available in aquatic environments rich in plant-derived lignin. Benzoate degradation is regulated at the transcriptional level in R. palustris in response to anoxia and the presence of benzoate and/or benzoyl-CoA (Bz-CoA). Here, we report evidence that anaerobic benzoate catabolism in this bacterium is also regulated at the posttranslational level. In this pathway, benzoate is activated to Bz-CoA by the AMP-forming Bz-CoA synthetase (BadA) enzyme. Mass spectrometry and mutational analysis data indicate that residue Lys512 is critical to BadA activity. Acetylation of Lys512 inactivated BadA; deacetylation reactivated BadA. Likewise, 4-hydroxybenzoyl-CoA (HbaA) and cyclohexanecarboxyl-CoA (AliA) synthetases were also reversibly acetylated. We identified one acetyltransferase that modified BadA, Hba, and AliA in vitro. The acetyltransferase enzyme is homologous to the protein acetyltransferase (Pat) enzyme of Salmonella enterica sv Typhimurium LT2, thus we refer to it as RpPat. RpPat also modified acetyl-CoA (Ac-CoA) synthetase (Acs) from R. palustris. In vivo data indicate that at least two deacetylases reactivate BadAAc. One is SrtN (encoded by srtN, formerly rpa2524), a sirtuin-type NAD+-dependent deacetylase (O-acetyl-ADP-ribose-forming); the other deacetylase is LdaA (encoded by ldaA, for lysine deacetylase A; formerly rpa0954), an acetate-forming protein deacetylase. LdaA reactivated HbaAc and AliAAc in vitro. PMID:20345662

  19. A special acyl carrier protein for transferring long hydroxylated fatty acids to lipid A in Rhizobium.

    PubMed

    Brozek, K A; Carlson, R W; Raetz, C R

    1996-12-13

    Lipid A, the hydrophobic anchor of lipopolysaccharides in the outer membranes of Gram-negative bacteria, varies in structure among different Rhizobiaceae. The Rhizobium meliloti lipid A backbone, like that of Escherichia coli, is a beta1'-6-linked glucosamine disaccharide that is phosphorylated at positions 1 and 4'. Rhizobium leguminosarum lipid A lacks both phosphates, but contains aminogluconate in place of the proximal glucosamine 1-phosphate, and galacturonic acid instead of the 4'-phosphate. A peculiar feature of the lipid As of all Rhizobiaceae is acylation with 27-hydroxyoctacosanoic acid, a long hydroxylated fatty acid not found in E. coli. We now describe an in vitro system, consisting of a membrane enzyme and a cytosolic acyl donor from R. leguminosarum, that transfers 27-hydroxyoctacosanoic acid to (Kdo)2-lipid IVA, a key lipid A precursor common to both E. coli and R. leguminosarum. The 27-hydroxyoctacosanoic acid moiety was detected in the lipid product by mass spectrometry. The membrane enzyme required the presence of Kdo residues in the acceptor substrate for activity. The cytosolic acyl donor was purified from wild-type R. leguminosarum using the acylation of (Kdo)2-[4'-32P]-lipid IVA as the assay. Amino-terminal sequencing of the purified acyl donor revealed an exact 19-amino acid match with a partially sequenced gene (orf*) of R. leguminosarum. Orf* contains the consensus sequence, DSLD, for attachment of 4'-phosphopantetheine. When the entire orf* gene was sequenced, it was found to encode a protein of 92 amino acids. Orf* is a new kind of acyl carrier protein because it is only approximately 25% identical both to the constitutive acyl carrier protein (AcpP) and to the inducible acyl carrier protein (NodF) of R. leguminosarum. Mass spectrometry of purified active Orf* confirmed the presence of 4'-phosphopantetheine and 27-hydroxyoctacosanoic acid in the major species. Smaller mass peaks indicative of Orf* acylation with hydroxylated 20, 22, 24

  20. Characteristics, protein engineering and applications of microbial thermostable pullulanases and pullulan hydrolases.

    PubMed

    Nisha, M; Satyanarayana, T

    2016-07-01

    Pullulan hydrolyzing enzymes are endoacting, classified based on the substrate specificity and hydrolysis products as pullulanases (type I and II) and pullulan hydrolases (type I, II and III). Pullulanases and pullulan hydrolase type I are produced by bacteria and archaea. Among bacteria, many mesophilic, thermophilic and hyperthermophilic bacteria produce pullulanases and neopullulanases. While pullulan hydrolase type II and type III are produced by fungi and archaea, respectively. These are multi-domain proteins with three conserved catalytic acidic residues of the glycosyl hydrolases. The recent advances in molecular biology and protein engineering via mutagenesis and truncation led to improvement in thermostability, catalytic activity and substrate specificity. Pullulanases are debranching enzymes, which are widely employed in starch saccharification that minimizes the use of glucoamylase (approx. 50 %) and reduces the total reaction time of the industrial starch conversion process. The thermostable amylopullulanases are useful in one-step starch liquefaction and saccharification, which replaces amylolytic enzymes like α-amylase and glucoamylase, thus resulting in the reduction in the cost of sugar production. The enzymes also find application in making resistant starches and as an antistale in bread making. Panose and isopanose containing syrups are useful as prebiotics, while panose has also been reported to display anticarcinogenic activity. This review focuses on the distinguishing features of these enzymes based on the analysis of amino acid sequences and domain structure, besides highlighting recent advances in the molecular biology and protein engineering for enhancing their thermostability, catalytic activity and substrate specificity. This review also briefly summarizes the potential applications of pullulanases and pullulan hydrolases.

  1. Different types of dienelactone hydrolase in 4-fluorobenzoate-utilizing bacteria.

    PubMed Central

    Schlömann, M; Schmidt, E; Knackmuss, H J

    1990-01-01

    Of various benzoate-utilizing bacteria tested, Alcaligenes eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, A. eutrophus JMP134, Alcaligenes strain A7, and Pseudomonas cepacia were able to grow with 4-fluorobenzoate as the sole source of carbon and energy. P. cepacia also utilizes 3-fluorobenzoate. Except for A. eutrophus JMP134, which is known to grow with 2,4-dichlorophenoxyacetate and 3-chlorobenzoate (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981), the strains were unable to grow at the expense of these compounds or 4-chlorobenzoate. Assays of cell extracts revealed that all strains express dienelactone hydrolase and maleylacetate reductase activities in addition to enzymes of the catechol branch of the 3-oxoadipate pathway when growing with 4-fluorobenzoate. Induction of dienelactone hydrolase and maleylacetate reductase apparently is not necessarily connected to synthesis of catechol 1,2-dioxygenase type II and chloromuconate cycloisomerase activities, which are indispensable for the degradation of chlorocatechols. Substrate specificities of the dienelactone hydrolases provisionally differentiate among three types of this activity. (i) Extracts of A. eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, and Alcaligenes strain A7 convert trans-4-carboxymethylenebut-2-en-4-olide (trans-dienelactone) much faster than the cis-isomer (type I). (ii) The enzyme present in P. cepacia shows the opposite preference for the isomeric substrates (type II). (iii) Cell extracts of A. eutrophus JMP134, as well as purified dienelactone hydrolase from Pseudomonas strain B13 (E. Schmidt and H.-J. Knackmuss, Biochem. J. 192:339-347, 1980), hydrolyze both dienelactones at rates that are of the same order of magnitude (type III). This classification implies that A. eutrophus JMP134 possesses at least two different dienelactone hydrolases, one of type III encoded by the plasmid pJP4 and one of type I, which is also present in the cured strain JMP222. PMID

  2. Acylated flavonol glycosides from the flower of Elaeagnus angustifolia L.

    PubMed

    Bendaikha, Sarah; Gadaut, Méredith; Harakat, Dominique; Magid, Alabdul

    2014-07-01

    Seven acylated flavonol glycosides named elaeagnosides A-G, in addition to seven known flavonoids were isolated from the flowers of Elaeagnus angustifolia. Their structures were elucidated by different spectroscopic methods including 1D, 2D NMR experiments and HR-ESI-MS analysis. In order to identify natural antioxidant and tyrosinase inhibitor agents, the abilities of these flavonoids to scavenge the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and to inhibit tyrosinase activity were evaluated. Results revealed that two of these compounds had significant anti-oxidant effect and one compound showed weak tyrosinase-inhibitory activity compared with kojic acid, quercetin, or ascorbic acid, which were used as positive control.

  3. A novel meta-cleavage product hydrolase from Flavobacterium sp. ATCC27551

    SciTech Connect

    Khajamohiddin, Syed; Babu, Pakala Suresh; Chakka, Deviprasanna; Merrick, Mike; Bhaduri, Anirban; Sowdhamini, Ramanathan; Siddavattam, Dayananda . E-mail: sdsl@uohyd.ernet.in

    2006-12-22

    The organophosphate degrading (opd) gene cluster of plasmid pPDL2 of Flavobacterium sp. ATCC27551 contains a novel open-reading frame, orf243. This was predicted to encode an {alpha}/{beta} hydrolase distantly related to the meta-fission product (MFP) hydrolases such as XylF, PhnD, and CumD. By homology modeling Orf243 has most of the structural features of MFP hydrolases including the characteristic active site catalytic triad. The purified protein (designated MfhA) is a homotetramer and shows similar affinity for 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD), 2-hydroxymuconic semialdehyde (HMSA), and 2-hydroxy-5-methylmuconic semialdehyde (HMMSA), the meta-fission products of 3-methyl catechol, catechol, and 4-methyl catechol. The unique catalytic properties of MfhA and the presence near its structural gene of cis-elements required for transposition suggest that mfhA has evolved towards encoding a common hydrolase that can act on meta-fission products containing either aldehyde or ketone groups.

  4. Supplementing with Non-Glycoside Hydrolase Proteins Enhances Enzymatic Deconstruction of Plant Biomass

    PubMed Central

    Su, Xiaoyun; Zhang, Jing; Mackie, Roderick I.; Cann, Isaac K. O.

    2012-01-01

    The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry. PMID:22952777

  5. In silico prediction of acyl glucuronide reactivity

    NASA Astrophysics Data System (ADS)

    Potter, Tim; Lewis, Richard; Luker, Tim; Bonnert, Roger; Bernstein, Michael A.; Birkinshaw, Timothy N.; Thom, Stephen; Wenlock, Mark; Paine, Stuart

    2011-11-01

    Drugs and drug candidates containing a carboxylic acid moiety, including many widely used non-steroidal anti-inflammatory drugs (NSAIDs) are often metabolized to form acyl glucuronides (AGs). NSAIDs such as Ibuprofen are amongst the most widely used drugs on the market, whereas similar carboxylic acid drugs such as Suprofen have been withdrawn due to adverse events. Although the link between these AG metabolites and toxicity is not proven, there is circumstantial literature evidence to suggest that more reactive acyl glucuronides may, in some cases, present a greater risk of exhibiting toxic effects. We wished therefore to rank the reactivity of potential new carboxylate-containing drug candidates, and performed kinetic studies on synthetic acyl glucuronides to benchmark our key compounds. Driven by the desire to quickly rank the reactivity of compounds without the need for lengthy synthesis of the acyl glucuronide, a correlation was established between the degradation half-life of the acyl glucuronide and the half life for the hydrolysis of the more readily available methyl ester derivative. This finding enabled a considerable broadening of chemical property space to be investigated. The need for kinetic measurements was subsequently eliminated altogether by correlating the methyl ester hydrolysis half-life with the predicted 13C NMR chemical shift of the carbonyl carbon together with readily available steric descriptors in a PLS model. This completely in silico prediction of acyl glucuronide reactivity is applicable within the earliest stages of drug design with low cost and acceptable accuracy to guide intelligent molecular design. This reactivity data will be useful alongside the more complex additional pharmacokinetic exposure and distribution data that is generated later in the drug discovery process for assessing the overall toxicological risk of acidic drugs.

  6. Characterization of intracellular pteroylpolyglutamate hydrolase (PPH) from human intestinal mucosa

    SciTech Connect

    Wang, T.T.Y.; Chandler, C.J.; Halsted, C.H.

    1986-03-01

    There are two forms of pteroylpolyglutamate hydrolase (PPH) in the human intestinal mucosa, one in the brush border membrane and the other intracellular; brush border PPH is an exopeptidase with optimal activity at pH 6.5 and a requirement for zinc. The presence study characterized human intracellular PPH and compared its properties to those of brush border PPH. Intracellular PPH was purified 30-fold. The enzyme had a MW of 75,000 by gel filtration, was optimally active at pH 4.5, and had an isoelectric point at pH 8.0. In contrast to brush border PPH, intracellular PPH was unstable at increasing temperatures, was unaffected by dialysis against chelating agents and showed no requirement for Zn/sup 2 +/. Using PteGlu/sub 2/(/sup 14/C)Glu as substrate, they demonstrated a K/sub m/ of 1.2 ..mu..M and increasing affinity for folates with longer glutamate chains. Intracellular PPH required the complete folic acid (PteGlu) moiety and a ..gamma..-glutamyl linkage for activity. Using ion exchange chromatography and an HPLC method to determine the hydrolytic products of the reaction, they found intracellular PPH could cleave both internal and terminal ..gamma..-glutamyl linkages, with PteGlu as an end product. After subcellular fractionation of the mucosa, PPH was found in the lysosomes. In summary, the distinct characteristics of brush border and intracellular PPH suggest that the two hydrolases serve different roles in folate metabolism.

  7. Acyl-coenzyme A:cholesterol acyltransferases

    PubMed Central

    Chang, Ta-Yuan; Li, Bo-Liang; Chang, Catherine C. Y.; Urano, Yasuomi

    2009-01-01

    The enzymes acyl-coenzyme A (CoA):cholesterol acyltransferases (ACATs) are membrane-bound proteins that utilize long-chain fatty acyl-CoA and cholesterol as substrates to form cholesteryl esters. In mammals, two isoenzymes, ACAT1 and ACAT2, encoded by two different genes, exist. ACATs play important roles in cellular cholesterol homeostasis in various tissues. This chapter summarizes the current knowledge on ACAT-related research in two areas: 1) ACAT genes and proteins and 2) ACAT enzymes as drug targets for atherosclerosis and for Alzheimer's disease. PMID:19141679

  8. Proteomic Analysis of a Poplar Cell Suspension Culture Suggests a Major Role of Protein S-Acylation in Diverse Cellular Processes

    PubMed Central

    Srivastava, Vaibhav; Weber, Joseph R.; Malm, Erik; Fouke, Bruce W.; Bulone, Vincent

    2016-01-01

    S-acylation is a reversible post-translational modification of proteins known to be involved in membrane targeting, subcellular trafficking, and the determination of a great variety of functional properties of proteins. The aim of this work was to identify S-acylated proteins in poplar. The use of an acyl-biotin exchange method and mass spectrometry allowed the identification of around 450 S-acylated proteins, which were subdivided into three major groups of proteins involved in transport, signal transduction, and response to stress, respectively. The largest group of S-acylated proteins was the protein kinase superfamily. Soluble N-ethylmaleimide-sensitive factor-activating protein receptors, band 7 family proteins and tetraspanins, all primarily related to intracellular trafficking, were also identified. In addition, cell wall related proteins, including cellulose synthases and other glucan synthases, were found to be S-acylated. Twenty four of the identified S-acylated proteins were also enriched in detergent-resistant membrane microdomains, suggesting S-acylation plays a key role in the localization of proteins to specialized plasma membrane subdomains. This dataset promises to enhance our current understanding of the various functions of S-acylated proteins in plants. PMID:27148305

  9. Sulfonyl Fluoride Inhibitors of Fatty Acid Amide Hydrolase

    PubMed Central

    Alapafuja, Shakiru O.; Nikas, Spyros P.; Bharatan, Indu; Shukla, Vidyanand G.; Nasr, Mahmoud L.; Bowman, Anna L.; Zvonok, Nikolai; Li, Jing; Shi, Xiaomeng; Engen, John R.; Makriyannis, Alexandros

    2013-01-01

    Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs. PMID:23083016

  10. The cytotoxic effect of 2-acylated-1,4-naphthohydroquinones on leukemia/lymphoma cells.

    PubMed

    Pedroza, Diego A; De Leon, Fernando; Varela-Ramirez, Armando; Lema, Carolina; Aguilera, Renato J; Mito, Shizue

    2014-01-15

    Here, we tested seven 2-acylated-1,4-hydronaphthoquinones for their cytotoxic effects on a panel of cancer lymphoma/leukemia cells and compared to a non-cancer origin cell line. Several naphthohydroquinones exhibited selective cytotoxic effects on lymphoma/leukemia cells with lowest activity on non-cancer cells. The mode of cell death induced by an acylated naphthohydroquinone, which has a long alkyl chain, was found to be via apoptosis. Furthermore, the naphthohydroquinone provoked mitochondria depolarization and activation of its downstream effector, caspase-3, thus implicating the intrinsic apoptotic pathway as its mechanism to exert cell death.

  11. The cytotoxic effect of 2-acylated-1,4-naphthohydroquinones on leukemia/lymphoma cells

    PubMed Central

    Pedroza, Diego A.; De Leon, Fernando; Varela-Ramirez, Armando; Lema, Carolina; Aguilera, Renato J.; Mito, Shizue

    2014-01-01

    Here, we tested seven 2-acylated-1,4-hydronaphthoquinones for their cytotoxic effects on a panel of cancer lymphoma/leukemia cells and compared to a non-cancer origin cell line. Several naphthohydroquinones exhibited selective cytotoxic effects on lymphoma/leukemia cells with lowest activity on non-cancer cells. The mode of cell death induced by an acylated naphthohydroquinone, which has a long alkyl chain, was found to be via apoptosis. Furthermore, the naphthohydroquinone provoked mitochondria depolarization and activation of its downstream effector, caspase-3, thus implicating the intrinsic apoptotic pathway as its mechanism to exert cell death. PMID:24368029

  12. Acyl anion free N-heterocyclic carbene organocatalysis.

    PubMed

    Ryan, Sarah J; Candish, Lisa; Lupton, David W

    2013-06-21

    Reaction discovery using N-heterocyclic carbene organocatalysis has been dominated by the chemistry of acyl anion equivalents. Recent studies demonstrate that NHCs are far more diverse catalysts, with a variety of reactions discovered that proceed without acyl anion equivalent formation. In this tutorial review selected examples of acyl anion free NHC catalysis using carbonyl compounds are presented.

  13. Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans

    PubMed Central

    Tuck, Laura R.; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D.; Campopiano, Dominic J.; Clarke, David J.; Marles-Wright, Jon

    2016-01-01

    The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD+. This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences w